Lars Steinmetz

All Publications

• Size-exclusion chromatography combined with DIA-MS enables deep proteome profiling of extracellular vesicles from melanoma plasma and serum. Cellular and molecular life sciences : CMLS Lattmann, E., Rass, L., Tognetti, M., Gomez, J. M., Lapaire, V., Bruderer, R., Reiter, L., Feng, Y., Steinmetz, L. M., Levesque, M. P. 2024; 81 (1): 90

  Abstract

  Extracellular vesicles (EVs) are important players in melanoma progression, but their use as clinical biomarkers has been limited by the difficulty of profiling blood-derived EV proteins with high depth of coverage, the requirement for large input amounts, and complex protocols. Here, we provide a streamlined and reproducible experimental workflow to identify plasma- and serum- derived EV proteins of healthy donors and melanoma patients using minimal amounts of sample input. SEC-DIA-MS couples size-exclusion chromatography to EV concentration and deep-proteomic profiling using data-independent acquisition. From as little as 200L of plasma per patient in a cohort of three healthy donors and six melanoma patients, we identified and quantified 2896 EV-associated proteins, achieving a 3.5-fold increase in depth compared to previously published melanoma studies. To compare the EV-proteome to unenriched blood, we employed an automated workflow to deplete the 14 most abundant proteins from plasma and serum and thereby approximately doubled protein group identifications versus native blood. The EV proteome diverged from corresponding unenriched plasma and serum, and unlike the latter, separated healthy donor and melanoma patient samples. Furthermore, known melanoma markers, such as MCAM, TNC, and TGFBI, were upregulated in melanoma EVs but not in depleted melanoma plasma, highlighting the specific information contained in EVs. Overall, EVs were significantly enriched in intact membrane proteins and proteins related to SNARE protein interactions and T-cell biology. Taken together, we demonstrated the increased sensitivity of an EV-based proteomic workflow that can be easily applied to larger melanoma cohorts and other indications.

  View details for DOI 10.1007/s00018-024-05137-y

  View details for PubMedID 38353833

• Dissecting quantitative trait nucleotides by saturation genome editing. bioRxiv : the preprint server for biology Roy, K. R., Smith, J. D., Li, S., Vonesch, S. C., Nguyen, M., Burnett, W. T., Orsley, K. M., Lee, C. S., Haber, J. E., St Onge, R. P., Steinmetz, L. M. 2024

  Abstract

  Genome editing technologies have the potential to transform our understanding of how genetic variation gives rise to complex traits through the systematic engineering and phenotypic characterization of genetic variants. However, there has yet to be a system with sufficient efficiency, fidelity, and throughput to comprehensively identify causal variants at the genome scale. Here we explored the ability of templated CRISPR editing systems to install natural variants genome-wide in budding yeast. We optimized several approaches to enhance homology-directed repair (HDR) with donor DNA templates, including donor recruitment to target sites, single-stranded donor production by bacterial retrons, and in vivo plasmid assembly. We uncovered unique advantages of each system that we integrated into a single superior system named MAGESTIC 3.0. We used MAGESTIC 3.0 to dissect causal variants residing in 112 quantitative trait loci across 32 environmental conditions, revealing an enrichment for missense variants and loci with multiple causal variants. MAGESTIC 3.0 will facilitate the functional analysis of the genome at single-nucleotide resolution and provides a roadmap for improving template-based genome editing systems in other organisms.

  View details for DOI 10.1101/2024.02.02.577784

  View details for PubMedID 38352467

  View details for PubMedCentralID PMC10862795

• Genome-scale analysis of interactions between genetic perturbations and natural variation. bioRxiv : the preprint server for biology Hale, J. J., Matsui, T., Goldstein, I., Mullis, M. N., Roy, K. R., Ville, C. N., Miller, D., Wang, C., Reynolds, T., Steinmetz, L. M., Levy, S. F., Ehrenreich, I. M. 2024

  Abstract

  Interactions between genetic perturbations and segregating loci can cause perturbations to show different phenotypic effects across genetically distinct individuals. To study these interactions on a genome scale in many individuals, we used combinatorial DNA barcode sequencing to measure the fitness effects of 7,700 CRISPRi perturbations targeting 1,712 distinct genes in 169 yeast cross progeny (or segregants). We identified 460 genes whose perturbation has different effects across segregants. Several factors caused perturbations to show variable effects, including baseline segregant fitness, the mean effect of a perturbation across segregants, and interacting loci. We mapped 234 interacting loci and found four hub loci that interact with many different perturbations. Perturbations that interact with a given hub exhibit similar epistatic relationships with the hub and show enrichment for cellular processes that may mediate these interactions. These results suggest that an individual's response to perturbations is shaped by a network of perturbation-locus interactions that cannot be measured by approaches that examine perturbations or natural variation alone.

  View details for DOI 10.1101/2023.05.06.539663

  View details for PubMedID 38293072

  View details for PubMedCentralID PMC10827069

• Uterine injury during diestrus leads to placental and embryonic defects in future pregnancies in mice. Biology of reproduction Zhang, E. T., Wells, K. L., Bergman, A. J., Ryan, E. E., Steinmetz, L. M., Baker, J. C. 2024

  Abstract

  Uterine injury from procedures such as Cesarean sections (C-sections) often have severe consequences on subsequent pregnancy outcomes, leading to disorders such as placenta previa, placenta accreta, and infertility. With rates of C-section at approximately 30% of deliveries in the US and projected to continue to climb, a deeper understanding of the mechanisms by which these pregnancy disorders arise and opportunities for intervention are needed. Here we describe a rodent model of uterine injury on subsequent in utero outcomes. We observed three distinct phenotypes: increased rates of resorption and death, embryo spacing defects, and placenta accreta-like features of reduced decidua and expansion of invasive trophoblasts. We show that the appearance of embryo spacing defects depends entirely on the phase of estrous cycle at the time of injury. Using RNA-seq, we identified perturbations in the expression of components of the COX/prostaglandin pathway after recovery from injury, a pathway that has previously been demonstrated to play an important role in embryo spacing. Therefore, we demonstrate that uterine damage in this mouse model causes morphological and molecular changes that ultimately lead to placental and embryonic developmental defects.

  View details for DOI 10.1093/biolre/ioae001

  View details for PubMedID 38206869

• An encyclopedia of enhancer-gene regulatory interactions in the human genome. bioRxiv : the preprint server for biology Gschwind, A. R., Mualim, K. S., Karbalayghareh, A., Sheth, M. U., Dey, K. K., Jagoda, E., Nurtdinov, R. N., Xi, W., Tan, A. S., Jones, H., Ma, X. R., Yao, D., Nasser, J., Avsec, Ž., James, B. T., Shamim, M. S., Durand, N. C., Rao, S. S., Mahajan, R., Doughty, B. R., Andreeva, K., Ulirsch, J. C., Fan, K., Perez, E. M., Nguyen, T. C., Kelley, D. R., Finucane, H. K., Moore, J. E., Weng, Z., Kellis, M., Bassik, M. C., Price, A. L., Beer, M. A., Guigó, R., Stamatoyannopoulos, J. A., Lieberman Aiden, E., Greenleaf, W. J., Leslie, C. S., Steinmetz, L. M., Kundaje, A., Engreitz, J. M. 2023

  Abstract

  Identifying transcriptional enhancers and their target genes is essential for understanding gene regulation and the impact of human genetic variation on disease1-6. Here we create and evaluate a resource of >13 million enhancer-gene regulatory interactions across 352 cell types and tissues, by integrating predictive models, measurements of chromatin state and 3D contacts, and largescale genetic perturbations generated by the ENCODE Consortium7. We first create a systematic benchmarking pipeline to compare predictive models, assembling a dataset of 10,411 elementgene pairs measured in CRISPR perturbation experiments, >30,000 fine-mapped eQTLs, and 569 fine-mapped GWAS variants linked to a likely causal gene. Using this framework, we develop a new predictive model, ENCODE-rE2G, that achieves state-of-the-art performance across multiple prediction tasks, demonstrating a strategy involving iterative perturbations and supervised machine learning to build increasingly accurate predictive models of enhancer regulation. Using the ENCODE-rE2G model, we build an encyclopedia of enhancer-gene regulatory interactions in the human genome, which reveals global properties of enhancer networks, identifies differences in the functions of genes that have more or less complex regulatory landscapes, and improves analyses to link noncoding variants to target genes and cell types for common, complex diseases. By interpreting the model, we find evidence that, beyond enhancer activity and 3D enhancer-promoter contacts, additional features guide enhancerpromoter communication including promoter class and enhancer-enhancer synergy. Altogether, these genome-wide maps of enhancer-gene regulatory interactions, benchmarking software, predictive models, and insights about enhancer function provide a valuable resource for future studies of gene regulation and human genetics.

  View details for DOI 10.1101/2023.11.09.563812

  View details for PubMedID 38014075

  View details for PubMedCentralID PMC10680627

• Consequences of a telomerase-related fitness defect and chromosome substitution technology in yeast synIX strains. Cell genomics McCulloch, L. H., Sambasivam, V., Hughes, A. L., Annaluru, N., Ramalingam, S., Fanfani, V., Lobzaev, E., Mitchell, L. A., Cai, J., Jiang, H., LaCava, J., Taylor, M. S., Bishai, W. R., Stracquadanio, G., Steinmetz, L. M., Bader, J. S., Zhang, W., Boeke, J. D., Chandrasegaran, S. 2023; 3 (11): 100419

  Abstract

  We describe the complete synthesis, assembly, debugging, and characterization of a synthetic 404,963 bp chromosome, synIX (synthetic chromosome IX). Combined chromosome construction methods were used to synthesize and integrate its left arm (synIXL) into a strain containing previously described synIXR. We identified and resolved a bug affecting expression of EST3, a crucial gene for telomerase function, producing a synIX strain with near wild-type fitness. To facilitate future synthetic chromosome consolidation and increase flexibility of chromosome transfer between distinct strains, we combined chromoduction, a method to transfer a whole chromosome between two strains, with conditional centromere destabilization to substitute a chromosome of interest for its native counterpart. Both steps of this chromosome substitution method were efficient. We observed that wild-type II tended to co-transfer with synIX and was co-destabilized with wild-type IX, suggesting a potential gene dosage compensation relationship between these chromosomes.

  View details for DOI 10.1016/j.xgen.2023.100419

  View details for PubMedID 38020974

  View details for PubMedCentralID PMC10667316

• Design, construction, and functional characterization of a tRNA neochromosome in yeast. Cell Schindler, D., Walker, R. S., Jiang, S., Brooks, A. N., Wang, Y., Muller, C. A., Cockram, C., Luo, Y., Garcia, A., Schraivogel, D., Mozziconacci, J., Pena, N., Assari, M., Sanchez Olmos, M. D., Zhao, Y., Ballerini, A., Blount, B. A., Cai, J., Ogunlana, L., Liu, W., Jonsson, K., Abramczyk, D., Garcia-Ruiz, E., Turowski, T. W., Swidah, R., Ellis, T., Pan, T., Antequera, F., Shen, Y., Nieduszynski, C. A., Koszul, R., Dai, J., Steinmetz, L. M., Boeke, J. D., Cai, Y. 2023

  Abstract

  Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the 190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-S.cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs.

  View details for DOI 10.1016/j.cell.2023.10.015

  View details for PubMedID 37944512

• Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions. Cell Zhao, Y., Coelho, C., Hughes, A. L., Lazar-Stefanita, L., Yang, S., Brooks, A. N., Walker, R. S., Zhang, W., Lauer, S., Hernandez, C., Cai, J., Mitchell, L. A., Agmon, N., Shen, Y., Sall, J., Fanfani, V., Jalan, A., Rivera, J., Liang, F. X., Bader, J. S., Stracquadanio, G., Steinmetz, L. M., Cai, Y., Boeke, J. D. 2023

  Abstract

  The Sc2.0 project is building a eukaryotic synthetic genome from scratch. A major milestone has been achieved with all individual Sc2.0 chromosomes assembled. Here, we describe the consolidation of multiple synthetic chromosomes using advanced endoreduplication intercrossing with tRNA expression cassettes to generate a strain with 6.5 synthetic chromosomes. The 3D chromosome organization and transcript isoform profiles were evaluated using Hi-C and long-read direct RNA sequencing. We developed CRISPR Directed Biallelic URA3-assisted Genome Scan, or "CRISPR D-BUGS," to map phenotypic variants caused by specific designer modifications, known as "bugs." We first fine-mapped a bug in synthetic chromosome II (synII) and then discovered a combinatorial interaction associated with synIII and synX, revealing an unexpected genetic interaction that links transcriptional regulation, inositol metabolism, and tRNASerCGA abundance. Finally, to expedite consolidation, we employed chromosome substitution to incorporate the largest chromosome (synIV), thereby consolidating >50% of the Sc2.0 genome in one strain.

  View details for DOI 10.1016/j.cell.2023.09.025

  View details for PubMedID 37944511

• Identification and quantification of small exon-containing isoforms in long-read RNA sequencing data. Nucleic acids research Liu, Z., Zhu, C., Steinmetz, L. M., Wei, W. 2023

  Abstract

  Small exons are pervasive in transcriptomes across organisms, and their quantification in RNA isoforms is crucial for understanding gene functions. Although long-read RNA-seq based on Oxford Nanopore Technologies (ONT) offers the advantage of covering transcripts in full length, its lower base accuracy poses challenges for identifying individual exons, particularly microexons (≤ 30 nucleotides). Here, we systematically assess small exons quantification in synthetic and human ONT RNA-seq datasets. We demonstrate that reads containing small exons are often not properly aligned, affecting the quantification of relevant transcripts. Thus, we develop a local-realignment method for misaligned exons (MisER), which remaps reads with misaligned exons to the transcript references. Using synthetic and simulated datasets, we demonstrate the high sensitivity and specificity of MisER for the quantification of transcripts containing small exons. Moreover, MisER enabled us to identify small exons with a higher percent spliced-in index (PSI) in neural, particularly neural-regulated microexons, when comparing 14 neural to 16 non-neural tissues in humans. Our work introduces an improved quantification method for long-read RNA-seq and especially facilitates studies using ONT long-reads to elucidate the regulation of genes involving small exons.

  View details for DOI 10.1093/nar/gkad810

  View details for PubMedID 37843096

• Gut barrier defects, intestinal immune hyperactivation and enhanced lipid catabolism drive lethality in NGLY1-deficient Drosophila. Nature communications Pandey, A., Galeone, A., Han, S. Y., Story, B. A., Consonni, G., Mueller, W. F., Steinmetz, L. M., Vaccari, T., Jafar-Nejad, H. 2023; 14 (1): 5667

  Abstract

  Intestinal barrier dysfunction leads to inflammation and associated metabolic changes. However, the relative impact of gut bacteria versus non-bacterial insults on animal health in the context of barrier dysfunction is not well understood. Here, we establish that loss of Drosophila N-glycanase 1 (Pngl) in a specific intestinal cell type leads to gut barrier defects, causing starvation and JNK overactivation. These abnormalities, along with loss of Pngl in enterocytes and fat body, result in Foxo overactivation, leading to hyperactive innate immune response and lipid catabolism and thereby contributing to lethality. Germ-free rearing of Pngl mutants rescued their developmental delay but not lethality. However, raising Pngl mutants on isocaloric, fat-rich diets partially rescued lethality. Our data indicate that Pngl functions in Drosophila larvae to establish the gut barrier, and that the lethality caused by loss of Pngl is primarily mediated through non-bacterial induction of immune and metabolic abnormalities.

  View details for DOI 10.1038/s41467-023-40910-w

  View details for PubMedID 37704604

• Digital assay for rapid electronic quantification of clinical pathogens using DNA nanoballs. Science advances Tayyab, M., Barrett, D., van Riel, G., Liu, S., Reinius, B., Scharfe, C., Griffin, P., Steinmetz, L. M., Javanmard, M., Pelechano, V. 2023; 9 (36): eadi4997

  Abstract

  Fast and accurate detection of nucleic acids is key for pathogen identification. Methods for DNA detection generally rely on fluorescent or colorimetric readout. The development of label-free assays decreases costs and test complexity. We present a novel method combining a one-pot isothermal generation of DNA nanoballs with their detection by electrical impedance. We modified loop-mediated isothermal amplification by using compaction oligonucleotides that self-assemble the amplified target into nanoballs. Next, we use capillary-driven flow to passively pass these nanoballs through a microfluidic impedance cytometer, thus enabling a fully compact system with no moving parts. The movement of individual nanoballs is detected by a change in impedance providing a quantized readout. This approach is flexible for the detection of DNA/RNA of numerous targets (severe acute respiratory syndrome coronavirus 2, HIV, β-lactamase gene, etc.), and we anticipate that its integration into a standalone device would provide an inexpensive (<$5), sensitive (10 target copies), and rapid test (<1 hour).

  View details for DOI 10.1126/sciadv.adi4997

  View details for PubMedID 37672583

  View details for PubMedCentralID PMC10482329

• Pooled Genome-Scale CRISPR Screens in Single Cells. Annual review of genetics Schraivogel, D., Steinmetz, L. M., Parts, L. 2023

  Abstract

  Assigning functions to genes and learning how to control their expression are part of the foundation of cell biology and therapeutic development. An efficient and unbiased method to accomplish this is genetic screening, which historically required laborious clone generation and phenotyping and is still limited by scale today. The rapid technological progress on modulating gene function with CRISPR-Cas and measuring it in individual cells has now relaxed the major experimental constraints and enabled pooled screening with complex readouts from single cells. Here, we review the principles and practical considerations for pooled single-cell CRISPR screening. We discuss perturbation strategies, experimental model systems, matching the perturbation to the individual cells, reading out cell phenotypes, and data analysis. Our focus is on single-cell RNA sequencing and cell sorting-based readouts, including image-enabled cell sorting. We expect this transformative approach to fuel biomedical research for the next several decades. Expected final online publication date for the Annual Review of Genetics, Volume 57 is November 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  View details for DOI 10.1146/annurev-genet-072920-013842

  View details for PubMedID 37562410

• Large scale microfluidic CRISPR screening for increased amylase secretion in yeast. Lab on a chip Johansson, S. A., Dulermo, T., Jann, C., Smith, J. D., Pryszlak, A., Pignede, G., Schraivogel, D., Colavizza, D., Desfougères, T., Rave, C., Farwick, A., Merten, C. A., Roy, K. R., Wei, W., Steinmetz, L. M. 2023

  Abstract

  Key to our ability to increase recombinant protein production through secretion is a better understanding of the pathways that interact to translate, process and export mature proteins to the surrounding environment, including the supporting cellular machinery that supplies necessary energy and building blocks. By combining droplet microfluidic screening with large-scale CRISPR libraries that perturb the expression of the majority of coding and non-coding genes in S. cerevisiae, we identified 345 genes for which an increase or decrease in gene expression resulted in increased secretion of α-amylase. Our results show that modulating the expression of genes involved in the trafficking of vesicles, endosome to Golgi transport, the phagophore assembly site, the cell cycle and energy supply improve α-amylase secretion. Besides protein-coding genes, we also find multiple long non-coding RNAs enriched in the vicinity of genes associated with endosomal, Golgi and vacuolar processes. We validated our results by overexpressing or deleting selected genes, which resulted in significant improvements in α-amylase secretion. The advantages, in terms of precision and speed, inherent to CRISPR based perturbations, enables iterative testing of new strains for increased protein secretion.

  View details for DOI 10.1039/d3lc00111c

  View details for PubMedID 37483015

• Mislocalization of pathogenic RBM20 variants in dilated cardiomyopathy is caused by loss-of-interaction with Transportin-3. Nature communications Kornienko, J., Rodríguez-Martínez, M., Fenzl, K., Hinze, F., Schraivogel, D., Grosch, M., Tunaj, B., Lindenhofer, D., Schraft, L., Kueblbeck, M., Smith, E., Mao, C., Brown, E., Owens, A., Saguner, A. M., Meder, B., Parikh, V., Gotthardt, M., Steinmetz, L. M. 2023; 14 (1): 4312

  Abstract

  Severe forms of dilated cardiomyopathy (DCM) are associated with point mutations in the alternative splicing regulator RBM20 that are frequently located in the arginine/serine-rich domain (RS-domain). Such mutations can cause defective splicing and cytoplasmic mislocalization, which leads to the formation of detrimental cytoplasmic granules. Successful development of personalized therapies requires identifying the direct mechanisms of pathogenic RBM20 variants. Here, we decipher the molecular mechanism of RBM20 mislocalization and its specific role in DCM pathogenesis. We demonstrate that mislocalized RBM20 RS-domain variants retain their splice regulatory activity, which reveals that aberrant cellular localization is the main driver of their pathological phenotype. A genome-wide CRISPR knockout screen combined with image-enabled cell sorting identified Transportin-3 (TNPO3) as the main nuclear importer of RBM20. We show that the direct RBM20-TNPO3 interaction involves the RS-domain, and is disrupted by pathogenic variants. Relocalization of pathogenic RBM20 variants to the nucleus restores alternative splicing and dissolves cytoplasmic granules in cell culture and animal models. These findings provide proof-of-principle for developing therapeutic strategies to restore RBM20's nuclear localization in RBM20-DCM patients.

  View details for DOI 10.1038/s41467-023-39965-6

  View details for PubMedID 37463913

  View details for PubMedCentralID PMC10353998

• Striated muscle-specific base editing enables correction of mutations causing dilated cardiomyopathy. Nature communications Grosch, M., Schraft, L., Chan, A., Kuchenhoff, L., Rapti, K., Ferreira, A., Kornienko, J., Li, S., Radke, M. H., Kramer, C., Clauder-Munster, S., Perlas, E., Backs, J., Gotthardt, M., Dieterich, C., van den Hoogenhof, M. M., Grimm, D., Steinmetz, L. M. 2023; 14 (1): 3714

  Abstract

  Dilated cardiomyopathy is the second most common cause for heart failure with no cure except a high-risk heart transplantation. Approximately 30% of patients harbor heritable mutations which are amenable to CRISPR-based gene therapy. However, challenges related to delivery of the editing complex and off-target concerns hamper the broad applicability of CRISPR agents in the heart. We employ a combination of the viral vector AAVMYO with superior targeting specificity of heart muscle tissue and CRISPR base editors to repair patient mutations in the cardiac splice factor Rbm20, which cause aggressive dilated cardiomyopathy. Using optimized conditions, we repair >70% of cardiomyocytes in two Rbm20 knock-in mouse models that we have generated to serve as an in vivo platform of our editing strategy. Treatment of juvenile mice restores the localization defect of RBM20 in 75% of cells and splicing of RBM20 targets including TTN. Three months after injection, cardiac dilation and ejection fraction reach wild-type levels. Single-nuclei RNA sequencing uncovers restoration of the transcriptional profile across all major cardiac cell types and whole-genome sequencing reveals no evidence for aberrant off-target editing. Our study highlights the potential of base editors combined with AAVMYO to achieve gene repair for treatment of hereditary cardiac diseases.

  View details for DOI 10.1038/s41467-023-39352-1

  View details for PubMedID 37349314

• Atlas of mRNA translation and decay for bacteria. Nature microbiology Huch, S., Nersisyan, L., Ropat, M., Barrett, D., Wu, M., Wang, J., Valeriano, V. D., Vardazaryan, N., Huerta-Cepas, J., Wei, W., Du, J., Steinmetz, L. M., Engstrand, L., Pelechano, V. 2023

  Abstract

  Regulation of messenger RNA stability is pivotal for programmed gene expression in bacteria and is achieved by a myriad of molecular mechanisms. By bulk sequencing of 5'monophosphorylated mRNA decay intermediates (5'P), we show that cotranslational mRNA degradation is conserved among both Gram-positive and -negative bacteria. We demonstrate that, in species with 5'-3' exonucleases, the exoribonuclease RNaseJ tracks the trailing ribosome to produce an in vivo single-nucleotide toeprint of the 5'position of the ribosome. In other species lacking 5'-3' exonucleases, ribosome positioning alters endonucleolytic cleavage sites. Using our metadegradome (5'P degradome) sequencing approach, we characterize 5'P mRNA decay intermediates in 96species including Bacillus subtilis, Escherichia coli, Synechocystis spp. and Prevotella copri and identify codon- and gene-level ribosome stalling responses to stress and drug treatment. We also apply 5'P sequencing to complex clinical and environmental microbiomes and demonstrate that metadegradome sequencing provides fast, species-specific posttranscriptional characterization of responses to drug or environmental perturbations. Finally we produce a degradome atlas for 96species to enable analysis of mechanisms of RNA degradation in bacteria. Our work paves the way for the application of metadegradome sequencing to investigation of posttranscriptional regulation in unculturable species and complex microbial communities.

  View details for DOI 10.1038/s41564-023-01393-z

  View details for PubMedID 37217719

• A scalable, GMP-compatible, autologous organotypic cell therapy for Dystrophic Epidermolysis Bullosa. bioRxiv : the preprint server for biology Neumayer, G., Torkelson, J. L., Li, S., McCarthy, K., Zhen, H. H., Vangipuram, M., Jackow, J., Rami, A., Hansen, C., Guo, Z., Gaddam, S., Pappalardo, A., Li, L., Cramer, A., Roy, K. R., Nguyen, T. M., Tanabe, K., McGrath, P. S., Bruckner, A., Bilousova, G., Roop, D., Bailey, I., Tang, J. Y., Christiano, A., Steinmetz, L. M., Wernig, M., Oro, A. E. 2023

  Abstract

  Gene editing in induced pluripotent stem (iPS) cells has been hailed to enable new cell therapies for various monogenetic diseases including dystrophic epidermolysis bullosa (DEB). However, manufacturing, efficacy and safety roadblocks have limited the development of genetically corrected, autologous iPS cell-based therapies.We developed Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a new generation GMP-compatible (cGMP), reproducible, and scalable platform to produce autologous clinical-grade iPS cell-derived organotypic induced skin composite (iSC) grafts to treat incurable wounds of patients lacking type VII collagen (C7). DEBCT uses a combined high-efficiency reprogramming and CRISPR-based genetic correction single step to generate genome scar-free, COL7A1 corrected clonal iPS cells from primary patient fibroblasts. Validated iPS cells are converted into epidermal, dermal and melanocyte progenitors with a novel 2D organoid differentiation protocol, followed by CD49f enrichment and expansion to minimize maturation heterogeneity. iSC product characterization by single cell transcriptomics was followed by mouse xenografting for disease correcting activity at 1 month and toxicology analysis at 1-6 months. Culture-acquired mutations, potential CRISPR-off targets, and cancer-driver variants were evaluated by targeted and whole genome sequencing.iPS cell-derived iSC grafts were reproducibly generated from four recessive DEB patients with different pathogenic mutations. Organotypic iSC grafts onto immune-compromised mice developed into stable stratified skin with functional C7 restoration. Single cell transcriptomic characterization of iSCs revealed prominent holoclone stem cell signatures in keratinocytes and the recently described Gibbin-dependent signature in dermal fibroblasts. The latter correlated with enhanced graftability. Multiple orthogonal sequencing and subsequent computational approaches identified random and non-oncogenic mutations introduced by the manufacturing process. Toxicology revealed no detectable tumors after 3-6 months in DEBCT-treated mice.DEBCT successfully overcomes previous roadblocks and represents a robust, scalable, and safe cGMP manufacturing platform for production of a CRISPR-corrected autologous organotypic skin graft to heal DEB patient wounds.

  View details for DOI 10.1101/2023.02.28.529447

  View details for PubMedID 36909618

  View details for PubMedCentralID PMC10002612

• Differential regulation of mRNA stability modulates transcriptional memory and facilitates environmental adaptation. Nature communications Li, B., Zeis, P., Zhang, Y., Alekseenko, A., Fürst, E., Sanchez, Y. P., Lin, G., Tekkedil, M. M., Piazza, I., Steinmetz, L. M., Pelechano, V. 2023; 14 (1): 910

  Abstract

  Transcriptional memory, by which cells respond faster to repeated stimuli, is key for cellular adaptation and organism survival. Chromatin organization has been shown to play a role in the faster response of primed cells. However, the contribution of post-transcriptional regulation is not yet explored. Here we perform a genome-wide screen to identify novel factors modulating transcriptional memory in S. cerevisiae in response to galactose. We find that depletion of the nuclear RNA exosome increases GAL1 expression in primed cells. Our work shows that gene-specific differences in intrinsic nuclear surveillance factor association can enhance both gene induction and repression in primed cells. Finally, we show that primed cells present altered levels of RNA degradation machinery and that both nuclear and cytoplasmic mRNA decay modulate transcriptional memory. Our results demonstrate that mRNA post-transcriptional regulation, and not only transcription regulation, should be considered when investigating gene expression memory.

  View details for DOI 10.1038/s41467-023-36586-x

  View details for PubMedID 36801853

  View details for PubMedCentralID 2779764

• Ehf and Fezf2 regulate late medullary thymic epithelial cell and thymic tuft cell development. Frontiers in immunology Lammers, S., Barrera, V., Brennecke, P., Miller, C., Yoon, J., Balolong, J., Anderson, M. S., Ho Sui, S., Steinmetz, L. M., von Andrian, U. H., Rattay, K. 2023; 14: 1277365

  Abstract

  Thymic epithelial cells are indispensable for T cell maturation and selection and the induction of central immune tolerance. The self-peptide repertoire expressed by medullary thymic epithelial cells is in part regulated by the transcriptional regulator Aire (Autoimmune regulator) and the transcription factor Fezf2. Due to the high complexity of mTEC maturation stages (i.e., post-Aire, Krt10+ mTECs, and Dclk1+ Tuft mTECs) and the heterogeneity in their gene expression profiles (i.e., mosaic expression patterns), it has been challenging to identify the additional factors complementing the transcriptional regulation. We aimed to identify the transcriptional regulators involved in the regulation of mTEC development and self-peptide expression in an unbiased and genome-wide manner. We used ATAC footprinting analysis as an indirect approach to identify transcription factors involved in the gene expression regulation in mTECs, which we validated by ChIP sequencing. This study identifies Fezf2 as a regulator of the recently described thymic Tuft cells (i.e., Tuft mTECs). Furthermore, we identify that transcriptional regulators of the ELF, ESE, ERF, and PEA3 subfamily of the ETS transcription factor family and members of the Krüppel-like family of transcription factors play a role in the transcriptional regulation of genes involved in late mTEC development and promiscuous gene expression.

  View details for DOI 10.3389/fimmu.2023.1277365

  View details for PubMedID 38420512

  View details for PubMedCentralID PMC10901246

• Cell sorters see things more clearly now. Molecular systems biology Schraivogel, D., Steinmetz, L. M. 2023: e11254

  Abstract

  Microscopy and fluorescence-activated cell sorting (FACS) are two of the most important tools for single-cell phenotyping in basic and biomedical research. Microscopy provides high-resolution snapshots of cell morphology and the inner workings of cells, while FACS isolates thousands of cells per second using simple parameters, such as the intensity of fluorescent protein labels. Recent technologies are now combining both methods to enable the fast isolation of cells with microscopic phenotypes of interest, thereby bridging a long-standing gap in the life sciences. In this Commentary, we discuss the technical advancements made by image-enabled cell sorting and highlight novel experimental strategies in functional genomics and single-cell research.

  View details for DOI 10.15252/msb.202211254

  View details for PubMedID 36779527

• Cardiac splicing as a diagnostic and therapeutic target. Nature reviews. Cardiology Gotthardt, M., Badillo-Lisakowski, V., Parikh, V. N., Ashley, E., Furtado, M., Carmo-Fonseca, M., Schudy, S., Meder, B., Grosch, M., Steinmetz, L., Crocini, C., Leinwand, L. 2023

  Abstract

  Despite advances in therapeutics for heart failure and arrhythmias, a substantial proportion of patients with cardiomyopathy do not respond to interventions, indicating a need to identify novel modifiable myocardial pathobiology. Human genetic variation associated with severe forms of cardiomyopathy and arrhythmias has highlighted the crucial role of alternative splicing in myocardial health and disease, given that it determines which mature RNA transcripts drive the mechanical, structural, signalling and metabolic properties of the heart. In this Review, we discuss how the analysis of cardiac isoform expression has been facilitated by technical advances in multiomics and long-read and single-cell sequencing technologies. The resulting insights into the regulation of alternative splicing - including theidentification of cardiac splice regulators as therapeutic targets and the development of a translational pipeline to evaluate splice modulators in human engineered heart tissue, animal models and clinical trials - provide a basis for improved diagnosis and therapy. Finally, we consider how the medical and scientific communities can benefit from facilitated acquisition and interpretation of splicing data towards improved clinical decision-making and patient care.

  View details for DOI 10.1038/s41569-022-00828-0

  View details for PubMedID 36653465

• RBM20 mislocalisation in dilated cardiomyopathy Kornienko, J., Martinez, M., Fenzl, K., Schraft, L., Clauder-Muenster, S., Grosch, M., Steinmetz, L. ELSEVIER SCI LTD. 2022: S102

  View details for Web of Science ID 000907054400200

• Deep phenotyping of two pre-clinical mouse models and a cohort of RBM20 mutation carriers reveals no sex-dependent disease severity in RBM20 cardiomyopathy. American journal of physiology. Heart and circulatory physiology Lennermann, D. C., Pepin, M. E., Grosch, M., Konrad, L., Kemmling, E., Hartmann, J., Nolte, J. L., Clauder-Munster, S., Kayvanpour, E., Sedaghat-Hamedani, F., Haas, J., Meder, B., van den Boogaard, M., Amin, A. S., Dewenter, M., Kruger, M., Steinmetz, L. M., Backs, J., van den Hoogenhof, M. M. 2022

  Abstract

  Aims RBM20 cardiomyopathy is an arrhythmogenic form of dilated cardiomyopathy caused by mutations in the splicing factor RBM20. A recent study found a more severe phenotype in male RBM20 cardiomyopathy patients than in female patients. Here, we aim to determine sex differences in an animal model of RBM20 cardiomyopathy, and investigate potential underlying mechanisms. Additionally, we aim to determine sex and gender differences in clinical parameters in a novel RBM20 cardiomyopathy patient cohort. Methods and Results We characterized a Rbm20 knockout (KO) mouse model, and show that splicing of key RBM20 targets, cardiac function, and arrhythmia susceptibility do not differ between sexes. Next, we performed deep phenotyping of these mice, and show that male and female Rbm20-KO mice possess transcriptomic and phosphoproteomic differences. Hypothesizing that these differences may influence the heart's ability to compensate for stress, we exposed Rbm20-KO mice to acute catecholaminergic stimulation, and again found no functional differences. We also replicate the lack of functional differences in a mouse model with the Rbm20-R636Q mutation. Lastly, we present a patient cohort of 33 RBM20 cardiomyopathy patients, and show that these patients do not possess sex and gender differences in disease severity. Conclusions Current mouse models of RBM20 cardiomyopathy show more pronounced changes in gene expression and phosphorylation of cardiac proteins in male mice, but no sex differences in cardiac morphology and function. Moreover, other than reported before, male RBM20 cardiomyopathy patients do not present with worse cardiac function in a patient cohort from Germany and the Netherlands.

  View details for DOI 10.1152/ajpheart.00328.2022

  View details for PubMedID 36367695

• Hyperactive innate immune response and altered metabolism in a Drosophila model of NGLY1 deficiency Pandey, A., Han, S., Galeone, A., Storey, B. A., Consonni, G., Mueller, W. F., Steinmetz, L., Vaccari, T., Jafar-Nejad, H. OXFORD UNIV PRESS INC. 2022: 1039

  View details for Web of Science ID 000878249700170

• NIMA-related kinase 9 regulates the phosphorylation of the essential myosin light chain in the heart. Nature communications Muller, M., Eghbalian, R., Boeckel, J., Frese, K. S., Haas, J., Kayvanpour, E., Sedaghat-Hamedani, F., Lackner, M. K., Tugrul, O. F., Ruppert, T., Tappu, R., Martins Bordalo, D., Kneuer, J. M., Piekarek, A., Herch, S., Schudy, S., Keller, A., Grammes, N., Bischof, C., Klinke, A., Cardoso-Moreira, M., Kaessmann, H., Katus, H. A., Frey, N., Steinmetz, L. M., Meder, B. 2022; 13 (1): 6209

  Abstract

  To adapt to changing hemodynamic demands, regulatory mechanisms modulate actin-myosin-kinetics by calcium-dependent and -independent mechanisms. We investigate the posttranslational modification of human essential myosin light chain (ELC) and identify NIMA-related kinase 9 (NEK9) to interact with ELC. NEK9 is highly expressed in the heart and the interaction with ELC is calcium-dependent. Silencing of NEK9 results in blunting of calcium-dependent ELC-phosphorylation. CRISPR/Cas9-mediated disruption of NEK9 leads to cardiomyopathy in zebrafish. Binding to ELC is mediated via the protein kinase domain of NEK9. A causal relationship between NEK9 activity and ELC-phosphorylation is demonstrated by genetic sensitizing in-vivo. Finally, we observe significantly upregulated ELC-phosphorylation in dilated cardiomyopathy patients and provide a unique map of human ELC-phosphorylation-sites. In summary, NEK9-mediated ELC-phosphorylation is a calcium-dependent regulatory system mediating cardiac contraction and inotropy.

  View details for DOI 10.1038/s41467-022-33658-2

  View details for PubMedID 36266340

• Genotype Complements the Phenotype: Identification of the Pathogenicity of an LMNA Splice Variant by Nanopore Long-Read Sequencing in a Large DCM Family. International journal of molecular sciences Sedaghat-Hamedani, F., Rebs, S., Kayvanpour, E., Zhu, C., Amr, A., Müller, M., Haas, J., Wu, J., Steinmetz, L. M., Ehlermann, P., Streckfuss-Bömeke, K., Frey, N., Meder, B. 2022; 23 (20)

  Abstract

  Dilated cardiomyopathy (DCM) is a common cause of heart failure (HF) and is of familial origin in 20-40% of cases. Genetic testing by next-generation sequencing (NGS) has yielded a definite diagnosis in many cases; however, some remain elusive. In this study, we used a combination of NGS, human-induced pluripotent-stem-cell-derived cardiomyocytes (iPSC-CMs) and nanopore long-read sequencing to identify the causal variant in a multi-generational pedigree of DCM. A four-generation family with familial DCM was investigated. Next-generation sequencing (NGS) was performed on 22 family members. Skin biopsies from two affected family members were used to generate iPSCs, which were then differentiated into iPSC-CMs. Short-read RNA sequencing was used for the evaluation of the target gene expression, and long-read RNA nanopore sequencing was used to evaluate the relevance of the splice variants. The pedigree suggested a highly penetrant, autosomal dominant mode of inheritance. The phenotype of the family was suggestive of laminopathy, but previous genetic testing using both Sanger and panel sequencing only yielded conflicting evidence for LMNA p.R644C (rs142000963), which was not fully segregated. By re-sequencing four additional affected family members, further non-coding LMNA variants could be detected: rs149339264, rs199686967, rs201379016, and rs794728589. To explore the roles of these variants, iPSC-CMs were generated. RNA sequencing showed the LMNA expression levels to be significantly lower in the iPSC-CMs of the LMNA variant carriers. We demonstrated a dysregulated sarcomeric structure and altered calcium homeostasis in the iPSC-CMs of the LMNA variant carriers. Using targeted nanopore long-read sequencing, we revealed the biological significance of the variant c.356+1G>A, which generates a novel 5' splice site in exon 1 of the cardiac isomer of LMNA, causing a nonsense mRNA product with almost complete RNA decay and haploinsufficiency. Using novel molecular analysis and nanopore technology, we demonstrated the pathogenesis of the rs794728589 (c.356+1G>A) splice variant in LMNA. This study highlights the importance of precise diagnostics in the clinical management and workup of cardiomyopathies.

  View details for DOI 10.3390/ijms232012230

  View details for PubMedID 36293084

• A new mechanism of ATP synthase subunit 6 and 9 translation regulation by their assembly in yeast mitochondria Kabala, A., Binko, K., Godard, F., Dautant, A., Baranowska, E., Skoczen, N., Gombeau, K., Bouhier, M., Becker, H., Ackerman, S., Steinmetz, L., Tribouillard-Tanvier, D., Kucharczyk, R., di Rago, J. ELSEVIER. 2022: 65

  View details for DOI 10.1016/j.bbabio.2022.148786

  View details for Web of Science ID 000854179100189

• Transcription Factor GATA4 Regulates Cell Type-Specific Splicing Through Direct Interaction With RNA in Human Induced Pluripotent Stem Cell-Derived Cardiac Progenitors. Circulation Zhu, L., Choudhary, K., Gonzalez-Teran, B., Ang, Y., Thomas, R., Stone, N. R., Liu, L., Zhou, P., Zhu, C., Ruan, H., Huang, Y., Jin, S., Pelonero, A., Koback, F., Padmanabhan, A., Sadagopan, N., Hsu, A., Costa, M. W., Gifford, C. A., van Bemmel, J., Huttenhain, R., Vedantham, V., Conklin, B. R., Black, B. L., Bruneau, B. G., Steinmetz, L., Krogan, N. J., Pollard, K. S., Srivastava, D. 2022: CIRCULATIONAHA121057620

  Abstract

  BACKGROUND: GATA4 (GATA-binding protein 4), a zinc finger-containing, DNA-binding transcription factor, is essential for normal cardiac development and homeostasis in mice and humans, and mutations in this gene have been reported in human heart defects. Defects in alternative splicing are associated with many heart diseases, yet relatively little is known about how cell type- or cell state-specific alternative splicing is achieved in the heart. Here, we show that GATA4 regulates cell type-specific splicing through direct interaction with RNA and the spliceosome in human induced pluripotent stem cell-derived cardiac progenitors.METHODS: We leveraged a combination of unbiased approaches including affinity purification of GATA4 and mass spectrometry, enhanced cross-linking with immunoprecipitation, electrophoretic mobility shift assays, in vitro splicing assays, and unbiased transcriptomic analysis to uncover GATA4's novel function as a splicing regulator in human induced pluripotent stem cell-derived cardiac progenitors.RESULTS: We found that GATA4 interacts with many members of the spliceosome complex in human induced pluripotent stem cell-derived cardiac progenitors. Enhanced cross-linking with immunoprecipitation demonstrated that GATA4 also directly binds to a large number of mRNAs through defined RNA motifs in a sequence-specific manner. In vitro splicing assays indicated that GATA4 regulates alternative splicing through direct RNA binding, resulting in functionally distinct protein products. Correspondingly, knockdown of GATA4 in human induced pluripotent stem cell-derived cardiac progenitors resulted in differential alternative splicing of genes involved in cytoskeleton organization and calcium ion import, with functional consequences associated with the protein isoforms.CONCLUSIONS: This study shows that in addition to its well described transcriptional function, GATA4 interacts with members of the spliceosome complex and regulates cell type-specific alternative splicing via sequence-specific interactions with RNA. Several genes that have splicing regulated by GATA4 have functional consequences and many are associated with dilated cardiomyopathy, suggesting a novel role for GATA4 in achieving the necessary cardiac proteome in normal and stress-responsive conditions.

  View details for DOI 10.1161/CIRCULATIONAHA.121.057620

  View details for PubMedID 35938400

• Transcriptomic diversity in human medullary thymic epithelial cells. Nature communications Carter, J. A., Stromich, L., Peacey, M., Chapin, S. R., Velten, L., Steinmetz, L. M., Brors, B., Pinto, S., Meyer, H. V. 2022; 13 (1): 4296

  Abstract

  The induction of central T cell tolerance in the thymus depends on the presentation of peripheral self-epitopes by medullary thymic epithelial cells (mTECs). This promiscuous gene expression (pGE) drives mTEC transcriptomic diversity, with non-canonical transcript initiation, alternative splicing, and expression of endogenous retroelements (EREs) representing important but incompletely understood contributors. Here we map the expression of genome-wide transcripts in immature and mature human mTECs using high-throughput 5' cap and RNA sequencing. Both mTEC populations show high splicing entropy, potentially driven by the expression of peripheral splicing factors. During mTEC maturation, rates of global transcript mis-initiation increase and EREs enriched in long terminal repeat retrotransposons are up-regulated, the latter often found in proximity to differentially expressed genes. As a resource, we provide an interactive public interface for exploring mTECtranscriptomic diversity. Our findings therefore help construct a map of transcriptomic diversity in the healthy human thymus and may ultimately facilitate the identification of thoseepitopes which contribute to autoimmunity and immune recognition of tumor antigens.

  View details for DOI 10.1038/s41467-022-31750-1

  View details for PubMedID 35918316

• KIR+CD8+ T cells suppress pathogenic T cells and are active in autoimmune diseases and COVID-19. Science (New York, N.Y.) Li, J., Zaslavsky, M., Su, Y., Guo, J., Sikora, M. J., van Unen, V., Christophersen, A., Chiou, S., Chen, L., Li, J., Ji, X., Wilhelmy, J., McSween, A. M., Palanski, B. A., Mallajosyula, V. V., Bracey, N. A., Dhondalay, G. K., Bhamidipati, K., Pai, J., Kipp, L. B., Dunn, J. E., Hauser, S. L., Oksenberg, J. R., Satpathy, A. T., Robinson, W. H., Dekker, C. L., Steinmetz, L. M., Khosla, C., Utz, P. J., Sollid, L. M., Chien, Y., Heath, J. R., Fernandez-Becker, N. Q., Nadeau, K. C., Saligrama, N., Davis, M. M. 2022: eabi9591

  Abstract

  Here we find that CD8+ T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49+CD8+ regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8+ T cells efficiently eliminated pathogenic gliadin-specific CD4+ T cells from celiac disease patients' leukocytes in vitro. We also find elevated levels of KIR+CD8+ T cells, but not CD4+ regulatory T cells, in COVID-19 patients, which correlated with disease severity and vasculitis. Selective ablation of Ly49+CD8+ T cells in virus-infected mice led to autoimmunity post infection. Our results indicate that in both species, these regulatory CD8+ T cells act uniquely to suppress pathogenic T cells in autoimmune and infectious diseases.

  View details for DOI 10.1126/science.abi9591

  View details for PubMedID 35258337

• Transcriptional neighborhoods regulate transcript isoform lengths and expression levels. Science (New York, N.Y.) Brooks, A. N., Hughes, A. L., Clauder-Munster, S., Mitchell, L. A., Boeke, J. D., Steinmetz, L. M. 2022; 375 (6584): 1000-1005

  Abstract

  Sequence features of genes and their flanking regulatory regions are determinants of RNA transcript isoform expression and have been used as context-independent plug-and-play modules in synthetic biology. However, genetic context-including the adjacent transcriptional environment-also influences transcript isoform expression levels and boundaries. We used synthetic yeast strains with stochastically repositioned genes to systematically disentangle the effects of sequence and context. Profiling 120 million full-length transcript molecules across 612 genomic perturbations, we observed sequence-independent alterations to gene expression levels and transcript isoform boundaries that were influenced by neighboring transcription. We identified features of transcriptional context that could predict these alterations and used these features to engineer a synthetic circuit where transcript length was controlled by neighboring transcription. This demonstrates how positional context can be leveraged in synthetic genome engineering.

  View details for DOI 10.1126/science.abg0162

  View details for PubMedID 35239377

• Assembly-dependent translation of subunits 6 (Atp6) and 9 (Atp9) of ATP synthase in yeast mitochondria. Genetics Kabala, A. M., Binko, K., Godard, F., Charles, C., Dautant, A., Baranowska, E., Skoczen, N., Gombeau, K., Bouhier, M., Becker, H. D., Ackerman, S. H., Steinmetz, L. M., Tribouillard-Tanvier, D., Kucharczyk, R., di Rago, J. P. 2022

  Abstract

  The yeast mitochondrial ATP synthase is an assembly of 28 subunits of 17 types of which 3 (subunits 6, 8, and 9) are encoded by mitochondrial genes while the 14 others have a nuclear genetic origin. Within the membrane domain (FO) of this enzyme, the subunit 6 and a ring of 10 identical subunits 9 transport protons across the mitochondrial inner membrane coupled to ATP synthesis in the extra-membrane structure (F1) of ATP synthase. As a result of their dual genetic origin, the ATP synthase subunits are synthesized in the cytosol and inside the mitochondrion. How they are produced in the proper stoichiometry from two different cellular compartments is still poorly understood. The experiments herein reported show that the rate of translation of the subunits 9 and 6 is enhanced in strains with mutations leading to specific defects in the assembly of these proteins. These translation modifications involve assembly intermediates interacting with subunits 6 and 9 within the final enzyme and cis-regulatory sequences that control gene expression in the organelle. In addition to enabling a balanced output of the ATP synthase subunits, these assembly-dependent feedback loops are presumably important to limit the accumulation of harmful assembly intermediates that have the potential to dissipate the mitochondrial membrane electrical potential and the main source of chemical energy of the cell.

  View details for DOI 10.1093/genetics/iyac007

  View details for PubMedID 35100419

• High-speed fluorescence image-enabled cell sorting. Science (New York, N.Y.) Schraivogel, D., Kuhn, T. M., Rauscher, B., Rodríguez-Martínez, M., Paulsen, M., Owsley, K., Middlebrook, A., Tischer, C., Ramasz, B., Ordoñez-Rueda, D., Dees, M., Cuylen-Haering, S., Diebold, E., Steinmetz, L. M. 2022; 375 (6578): 315-320

  Abstract

  Fast and selective isolation of single cells with unique spatial and morphological traits remains a technical challenge. Here, we address this by establishing high-speed image-enabled cell sorting (ICS), which records multicolor fluorescence images and sorts cells based on measurements from image data at speeds up to 15,000 events per second. We show that ICS quantifies cell morphology and localization of labeled proteins and increases the resolution of cell cycle analyses by separating mitotic stages. We combine ICS with CRISPR-pooled screens to identify regulators of the nuclear factor κB (NF-κB) pathway, enabling the completion of genome-wide image-based screens in about 9 hours of run time. By assessing complex cellular phenotypes, ICS substantially expands the phenotypic space accessible to cell-sorting applications and pooled genetic screening.

  View details for DOI 10.1126/science.abj3013

  View details for PubMedID 35050652

• Patient-derived gene and protein expression signatures of NGLY1 deficiency. Journal of biochemistry Rauscher, B., Mueller, W. F., Clauder-Munster, S., Jakob, P., Islam, M. S., Sun, H., Ghidelli-Disse, S., Boesche, M., Bantscheff, M., Pflaumer, H., Collier, P., Haase, B., Chen, S., Hoffman, R., Wang, G., Benes, V., Drewes, G., Snyder, M., Steinmetz, L. M. 2021

  Abstract

  N-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing 4 different cell types derived from 14 NGLY1 deficient patients and 17 controls. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared to parents, residual and likely non-functional NGLY1 protein was detectable in all patient-derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosome biogenesis and mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cysteine ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients.

  View details for DOI 10.1093/jb/mvab131

  View details for PubMedID 34878535

• Evidence for hyperactivation of innate immunity in a Drosophila model of NGLY1 deficiency Pandey, A., Han, S., Mueller, W. F., Story, B. A., Galeone, A., Steinmetz, L., Jafar-Nejad, H. OXFORD UNIV PRESS INC. 2021: 1715-1716

  View details for Web of Science ID 000754737200090

• High-throughput functional characterization of protein phosphorylation sites in yeast. Nature biotechnology Vieitez, C., Busby, B. P., Ochoa, D., Mateus, A., Memon, D., Galardini, M., Yildiz, U., Trovato, M., Jawed, A., Geiger, A. G., Oborska-Oplova, M., Potel, C. M., Vonesch, S. C., Szu Tu, C., Shahraz, M., Stein, F., Steinmetz, L. M., Panse, V. G., Noh, K., Savitski, M. M., Typas, A., Beltrao, P. 2021

  Abstract

  Phosphorylation is a critical post-translational modification involved in the regulation of almost all cellular processes. However, fewer than 5% of thousands of recently discovered phosphosites have been functionally annotated. In this study, we devised a chemical genetic approach to study the functional relevance of phosphosites in Saccharomyces cerevisiae. We generated 474 yeast strains with mutations in specific phosphosites that were screened for fitness in 102 conditions, along with a gene deletion library. Of these phosphosites, 42% exhibited growth phenotypes, suggesting that these are more likely functional. We inferred their function based on the similarity of their growth profiles with that of gene deletions and validated a subset by thermal proteome profiling and lipidomics. A high fraction exhibited phenotypes not seen in the corresponding gene deletion, suggestive of a gain-of-function effect. For phosphosites conserved in humans, the severity of the yeast phenotypes is indicative of their human functional relevance. This high-throughput approach allows for functionally characterizing individual phosphosites at scale.

  View details for DOI 10.1038/s41587-021-01051-x

  View details for PubMedID 34663920

• Regulation of Msh4-Msh5 association with meiotic chromosomes in budding yeast. Genetics Nandanan, K. G., Salim, S., Pankajam, A. V., Shinohara, M., Lin, G., Chakraborty, P., Farnaz, A., Steinmetz, L. M., Shinohara, A., Nishant, K. T. 2021; 219 (2)

  Abstract

  In the baker's yeast Saccharomyces cerevisiae, most of the meiotic crossovers are generated through a pathway involving the highly conserved mismatch repair related Msh4-Msh5 complex. To understand the role of Msh4-Msh5 in meiotic crossing over, we determined its genome wide in vivo binding sites in meiotic cells. We show that Msh5 specifically associates with DSB hotspots, chromosome axes, and centromeres on chromosomes. A basal level of Msh5 association with these chromosomal features is observed even in the absence of DSB formation (spo11Δ mutant) at the early stages of meiosis. But efficient binding to DSB hotspots and chromosome axes requires DSB formation and resection and is enhanced by double Holliday junction structures. Msh5 binding is also correlated to DSB frequency and enhanced on small chromosomes with higher DSB and crossover density. The axis protein Red1 is required for Msh5 association with the chromosome axes and DSB hotspots but not centromeres. Although binding sites of Msh5 and other pro-crossover factors like Zip3 show extensive overlap, Msh5 associates with centromeres independent of Zip3. These results on Msh5 localization in wild type and meiotic mutants have implications for how Msh4-Msh5 works with other pro-crossover factors to ensure crossover formation.

  View details for DOI 10.1093/genetics/iyab102

  View details for PubMedID 34849874

• Regulation of Msh4-Msh5 association with meiotic chromosomes in budding yeast GENETICS Nandanan, K. G., Salim, S., Pankajam, A., Shinohara, M., Lin, G., Chakraborty, P., Farnaz, A., Steinmetz, L. M., Shinohara, A., Nishant, K. T. 2021; 219 (2)

  View details for DOI 10.1093/genetics/iyab102

  View details for Web of Science ID 000708515000005

• High-Throughput Nucleotide Resolution Predictions of Assay Limitations Increase the Reliability and Concordance of Clinical Tests. JCO clinical cancer informatics Bieler, J., Pozzorini, C., Garcia, J., Tuck, A. C., Macheret, M., Willig, A., Couraud, S., Xing, X., Menu, P., Steinmetz, L. M., Payen, L., Xu, Z. 2021; 5: 1085-1095

  Abstract

  PURPOSE: The ability of next-generation sequencing (NGS) assays to interrogate thousands of genomic loci has revolutionized genetic testing. However, translation to the clinic is impeded by false-negative results that pose a risk to patients. In response, regulatory bodies are calling for reliability measures to be reported alongside NGS results. Existing methods to estimate reliability do not account for sample- and position-specific variability, which can be significant. Here, we report an approach that computes reliability metrics for every genomic position and sample interrogated by an NGS assay.METHODS: Our approach predicts the limit of detection (LOD), the lowest reliably detectable variant fraction, by taking technical factors into account. We initially explored how LOD is affected by input material amount, library conversion rate, sequencing coverage, and sequencing error rate. This revealed that LOD depends heavily on genomic context and sample properties. Using these insights, we developed a computational approach to predict LOD on the basis of a biophysical model of the NGS workflow. We focused on targeted assays for cell-free DNA, but, in principle, this approach applies to any NGS assay.RESULTS: We validated our approach by showing that it accurately predicts LOD and distinguishes reliable from unreliable results when screening 580 lung cancer samples for actionable mutations. Compared with a standard variant calling workflow, our approach avoided most false negatives and improved interassay concordance from 94% to 99%.CONCLUSION: Our approach, which we name LAVA (LOD-aware variant analysis), reports the LOD for every position and sample interrogated by an NGS assay. This enables reliable results to be identified and improves the transparency and safety of genetic tests.

  View details for DOI 10.1200/CCI.21.00057

  View details for PubMedID 34731027

• Case-control study evaluating risk factors for SARS-CoV-2 outbreak amongst healthcare personnel at a tertiary care center. American journal of infection control Rosser, J. I., Tayyar, R., Giardina, R., Kolonoski, P., Kenski, D., Shen, P., Steinmetz, L. M., Hung, L., Xiao, W., Bains, K., Morrison, T., Madison, A., Chang, S., Tompkins, L., Pinsky, B. A., Holubar, M. 2021

  Abstract

  BACKGROUND: Despite several outbreaks of SARS-CoV-2 amongst healthcare personnel (HCP) exposed to COVID-19 patients globally, risk factors for transmission remain poorly understood.METHODS: We conducted an outbreak investigation and case-control study to evaluate SARS-CoV-2 transmission risk in an outbreak among HCP at an academic medical center in California that was confirmed by whole genome sequencing.RESULTS: A total of 7/9 cases and 93/182 controls completed a voluntary survey about risk factors. Compared to controls, cases reported significantly more patient contact time. Cases were also significantly more likely to have performed airway procedures on the index patient, particularly placing the patient on high flow nasal cannula, continuous positive airway pressure (CPAP), or bilevel positive airway pressure (BiPAP) (OR=11.6; 95% CI=1.7-132.1).DISCUSSION: This study highlights the risk of nosocomial infection of SARS-CoV-2 from patients who become infectious midway into their hospitalization. Our findings also reinforce the importance of patient contact and aerosol-generating procedures as key risk factors for HCP infection with SARS-CoV-2.CONCLUSIONS: Re-testing patients for SARS-CoV-2 after admission in suspicious cases and using N95 masks for all aerosol-generating procedures regardless of initial patient SARS-CoV-2 test results can help reduce the risk of SARS-COV-2 transmission to HCP.

  View details for DOI 10.1016/j.ajic.2021.09.004

  View details for PubMedID 34536502

• Dysregulated ribonucleoprotein granules promote cardiomyopathy in RBM20 gene-edited pigs (vol 26, pg 1788, 2020) NATURE MEDICINE Schneider, J. W., Oommen, S., Qureshi, M. Y., Goetsch, S. C., Pease, D. R., Sundsbak, R. S., Guo, W., Sun, M., Sun, H., Kuroyanagi, H., Webster, D. A., Coutts, A. W., Holst, K. A., Edwards, B. S., Newville, N., Hathcock, M. A., Melkamu, T., Briganti, F., Wei, W., Romanelli, M. G., Fahrenkrug, S. C., Frantz, D. E., Olson, T. M., Steinmetz, L. M., Carlson, D. F., Nelson, T. J., Wanek Program Preclinical Pipeline 2021

  View details for DOI 10.1038/s41591-021-01412-8

  View details for Web of Science ID 000657340000002

  View details for PubMedID 34079106

• The chaperone-binding activity of the mitochondrial surface receptor Tom70 protects the cytosol against mitoprotein-induced stress. Cell reports Backes, S., Bykov, Y. S., Flohr, T., Raschle, M., Zhou, J., Lenhard, S., Kramer, L., Muhlhaus, T., Bibi, C., Jann, C., Smith, J. D., Steinmetz, L. M., Rapaport, D., Storchova, Z., Schuldiner, M., Boos, F., Herrmann, J. M. 2021; 35 (1): 108936

  Abstract

  Most mitochondrial proteins are synthesized as precursors in the cytosol and post-translationally transported into mitochondria. The mitochondrial surface protein Tom70 acts at the interface of the cytosol and mitochondria. Invitro import experiments identified Tom70 as targeting receptor, particularly for hydrophobic carriers. Using invivo methods and high-content screens, we revisit the question of Tom70 function and considerably expand the set of Tom70-dependent mitochondrial proteins. We demonstrate that the crucial activity of Tom70 is its ability to recruit cytosolic chaperones to the outer membrane. Indeed, tethering an unrelated chaperone-binding domain onto the mitochondrial surface complements most of the defects caused by Tom70 deletion. Tom70-mediated chaperone recruitment reduces the proteotoxicity of mitochondrial precursor proteins, particularly of hydrophobic inner membrane proteins. Thus, our work suggests that the predominant function of Tom70 is to tether cytosolic chaperones to the outer mitochondrial membrane, rather than to serve as a mitochondrion-specifying targeting receptor.

  View details for DOI 10.1016/j.celrep.2021.108936

  View details for PubMedID 33826901

• Recommendations for accurate genotyping of SARS-CoV-2 using amplicon-based sequencing of clinical samples. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases Kubik, S., Marques, A. C., Xing, X., Silvery, J., Bertelli, C., De Maio, F., Pournaras, S., Burr, T., Duffourd, Y., Siemens, H., Alloui, C., Song, L., Wenger, Y., Saitta, A., Macheret, M., Smith, E. W., Menu, P., Brayer, M., Steinmetz, L. M., Si-Mohammed, A., Chuisseu, J., Stevens, R., Constantoulakis, P., Sali, M., Greub, G., Tiemann, C., Pelechano, V., Willig, A., Xu, Z. 2021

  Abstract

  OBJECTIVES: SARS-CoV-2 genotyping has been instrumental to monitor viral evolution and transmission during the pandemic. The quality of the sequence data obtained from these genotyping efforts depends on several factors, including the quantity/integrity of the input material, the technology as well as laboratory-specific implementation. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in genomic epidemiology studies. We aimed at establishing clear and broadly applicable recommendations for reliable virus genotyping.METHODS: We established and used a sequencing data analysis workflow that reliably identifies and removes technical artifacts, which can result in miscalls when using alternative pipelines, to process clinical samples and synthetic viral genomes with an amplicon-based genotyping approach. We evaluated the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination.RESULTS: We found that at least 1000 viral genomes are necessary to confidently detect variants in the SARS-CoV-2 genome at frequencies of 10% or higher. The broad applicability of our recommendations was validated in over 200 clinical samples from six independent laboratories. The genotypes we determined for clinical isolates with sufficient quality cluster by sampling location and period. Our analysis also supports the rise in frequency of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was facilitated by travelling during the summer of 2020.CONCLUSIONS: We present much-needed recommendations for reliable determination of SARS-CoV-2 genome sequence and demonstrate their broad applicability in a large cohort of clinical samples.

  View details for DOI 10.1016/j.cmi.2021.03.029

  View details for PubMedID 33813118

• Single-cell analyses reveal SARS-CoV-2 interference with intrinsic immune response in the human gut. Molecular systems biology Triana, S., Metz-Zumaran, C., Ramirez, C., Kee, C., Doldan, P., Shahraz, M., Schraivogel, D., Gschwind, A. R., Sharma, A. K., Steinmetz, L. M., Herrmann, C., Alexandrov, T., Boulant, S., Stanifer, M. L. 2021; 17 (4): e10232

  Abstract

  Exacerbated pro-inflammatory immune response contributes to COVID-19 pathology. However, despite the mounting evidence about SARS-CoV-2 infecting the human gut, little is known about the antiviral programs triggered in this organ. To address this gap, we performed single-cell transcriptomics of SARS-CoV-2-infected intestinal organoids. We identified a subpopulation of enterocytes as the prime target of SARS-CoV-2 and, interestingly, found the lack of positive correlation between susceptibility to infection and the expression of ACE2. Infected cells activated strong pro-inflammatory programs and produced interferon, while expression of interferon-stimulated genes was limited to bystander cells due to SARS-CoV-2 suppressing the autocrine action of interferon. These findings reveal that SARS-CoV-2 curtails the immune response and highlights the gut as a pro-inflammatory reservoir that should be considered to fully understand SARS-CoV-2 pathogenesis.

  View details for DOI 10.15252/msb.202110232

  View details for PubMedID 33904651

• CRISPRi screens reveal genes modulating yeast growth in lignocellulose hydrolysate. Biotechnology for biofuels Gutmann, F., Jann, C., Pereira, F., Johansson, A., Steinmetz, L. M., Patil, K. R. 2021; 14 (1): 41

  Abstract

  BACKGROUND: Baker's yeast is a widely used eukaryotic cell factory, producing a diverse range of compounds including biofuels and fine chemicals. The use of lignocellulose as feedstock offers the opportunity to run these processes in an environmentally sustainable way. However, the required hydrolysis pretreatment of lignocellulosic material releases toxic compounds that hamper yeast growth and consequently productivity.RESULTS: Here, we employ CRISPR interference in S. cerevisiae to identify genes modulating fermentative growth in plant hydrolysate and in presence of lignocellulosic toxins. We find that at least one-third of hydrolysate-associated gene functions are explained by effects of known toxic compounds, such as the decreased growth of YAP1 or HAA1, or increased growth of DOT6 knock-down strains in hydrolysate.CONCLUSION: Our study confirms previously known genetic elements and uncovers new targets towards designing more robust yeast strains for the utilization of lignocellulose hydrolysate as sustainable feedstock, and, more broadly, paves the way for applying CRISPRi screens to improve industrial fermentation processes.

  View details for DOI 10.1186/s13068-021-01880-7

  View details for PubMedID 33568224

• Fast and inexpensive whole-genome sequencing library preparation from intact yeast cells. G3 (Bethesda, Md.) Vonesch, S. C., Li, S., Szu Tu, C., Hennig, B. P., Dobrev, N., Steinmetz, L. M. 2021; 11 (1)

  Abstract

  Through the increase in the capacity of sequencing machines massively parallel sequencing of thousands of samples in a single run is now possible. With the improved throughput and resulting drop in the price of sequencing, the cost and time for preparation of sequencing libraries have become the major bottleneck in large-scale experiments. Methods using a hyperactive variant of the Tn5 transposase efficiently generate libraries starting from cDNA or genomic DNA in a few hours and are highly scalable. For genome sequencing, however, the time and effort spent on genomic DNA isolation limit the practicability of sequencing large numbers of samples. Here, we describe a highly scalable method for preparing high-quality whole-genome sequencing libraries directly from Saccharomyces cerevisiae cultures in less than 3 h at 34 cents per sample. We skip the rate-limiting step of genomic DNA extraction by directly tagmenting lysed yeast spheroplasts and add a nucleosome release step prior to enrichment PCR to improve the evenness of genomic coverage. Resulting libraries do not show any GC bias and are comparable in quality to libraries processed from genomic DNA with a commercially available Tn5-based kit. We use our protocol to investigate CRISPR/Cas9 on- and off-target edits and reliably detect edited variants and shared polymorphisms between strains. Our protocol enables rapid preparation of unbiased and high-quality, sequencing-ready indexed libraries for hundreds of yeast strains in a single day at a low price. By adjusting individual steps of our workflow, we expect that our protocol can be adapted to other organisms.

  View details for DOI 10.1093/g3journal/jkaa009

  View details for PubMedID 33561223

• Identification of leukemic and pre-leukemic stem cells by clonal tracking from single-cell transcriptomics. Nature communications Velten, L., Story, B. A., Hernandez-Malmierca, P., Raffel, S., Leonce, D. R., Milbank, J., Paulsen, M., Demir, A., Szu-Tu, C., Fromel, R., Lutz, C., Nowak, D., Jann, J., Pabst, C., Boch, T., Hofmann, W., Muller-Tidow, C., Trumpp, A., Haas, S., Steinmetz, L. M. 2021; 12 (1): 1366

  Abstract

  Cancer stem cells drive disease progression and relapse in many types of cancer. Despite this, a thorough characterization of these cells remains elusive and with it the ability to eradicate cancer at its source. In acute myeloid leukemia (AML), leukemic stem cells (LSCs) underlie mortality but are difficult to isolate due to their low abundance and high similarity to healthy hematopoietic stem cells (HSCs). Here, we demonstrate that LSCs, HSCs, and pre-leukemic stem cells can be identified and molecularly profiled by combining single-cell transcriptomics with lineage tracing using both nuclear and mitochondrial somatic variants. While mutational status discriminates between healthy and cancerous cells, gene expression distinguishes stem cells and progenitor cell populations. Our approach enables the identification of LSC-specific gene expression programs and the characterization of differentiation blocks induced by leukemic mutations. Taken together, we demonstrate the power of single-cell multi-omic approaches in characterizing cancer stem cells.

  View details for DOI 10.1038/s41467-021-21650-1

  View details for PubMedID 33649320

• Integrated single-cell transcriptomics and epigenomics reveals strong germinal center-associated etiology of autoimmune risk loci. Science immunology King, H. W., Wells, K. L., Shipony, Z., Kathiria, A. S., Wagar, L. E., Lareau, C., Orban, N., Capasso, R., Davis, M. M., Steinmetz, L. M., James, L. K., Greenleaf, W. J. 2021; 6 (64): eabh3768

  Abstract

  [Figure: see text].

  View details for DOI 10.1126/sciimmunol.abh3768

  View details for PubMedID 34623901

• RBM20-Related Cardiomyopathy: Current Understanding and Future Options. Journal of clinical medicine Koelemen, J., Gotthardt, M., Steinmetz, L. M., Meder, B. 2021; 10 (18)

  Abstract

  Splice regulators play an essential role in the transcriptomic diversity of all eukaryotic cell types and organ systems. Recent evidence suggests a contribution of splice-regulatory networks in many diseases, such as cardiomyopathies. Adaptive splice regulators, such as RNA-binding motif protein 20 (RBM20) determine the physiological mRNA landscape formation, and rare variants in the RBM20 gene explain up to 6% of genetic dilated cardiomyopathy (DCM) cases. With ample knowledge from RBM20-deficient mice, rats, swine and induced pluripotent stem cells (iPSCs), the downstream targets and quantitative effects on splicing are now well-defined and the prerequisites for corrective therapeutic approaches are set. This review article highlights some of the recent advances in the field, ranging from aspects of granule formation to 3D genome architectures underlying RBM20-related cardiomyopathy. Promising therapeutic strategies are presented and put into context with the pathophysiological characteristics of RBM20-related diseases.

  View details for DOI 10.3390/jcm10184101

  View details for PubMedID 34575212

• Single-molecule, full-length transcript isoform sequencing reveals disease-associated RNA isoforms in cardiomyocytes. Nature communications Zhu, C., Wu, J., Sun, H., Briganti, F., Meder, B., Wei, W., Steinmetz, L. M. 2021; 12 (1): 4203

  Abstract

  Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we establish an experimental and computational pipeline that performs de novo transcript annotation and accurately quantifies transcript isoforms from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generate a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms ( http://steinmetzlab.embl.de/iBrowser/ ). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identify 121 differentially expressed transcript isoforms in 107 cardiac genes. Our approach enables quantitative dissection of complex transcript architecture instead of mere identification of inclusion or exclusion of individual exons, as exemplified by the discovery of IMMT isoforms mis-spliced by RBM20 mutations. Thereby we achieve a path to direct differential expression testing independent of an existing annotation of transcript isoforms, providing more immediate biological interpretation and higher resolution transcriptome comparisons.

  View details for DOI 10.1038/s41467-021-24484-z

  View details for PubMedID 34244519

• A functional connection between translation elongation and protein folding at the ribosome exit tunnel in Saccharomyces cerevisiae. Nucleic acids research Rodriguez-Galan, O., Garcia-Gomez, J. J., Rosado, I. V., Wei, W., Mendez-Godoy, A., Pillet, B., Alekseenko, A., Steinmetz, L. M., Pelechano, V., Kressler, D., de la Cruz, J. 2020

  Abstract

  Proteostasis needs to be tightly controlled to meet the cellular demand for correctly de novo folded proteins and to avoid protein aggregation. While a coupling between translation rate and co-translational folding, likely involving an interplay between the ribosome and its associated chaperones, clearly appears to exist, the underlying mechanisms and the contribution of ribosomal proteins remain to be explored. The ribosomal protein uL3 contains a long internal loop whose tip region is in close proximity to the ribosomal peptidyl transferase center. Intriguingly, the rpl3[W255C] allele, in which the residue making the closest contact to this catalytic site is mutated, affects diverse aspects of ribosome biogenesis and function. Here, we have uncovered, by performing a synthetic lethal screen with this allele, an unexpected link between translation and the folding of nascent proteins by the ribosome-associated Ssb-RAC chaperone system. Our results reveal that uL3 and Ssb-RAC cooperate to prevent 80S ribosomes from piling up within the 5' region of mRNAs early on during translation elongation. Together, our study provides compelling in vivo evidence for a functional connection between peptide bond formation at the peptidyl transferase center and chaperone-assisted de novo folding of nascent polypeptides at the solvent-side of the peptide exit tunnel.

  View details for DOI 10.1093/nar/gkaa1200

  View details for PubMedID 33330942

• Exploring the mechanisms underlying the NGLY1 loss-of-function phenotypes in Drosophila Pandey, A., Han, S., Mueller, W. F., Story, B. A., Moore, T., Galeone, A., Cowan, T. M., Steinmetz, L. OXFORD UNIV PRESS INC. 2020: 1026

  View details for Web of Science ID 000606577100018

• Combined transient ablation and single cell RNA sequencing reveals the development of medullary thymic epithelial cells. eLife Wells, K. L., Miller, C. N., Gschwind, A. R., Wei, W., Phipps, J. D., Anderson, M. S., Steinmetz, L. M. 2020; 9

  Abstract

  Medullary thymic epithelial cells (mTECs) play a critical role in central immune tolerance by mediating negative selection of autoreactive T cells through the collective expression of the peripheral self-antigen compartment, including tissue-specific antigens (TSAs). Recent work has shown that gene expression patterns within the mTEC compartment are remarkably heterogenous and include multiple differentiated cell states. To further define mTEC development and medullary epithelial lineage relationships, we combined lineage tracing and recovery from transient in vivo mTEC ablation with single cell RNA-sequencing in Mus musculus. The combination of bioinformatic and experimental approaches revealed a non-stem transit-amplifying population of cycling mTECs that preceded Aire expression. Based on our findings, we propose a branching model of mTEC development wherein a heterogeneous pool of transit-amplifying cells gives rise to Aire- and Ccl21a-expressing mTEC subsets. We further use experimental techniques to show that within the Aire-expressing developmental branch, TSA expression peaked as Aire expression decreased, implying Aire expression must be established before TSA expression can occur. Collectively, these data provide a higher order roadmap of mTEC development and demonstrate the power of combinatorial approaches leveraging both in vivo models and high-dimensional datasets.

  View details for DOI 10.7554/eLife.60188

  View details for PubMedID 33226342

• Rpb4 and Puf3 imprint and post-transcriptionally control the stability of a common set of mRNAs in yeast. RNA biology Garrido-Godino, A. I., Gupta, I., Gutierrez-Santiago, F., Martinez-Padilla, A. B., Alekseenko, A., Steinmetz, L. M., Perez-Ortin, J. E., Pelechano, V., Navarro, F. 2020

  Abstract

  Gene expression involving RNA polymerase II is regulated by the concerted interplay between mRNA synthesis and degradation, crosstalk in which mRNA decay machinery and transcription machinery respectively impact transcription and mRNA stability. Rpb4, and likely dimer Rpb4/7, seem the central components of the RNA pol II governing these processes. In this work we unravel the molecular mechanisms participated by Rpb4 that mediate the posttranscriptional events regulating mRNA imprinting and stability. By RIP-Seq, we analysed genome-wide the association of Rpb4 with mRNAs and demonstrated that it targeted a large population of more than 1400 transcripts. A group of these mRNAs was also the target of the RNA binding protein, Puf3. We demonstrated that Rpb4 and Puf3 physically, genetically, and functionally interact and also affect mRNA stability, and likely the imprinting, of a common group of mRNAs. Furthermore, the Rpb4 and Puf3 association with mRNAs depends on one another. We also demonstrated, for the first time, that Puf3 associates with chromatin in an Rpb4-dependent manner. Our data also suggest that Rpb4 could be a key element of the RNA pol II that coordinates mRNA synthesis, imprinting and stability in cooperation with RBPs.

  View details for DOI 10.1080/15476286.2020.1839229

  View details for PubMedID 33094674

• MULTI-LAYERED SINGLE-CELL TRANSCRIPTIONAL PROFILING OF ALL BONE AND BONE MARROW POPULATIONS PROVIDES A SYSTEMS VIEW OF THE MESENCHYMALAND HEMATOPOIETIC STEM CELL NICHE Haas, S., Velten, L., Al-Sabah, J., Baccin, C., Steinmetz, L., Trumpp, A. ELSEVIER SCIENCE INC. 2018: S47-S48

  View details for Web of Science ID 000975534800053

• Thymic tuft cells promote an IL-4-enriched medulla and shape thymocyte development NATURE Miller, C. N., Proekt, I., von Moltke, J., Wells, K. L., Rajpurkar, A. R., Wang, H., Rattay, K., Khan, I. S., Metzger, T. C., Pollack, J. L., Fries, A. C., Lwin, W. W., Wigton, E. J., Parent, A. V., Kyewski, B., Erle, D. J., Hogquist, K. A., Steinmetz, L. M., Locksley, R. M., Anderson, M. S. 2018; 559 (7715): 627-+

  Abstract

  The thymus is responsible for generating a diverse yet self-tolerant pool of T cells1. Although the thymic medulla consists mostly of developing and mature AIRE+ epithelial cells, recent evidence has suggested that there is far greater heterogeneity among medullary thymic epithelial cells than was previously thought2. Here we describe in detail an epithelial subset that is remarkably similar to peripheral tuft cells that are found at mucosal barriers3. Similar to the periphery, thymic tuft cells express the canonical taste transduction pathway and IL-25. However, they are unique in their spatial association with cornified aggregates, ability to present antigens and expression of a broad diversity of taste receptors. Some thymic tuft cells pass through an Aire-expressing stage and depend on a known AIRE-binding partner, HIPK2, for their development. Notably, the taste chemosensory protein TRPM5 is required for their thymic function through which they support the development and polarization of thymic invariant natural killer T cells and act to establish a medullary microenvironment that is enriched in the type 2 cytokine, IL-4. These findings indicate that there is a compartmentalized medullary environment in which differentiation of a minor and highly specialized epithelial subset has a non-redundant role in shaping thymic function.

  View details for PubMedID 30022164

  View details for PubMedCentralID PMC6062473

• Multiplexed precision genome editing with trackable genomic barcodes in yeast. Nature biotechnology Roy, K. R., Smith, J. D., Vonesch, S. C., Lin, G., Tu, C. S., Lederer, A. R., Chu, A., Suresh, S., Nguyen, M., Horecka, J., Tripathi, A., Burnett, W. T., Morgan, M. A., Schulz, J., Orsley, K. M., Wei, W., Aiyar, R. S., Davis, R. W., Bankaitis, V. A., Haber, J. E., Salit, M. L., St Onge, R. P., Steinmetz, L. M. 2018

  Abstract

  Our understanding of how genotype controls phenotype is limited by the scale at which we can precisely alter the genome and assess the phenotypic consequences of each perturbation. Here we describe a CRISPR-Cas9-based method for multiplexed accurate genome editing with short, trackable, integrated cellular barcodes (MAGESTIC) in Saccharomyces cerevisiae. MAGESTIC uses array-synthesized guide-donor oligos for plasmid-based high-throughput editing and features genomic barcode integration to prevent plasmid barcode loss and to enable robust phenotyping. We demonstrate that editing efficiency can be increased more than fivefold by recruiting donor DNA to the site of breaks using the LexA-Fkh1p fusion protein. We performed saturation editing of the essential gene SEC14 and identified amino acids critical for chemical inhibition of lipid signaling. We also constructed thousands of natural genetic variants, characterized guide mismatch tolerance at the genome scale, and ascertained that cryptic Pol III termination elements substantially reduce guide efficacy. MAGESTIC will be broadly useful to uncover the genetic basis of phenotypes in yeast.

  View details for PubMedID 29734294

• Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol. G3 (Bethesda, Md.) Hennig, B. P., Velten, L. n., Racke, I. n., Tu, C. S., Thoms, M. n., Rybin, V. n., Besir, H. n., Remans, K. n., Steinmetz, L. M. 2018; 8 (1): 79–89

  Abstract

  Efficient preparation of high-quality sequencing libraries that well represent the biological sample is a key step for using next-generation sequencing in research. Tn5 enables fast, robust, and highly efficient processing of limited input material while scaling to the parallel processing of hundreds of samples. Here, we present a robust Tn5 transposase purification strategy based on an N-terminal His6-Sumo3 tag. We demonstrate that libraries prepared with our in-house Tn5 are of the same quality as those processed with a commercially available kit (Nextera XT), while they dramatically reduce the cost of large-scale experiments. We introduce improved purification strategies for two versions of the Tn5 enzyme. The first version carries the previously reported point mutations E54K and L372P, and stably produces libraries of constant fragment size distribution, even if the Tn5-to-input molecule ratio varies. The second Tn5 construct carries an additional point mutation (R27S) in the DNA-binding domain. This construct allows for adjustment of the fragment size distribution based on enzyme concentration during tagmentation, a feature that opens new opportunities for use of Tn5 in customized experimental designs. We demonstrate the versatility of our Tn5 enzymes in different experimental settings, including a novel single-cell polyadenylation site mapping protocol as well as ultralow input DNA sequencing.

  View details for PubMedID 29118030

  View details for PubMedCentralID PMC5765368

• HUMAN HAEMATOPOIETIC STEM CELL DIFFERENTIATION FOLLOWS A CONTINUOUS WADDINGTON-LIKE LANDSCAPE Haas, S., Velten, L., Raffel, S., Trumpp, A., Essers, M., Steinmetz, L. ELSEVIER SCIENCE INC. 2017: S101

  View details for Web of Science ID 000972742900223

• Human haematopoietic stem cell lineage commitment is a continuous process NATURE CELL BIOLOGY Velten, L., Haas, S. F., Raffel, S., Blaszkiewicz, S., Islam, S., Hennig, B. P., Hirche, C., Lutz, C., Buss, E. C., Nowak, D., Boch, T., Hofmann, W., Ho, A. D., Huber, W., Trumpp, A., Essers, M. A., Steinmetz, L. M. 2017; 19 (4): 271-?

  Abstract

  Blood formation is believed to occur through stepwise progression of haematopoietic stem cells (HSCs) following a tree-like hierarchy of oligo-, bi- and unipotent progenitors. However, this model is based on the analysis of predefined flow-sorted cell populations. Here we integrated flow cytometric, transcriptomic and functional data at single-cell resolution to quantitatively map early differentiation of human HSCs towards lineage commitment. During homeostasis, individual HSCs gradually acquire lineage biases along multiple directions without passing through discrete hierarchically organized progenitor populations. Instead, unilineage-restricted cells emerge directly from a 'continuum of low-primed undifferentiated haematopoietic stem and progenitor cells' (CLOUD-HSPCs). Distinct gene expression modules operate in a combinatorial manner to control stemness, early lineage priming and the subsequent progression into all major branches of haematopoiesis. These data reveal a continuous landscape of human steady-state haematopoiesis downstream of HSCs and provide a basis for the understanding of haematopoietic malignancies.

  View details for DOI 10.1038/ncb3493

  View details for Web of Science ID 000397917000006

  View details for PubMedID 28319093

• Single-cell transcriptome analysis reveals coordinated ectopic gene-expression patterns in medullary thymic epithelial cells NATURE IMMUNOLOGY Brennecke, P., Reyes, A., Pinto, S., Rattay, K., Nguyen, M., Kuechlert, R., Huber, W., Kyewski, B., Steinmetz, L. M. 2015; 16 (9): 933-941

  Abstract

  Expression of tissue-restricted self antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for the induction of self-tolerance and prevents autoimmunity, with each TRA being expressed in only a few mTECs. How this process is regulated in single mTECs and is coordinated at the population level, such that the varied single-cell patterns add up to faithfully represent TRAs, is poorly understood. Here we used single-cell RNA sequencing and obtained evidence of numerous recurring TRA-co-expression patterns, each present in only a subset of mTECs. Co-expressed genes clustered in the genome and showed enhanced chromatin accessibility. Our findings characterize TRA expression in mTECs as a coordinated process that might involve local remodeling of chromatin and thus ensures a comprehensive representation of the immunological self.

  View details for DOI 10.1038/ni.3246

  View details for Web of Science ID 000359876900011

  View details for PubMedCentralID PMC4675844

• Widespread Co-translational RNA Decay Reveals Ribosome Dynamics. Cell Pelechano, V., Wei, W., Steinmetz, L. M. 2015; 161 (6): 1400-1412

  Abstract

  It is generally assumed that mRNAs undergoing translation are protected from decay. Here, we show that mRNAs are, in fact, co-translationally degraded. This is a widespread and conserved process affecting most genes, where 5'-3' transcript degradation follows the last translating ribosome, producing an in vivo ribosomal footprint. By sequencing the ends of 5' phosphorylated mRNA degradation intermediates, we obtain a genome-wide drug-free measurement of ribosome dynamics. We identify general translation termination pauses in both normal and stress conditions. In addition, we describe novel codon-specific ribosomal pausing sites in response to oxidative stress that are dependent on the RNase Rny1. Our approach is simple and straightforward and does not require the use of translational inhibitors or in vitro RNA footprinting that can alter ribosome protection patterns.

  View details for DOI 10.1016/j.cell.2015.05.008

  View details for PubMedID 26046441

• Genotype-environment interactions reveal causal pathways that mediate genetic effects on phenotype. PLoS genetics Gagneur, J., Stegle, O., Zhu, C., Jakob, P., Tekkedil, M. M., Aiyar, R. S., Schuon, A., Pe'er, D., Steinmetz, L. M. 2013; 9 (9)

  Abstract

  Unraveling the molecular processes that lead from genotype to phenotype is crucial for the understanding and effective treatment of genetic diseases. Knowledge of the causative genetic defect most often does not enable treatment; therefore, causal intermediates between genotype and phenotype constitute valuable candidates for molecular intervention points that can be therapeutically targeted. Mapping genetic determinants of gene expression levels (also known as expression quantitative trait loci or eQTL studies) is frequently used for this purpose, yet distinguishing causation from correlation remains a significant challenge. Here, we address this challenge using extensive, multi-environment gene expression and fitness profiling of hundreds of genetically diverse yeast strains, in order to identify truly causal intermediate genes that condition fitness in a given environment. Using functional genomics assays, we show that the predictive power of eQTL studies for inferring causal intermediate genes is poor unless performed across multiple environments. Surprisingly, although the effects of genotype on fitness depended strongly on environment, causal intermediates could be most reliably predicted from genetic effects on expression present in all environments. Our results indicate a mechanism explaining this apparent paradox, whereby immediate molecular consequences of genetic variation are shared across environments, and environment-dependent phenotypic effects result from downstream integration of environmental signals. We developed a statistical model to predict causal intermediates that leverages this insight, yielding over 400 transcripts, for the majority of which we experimentally validated their role in conditioning fitness. Our findings have implications for the design and analysis of clinical omics studies aimed at discovering personalized targets for molecular intervention, suggesting that inferring causation in a single cellular context can benefit from molecular profiling in multiple contexts.

  View details for DOI 10.1371/journal.pgen.1003803

  View details for PubMedID 24068968

  View details for PubMedCentralID PMC3778020

• The Genomic and Transcriptomic Landscape of a HeLa Cell Line G3-GENES GENOMES GENETICS Landry, J. J., Pyl, P. T., Rausch, T., Zichner, T., Tekkedil, M. M., Stuetz, A. M., Jauch, A., Aiyar, R. S., Pau, G., Delhomme, N., Gagneur, J., Korbel, J. O., Huber, W., Steinmetz, L. M. 2013; 3 (8): 1213-1224

  Abstract

  HeLa is the most widely used model cell line for studying human cellular and molecular biology. To date, no genomic reference for this cell line has been released, and experiments have relied on the human reference genome. Effective design and interpretation of molecular genetic studies performed using HeLa cells require accurate genomic information. Here we present a detailed genomic and transcriptomic characterization of a HeLa cell line. We performed DNA and RNA sequencing of a HeLa Kyoto cell line and analyzed its mutational portfolio and gene expression profile. Segmentation of the genome according to copy number revealed a remarkably high level of aneuploidy and numerous large structural variants at unprecedented resolution. Some of the extensive genomic rearrangements are indicative of catastrophic chromosome shattering, known as chromothripsis. Our analysis of the HeLa gene expression profile revealed that several pathways, including cell cycle and DNA repair, exhibit significantly different expression patterns from those in normal human tissues. Our results provide the first detailed account of genomic variants in the HeLa genome, yielding insight into their impact on gene expression and cellular function as well as their origins. This study underscores the importance of accounting for the strikingly aberrant characteristics of HeLa cells when designing and interpreting experiments, and has implications for the use of HeLa as a model of human biology.

  View details for DOI 10.1534/g3.113.005777

  View details for Web of Science ID 000322822300005

  View details for PubMedID 23550136

• Extensive transcriptional heterogeneity revealed by isoform profiling. Nature Pelechano, V., Wei, W., Steinmetz, L. M. 2013; 497 (7447): 127-131

  Abstract

  Transcript function is determined by sequence elements arranged on an individual RNA molecule. Variation in transcripts can affect messenger RNA stability, localization and translation, or produce truncated proteins that differ in localization or function. Given the existence of overlapping, variable transcript isoforms, determining the functional impact of the transcriptome requires identification of full-length transcripts, rather than just the genomic regions that are transcribed. Here, by jointly determining both transcript ends for millions of RNA molecules, we reveal an extensive layer of isoform diversity previously hidden among overlapping RNA molecules. Variation in transcript boundaries seems to be the rule rather than the exception, even within a single population of yeast cells. Over 26 major transcript isoforms per protein-coding gene were expressed in yeast. Hundreds of short coding RNAs and truncated versions of proteins are concomitantly encoded by alternative transcript isoforms, increasing protein diversity. In addition, approximately 70% of genes express alternative isoforms that vary in post-transcriptional regulatory elements, and tandem genes frequently produce overlapping or even bicistronic transcripts. This extensive transcript diversity is generated by a relatively simple eukaryotic genome with limited splicing, and within a genetically homogeneous population of cells. Our findings have implications for genome compaction, evolution and phenotypic diversity between single cells. These data also indicate that isoform diversity as well as RNA abundance should be considered when assessing the functional repertoire of genomes.

  View details for DOI 10.1038/nature12121

  View details for PubMedID 23615609

• Bidirectional promoters generate pervasive transcription in yeast NATURE Xu, Z., Wei, W., Gagneur, J., Perocchi, F., Clauder-Muenster, S., Camblong, J., Guffanti, E., Stutz, F., Huber, W., Steinmetz, L. M. 2009; 457 (7232): 1033-U7

  Abstract

  Genome-wide pervasive transcription has been reported in many eukaryotic organisms, revealing a highly interleaved transcriptome organization that involves hundreds of previously unknown non-coding RNAs. These recently identified transcripts either exist stably in cells (stable unannotated transcripts, SUTs) or are rapidly degraded by the RNA surveillance pathway (cryptic unstable transcripts, CUTs). One characteristic of pervasive transcription is the extensive overlap of SUTs and CUTs with previously annotated features, which prompts questions regarding how these transcripts are generated, and whether they exert function. Single-gene studies have shown that transcription of SUTs and CUTs can be functional, through mechanisms involving the generated RNAs or their generation itself. So far, a complete transcriptome architecture including SUTs and CUTs has not been described in any organism. Knowledge about the position and genome-wide arrangement of these transcripts will be instrumental in understanding their function. Here we provide a comprehensive analysis of these transcripts in the context of multiple conditions, a mutant of the exosome machinery and different strain backgrounds of Saccharomyces cerevisiae. We show that both SUTs and CUTs display distinct patterns of distribution at specific locations. Most of the newly identified transcripts initiate from nucleosome-free regions (NFRs) associated with the promoters of other transcripts (mostly protein-coding genes), or from NFRs at the 3' ends of protein-coding genes. Likewise, about half of all coding transcripts initiate from NFRs associated with promoters of other transcripts. These data change our view of how a genome is transcribed, indicating that bidirectionality is an inherent feature of promoters. Such an arrangement of divergent and overlapping transcripts may provide a mechanism for local spreading of regulatory signals-that is, coupling the transcriptional regulation of neighbouring genes by means of transcriptional interference or histone modification.

  View details for DOI 10.1038/nature07728

  View details for Web of Science ID 000263425400047

  View details for PubMedID 19169243

• High-resolution mapping of meiotic crossovers and non-crossovers in yeast NATURE Mancera, E., Bourgon, R., Brozzi, A., Huber, W., Steinmetz, L. M. 2008; 454 (7203): 479-U1

  Abstract

  Meiotic recombination has a central role in the evolution of sexually reproducing organisms. The two recombination outcomes, crossover and non-crossover, increase genetic diversity, but have the potential to homogenize alleles by gene conversion. Whereas crossover rates vary considerably across the genome, non-crossovers and gene conversions have only been identified in a handful of loci. To examine recombination genome wide and at high spatial resolution, we generated maps of crossovers, crossover-associated gene conversion and non-crossover gene conversion using dense genetic marker data collected from all four products of fifty-six yeast (Saccharomyces cerevisiae) meioses. Our maps reveal differences in the distributions of crossovers and non-crossovers, showing more regions where either crossovers or non-crossovers are favoured than expected by chance. Furthermore, we detect evidence for interference between crossovers and non-crossovers, a phenomenon previously only known to occur between crossovers. Up to 1% of the genome of each meiotic product is subject to gene conversion in a single meiosis, with detectable bias towards GC nucleotides. To our knowledge the maps represent the first high-resolution, genome-wide characterization of the multiple outcomes of recombination in any organism. In addition, because non-crossover hotspots create holes of reduced linkage within haplotype blocks, our results stress the need to incorporate non-crossovers into genetic linkage analysis.

  View details for DOI 10.1038/nature07135

  View details for Web of Science ID 000257860300043

  View details for PubMedID 18615017

• Genome sequencing and comparative analysis of Saccharomyces cerevisiae strain YJM789 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wei, W., McCusker, J. H., Hyman, R. W., Jones, T., Ning, Y., Cao, Z., Gu, Z., Bruno, D., Miranda, M., Nguyen, M., Wilhelmy, J., Komp, C., Tamse, R., Wang, X., Jia, P., Luedi, P., Oefner, P. J., David, L., Dietrich, F. S., Li, Y., Davis, R. W., Steinmetz, L. M. 2007; 104 (31): 12825-12830

  Abstract

  We sequenced the genome of Saccharomyces cerevisiae strain YJM789, which was derived from a yeast isolated from the lung of an AIDS patient with pneumonia. The strain is used for studies of fungal infections and quantitative genetics because of its extensive phenotypic differences to the laboratory reference strain, including growth at high temperature and deadly virulence in mouse models. Here we show that the approximately 12-Mb genome of YJM789 contains approximately 60,000 SNPs and approximately 6,000 indels with respect to the reference S288c genome, leading to protein polymorphisms with a few known cases of phenotypic changes. Several ORFs are found to be unique to YJM789, some of which might have been acquired through horizontal transfer. Localized regions of high polymorphism density are scattered over the genome, in some cases spanning multiple ORFs and in others concentrated within single genes. The sequence of YJM789 contains clues to pathogenicity and spurs the development of more powerful approaches to dissecting the genetic basis of complex hereditary traits.

  View details for DOI 10.1073/pnas.0701291104

  View details for Web of Science ID 000248603900043

  View details for PubMedID 17652520

  View details for PubMedCentralID PMC1933262

• A high-resolution map of transcription in the yeast genome PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA David, L., Huber, W., Granovskaia, M., Toedling, J., Palm, C. J., Bofkin, L., Jones, T., Davis, R. W., Steinmetz, L. M. 2006; 103 (14): 5320-5325

  Abstract

  There is abundant transcription from eukaryotic genomes unaccounted for by protein coding genes. A high-resolution genome-wide survey of transcription in a well annotated genome will help relate transcriptional complexity to function. By quantifying RNA expression on both strands of the complete genome of Saccharomyces cerevisiae using a high-density oligonucleotide tiling array, this study identifies the boundary, structure, and level of coding and noncoding transcripts. A total of 85% of the genome is expressed in rich media. Apart from expected transcripts, we found operon-like transcripts, transcripts from neighboring genes not separated by intergenic regions, and genes with complex transcriptional architecture where different parts of the same gene are expressed at different levels. We mapped the positions of 3' and 5' UTRs of coding genes and identified hundreds of RNA transcripts distinct from annotated genes. These nonannotated transcripts, on average, have lower sequence conservation and lower rates of deletion phenotype than protein coding genes. Many other transcripts overlap known genes in antisense orientation, and for these pairs global correlations were discovered: UTR lengths correlated with gene function, localization, and requirements for regulation; antisense transcripts overlapped 3' UTRs more than 5' UTRs; UTRs with overlapping antisense tended to be longer; and the presence of antisense associated with gene function. These findings may suggest a regulatory role of antisense transcription in S. cerevisiae. Moreover, the data show that even this well studied genome has transcriptional complexity far beyond current annotation.

  View details for DOI 10.1073/pnas.0601091103

  View details for PubMedID 16569694

• Targeted Perturb-seq enables genome-scale genetic screens in single cells. Nature methods Schraivogel, D., Gschwind, A. R., Milbank, J. H., Leonce, D. R., Jakob, P., Mathur, L., Korbel, J. O., Merten, C. A., Velten, L., Steinmetz, L. M. 2020

  Abstract

  The transcriptome contains rich information on molecular, cellular and organismal phenotypes. However, experimental and statistical limitations constrain sensitivity and throughput of genetic screening with single-cell transcriptomics readout. To overcome these limitations, we introduce targeted Perturb-seq (TAP-seq), a sensitive, inexpensive and platform-independent method focusing single-cell RNA-seq coverage on genes of interest, thereby increasing the sensitivity and scale of genetic screens by orders of magnitude. TAP-seq permits routine analysis of thousands of CRISPR-mediated perturbations within a single experiment, detects weak effects and lowly expressed genes, and decreases sequencing requirements by up to 50-fold. We apply TAP-seq to generate perturbation-based enhancer-target gene maps for 1,778 enhancers within 2.5% of the human genome. We thereby show that enhancer-target association is jointly determined by three-dimensional contact frequency and epigenetic states, allowing accurate prediction of enhancer targets throughout the genome. In addition, we demonstrate that TAP-seq can identify cell subtypes with only 100 sequencing reads per cell.

  View details for DOI 10.1038/s41592-020-0837-5

  View details for PubMedID 32483332

• A Circulating Bioreactor Reprograms Cancer Cells Toward a More Mesenchymal Niche. Advanced biosystems Calamak, S., Ermis, M., Sun, H., Islam, S., Sikora, M., Nguyen, M., Hasirci, V., Steinmetz, L. M., Demirci, U. 2020; 4 (2): e1900139

  Abstract

  Cancer is a complex and heterogeneous disease, and cancer cells dynamically interact with the mechanical microenvironment such as hydrostatic pressure, fluid shear, and interstitial flow. These factors play an essential role in cell fate and circulating tumor cell heterogeneity, and can influence the cellular phenotype. In this study, a peristaltic continuous flow reactor is designed and applied to HCT-116 colorectal carcinoma cells to mimic the fluid dynamics of circulation. With this intervention, a CD44/CD24-cell subpopulation emerges, and 100 genes are significantly regulated. The expression of cells at 4 h in the flow reactor is very similar to TGF-ß treatment, which is an inducer of epithelial-mesenchymal transition. ATF3 and SERPINE1 are significantly upregulated in these groups, suggesting that the mesenchymal transition is induced through this signaling pathway. This flow reactor model is satisfactory on its own to reprogram colorectal cancer cells toward a more mesenchymal niche mimicking circulation of the blood.

  View details for DOI 10.1002/adbi.201900139

  View details for PubMedID 32293132

• A Circulating Bioreactor Reprograms Cancer Cells Toward a More Mesenchymal Niche ADVANCED BIOSYSTEMS Calamak, S., Ermis, M., Sun, H., Islam, S., Sikora, M., Nguyen, M., Hasirci, V., Steinmetz, L. M., Demirci, U. 2020

  View details for DOI 10.1002/adbi.201900139

  View details for Web of Science ID 000506961500001

• iPSC Modeling of RBM20-Deficient DCM Identifies Upregulation of RBM20 as a Therapeutic Strategy. Cell reports Briganti, F. n., Sun, H. n., Wei, W. n., Wu, J. n., Zhu, C. n., Liss, M. n., Karakikes, I. n., Rego, S. n., Cipriano, A. n., Snyder, M. n., Meder, B. n., Xu, Z. n., Millat, G. n., Gotthardt, M. n., Mercola, M. n., Steinmetz, L. M. 2020; 32 (10): 108117

  Abstract

  Recent advances in induced pluripotent stem cell (iPSC) technology and directed differentiation of iPSCs into cardiomyocytes (iPSC-CMs) make it possible to model genetic heart disease in vitro. We apply CRISPR/Cas9 genome editing technology to introduce three RBM20 mutations in iPSCs and differentiate them into iPSC-CMs to establish an in vitro model of RBM20 mutant dilated cardiomyopathy (DCM). In iPSC-CMs harboring a known causal RBM20 variant, the splicing of RBM20 target genes, calcium handling, and contractility are impaired consistent with the disease manifestation in patients. A variant (Pro633Leu) identified by exome sequencing of patient genomes displays the same disease phenotypes, thus establishing this variant as disease causing. We find that all-trans retinoic acid upregulates RBM20 expression and reverts the splicing, calcium handling, and contractility defects in iPSC-CMs with different causal RBM20 mutations. These results suggest that pharmacological upregulation of RBM20 expression is a promising therapeutic strategy for DCM patients with a heterozygous mutation in RBM20.

  View details for DOI 10.1016/j.celrep.2020.108117

  View details for PubMedID 32905764

• Loss of N-glycanase 1 Alters Transcriptional and Translational Regulation in K562 Cell Lines. G3 (Bethesda, Md.) Mueller, W. F., Jakob, P. n., Sun, H. n., Clauder-Münster, S. n., Ghidelli-Disse, S. n., Ordonez, D. n., Boesche, M. n., Bantscheff, M. n., Collier, P. n., Haase, B. n., Benes, V. n., Paulsen, M. n., Sehr, P. n., Lewis, J. n., Drewes, G. n., Steinmetz, L. M. 2020

  Abstract

  N-Glycanase 1 (NGLY1) deficiency is an ultra-rare, complex and devastating neuromuscular disease, with multi-organ symptoms. NGLY1 is a deglycosylating protein involved in the degradation of misfolded proteins retrotranslocated from the endoplasmic reticulum (ER). We show that the loss of NGLY1 causes substantial changes in the RNA and protein landscape of K562 cells. We employed the CMap database to predict compounds that can modulate NGLY1 activity. Utilizing our robust K562 screening system, we demonstrate that the compound NVP-BEZ235 promotes degradation of NGLY1-dependent substrates, concurrent with increased autophagic flux, suggesting that autophagy may assist in clearing aberrant substrates during NGLY1 deficiency.

  View details for DOI 10.1534/g3.119.401031

  View details for PubMedID 32265286

• TIF-Seq2 disentangles overlapping isoforms in complex human transcriptomes. Nucleic acids research Wang, J. n., Li, B. n., Marques, S. n., Steinmetz, L. M., Wei, W. n., Pelechano, V. n. 2020

  Abstract

  Eukaryotic transcriptomes are complex, involving thousands of overlapping transcripts. The interleaved nature of the transcriptomes limits our ability to identify regulatory regions, and in some cases can lead to misinterpretation of gene expression. To improve the understanding of the overlapping transcriptomes, we have developed an optimized method, TIF-Seq2, able to sequence simultaneously the 5' and 3' ends of individual RNA molecules at single-nucleotide resolution. We investigated the transcriptome of a well characterized human cell line (K562) and identified thousands of unannotated transcript isoforms. By focusing on transcripts which are challenging to be investigated with RNA-Seq, we accurately defined boundaries of lowly expressed unannotated and read-through transcripts putatively encoding fusion genes. We validated our results by targeted long-read sequencing and standard RNA-Seq for chronic myeloid leukaemia patient samples. Taking the advantage of TIF-Seq2, we explored transcription regulation among overlapping units and investigated their crosstalk. We show that most overlapping upstream transcripts use poly(A) sites within the first 2 kb of the downstream transcription units. Our work shows that, by paring the 5' and 3' end of each RNA, TIF-Seq2 can improve the annotation of complex genomes, facilitate accurate assignment of promoters to genes and easily identify transcriptionally fused genes.

  View details for DOI 10.1093/nar/gkaa691

  View details for PubMedID 32816037

• Dysregulated ribonucleoprotein granules promote cardiomyopathy in RBM20 gene-edited pigs. Nature medicine Schneider, J. W., Oommen, S. n., Qureshi, M. Y., Goetsch, S. C., Pease, D. R., Sundsbak, R. S., Guo, W. n., Sun, M. n., Sun, H. n., Kuroyanagi, H. n., Webster, D. A., Coutts, A. W., Holst, K. A., Edwards, B. S., Newville, N. n., Hathcock, M. A., Melkamu, T. n., Briganti, F. n., Wei, W. n., Romanelli, M. G., Fahrenkrug, S. C., Frantz, D. E., Olson, T. M., Steinmetz, L. M., Carlson, D. F., Nelson, T. J. 2020

  Abstract

  Ribonucleoprotein (RNP) granules are biomolecular condensates-liquid-liquid phase-separated droplets that organize and manage messenger RNA metabolism, cell signaling, biopolymer assembly, biochemical reactions and stress granule responses to cellular adversity. Dysregulated RNP granules drive neuromuscular degenerative disease but have not previously been linked to heart failure. By exploring the molecular basis of congenital dilated cardiomyopathy (DCM) in genome-edited pigs homozygous for an RBM20 allele encoding the pathogenic R636S variant of human RNA-binding motif protein-20 (RBM20), we discovered that RNP granules accumulated abnormally in the sarcoplasm, and we confirmed this finding in myocardium and reprogrammed cardiomyocytes from patients with DCM carrying the R636S allele. Dysregulated sarcoplasmic RBM20 RNP granules displayed liquid-like material properties, docked at precisely spaced intervals along cytoskeletal elements, promoted phase partitioning of cardiac biomolecules and fused with stress granules. Our results link dysregulated RNP granules to myocardial cellular pathobiology and heart failure in gene-edited pigs and patients with DCM caused by RBM20 mutation.

  View details for DOI 10.1038/s41591-020-1087-x

  View details for PubMedID 33188278

• GOTI, a method to identify genome-wide off-target effects of genome editing in mouse embryos. Nature protocols Zuo, E. n., Sun, Y. n., Wei, W. n., Yuan, T. n., Ying, W. n., Sun, H. n., Yuan, L. n., Steinmetz, L. M., Li, Y. n., Yang, H. n. 2020

  Abstract

  Genome editing holds great potential for correcting pathogenic mutations. We developed a method called GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. GOTI directly compares edited and non-edited cells without the interference of genetic background and thus could detect potential off-target variants with high sensitivity. Notably, the GOTI method was designed to detect potential off-target variants of any genome editing tools by the combination of experimental and computational approaches, which is critical for accurate evaluation of the safety of genome editing tools. Here we provide a detailed protocol for GOTI, including mice mating, two-cell embryo injection, embryonic day 14.5 embryo digestion, fluorescence-activated cell sorting, whole-genome sequencing and data analysis. To enhance the utility of GOTI, we also include a computational workflow called GOTI-seq (https://github.com/sydaileen/GOTI-seq) for the sequencing data analysis, which can generate the final genome-wide off-target variants from raw sequencing data directly. The protocol typically takes 20 d from the mice mating to sequencing and 7 d for sequencing data analysis.

  View details for DOI 10.1038/s41596-020-0361-1

  View details for PubMedID 32796939

• Chromatin-sensitive cryptic promoters putatively drive expression of alternative protein isoforms in yeast. Genome research Wei, W., Hennig, B. P., Wang, J., Zhang, Y., Piazza, I., Pareja Sanchez, Y., Chabbert, C. D., Adjalley, S. H., Steinmetz, L. M., Pelechano, V. 2019

  Abstract

  Cryptic transcription is widespread and generates a heterogeneous group of RNA molecules of unknown function. To improve our understanding of cryptic transcription, we investigated their transcription start site (TSS) usage, chromatin organization, and posttranscriptional consequences in Saccharomyces cerevisiae We show that TSSs of chromatin-sensitive internal cryptic transcripts retain comparable features of canonical TSSs in terms of DNA sequence, directionality, and chromatin accessibility. We define the 5' and 3' boundaries of cryptic transcripts and show that, contrary to RNA degradation-sensitive ones, they often overlap with the end of the gene, thereby using the canonical polyadenylation site, and associate to polyribosomes. We show that chromatin-sensitive cryptic transcripts can be recognized by ribosomes and may produce truncated polypeptides from downstream, in-frame start codons. Finally, we confirm the presence of the predicted polypeptides by reanalyzing N-terminal proteomic data sets. Our work suggests that a fraction of chromatin-sensitive internal cryptic promoters initiates the transcription of alternative truncated mRNA isoforms. The expression of these chromatin-sensitive isoforms is conserved from yeast to human, expanding the functional consequences of cryptic transcription and proteome complexity.

  View details for DOI 10.1101/gr.243378.118

  View details for PubMedID 31740578

• Yeast Single-cell RNA-seq, Cell by Cell and Step by Step. Bio-protocol Nadal-Ribelles, M., Islam, S., Wei, W., Latorre, P., Nguyen, M., de Nadal, E., Posas, F., Steinmetz, L. M. 2019; 9 (17): e3359

  Abstract

  Single-cell RNA-seq (scRNA-seq) has become an established method for uncovering the intrinsic complexity within populations. Even within seemingly homogenous populations of isogenic yeast cells, there is a high degree of heterogeneity that originates from a compact and pervasively transcribed genome. Research with microorganisms such as yeast represents a major challenge for single-cell transcriptomics, due to their small size, rigid cell wall, and low RNA content per cell. Because of these technical challenges, yeast-specific scRNA-seq methodologies have recently started to appear, each one of them relying on different cell-isolation and library-preparation methods. Consequently, each approach harbors unique strengths and weaknesses that need to be considered. We have recently developed a yeast single-cell RNA-seq protocol (yscRNA-seq), which is inexpensive, high-throughput and easy-to-implement, tailored to the unique needs of yeast. yscRNA-seq provides a unique platform that combines single-cell phenotyping via index sorting with the incorporation of unique molecule identifiers on transcripts that allows to digitally count the number of molecules in a strand- and isoform-specific manner. Here, we provide a detailed, step-by-step description of the experimental and computational steps of yscRNA-seq protocol. This protocol will ease the implementation of yscRNA-seq in other laboratories and provide guidelines for the development of novel technologies.

  View details for DOI 10.21769/BioProtoc.3359

  View details for PubMedID 33654857

  View details for PubMedCentralID PMC7854150

• Select sequencing of clonally expanded CD8+ T cells reveals limits to clonal expansion. Proceedings of the National Academy of Sciences of the United States of America Huang, H., Sikora, M. J., Islam, S., Chowdhury, R. R., Chien, Y., Scriba, T. J., Davis, M. M., Steinmetz, L. M. 2019

  Abstract

  To permit the recognition of antigens, T cells generate a vast diversity of T cell receptor (TCR) sequences. Upon binding of the TCR to an antigen-MHC complex, T cells clonally expand to establish an immune response. To study antigen-specific T cell clonality, we have developed a method that allows selection of rare cells, based on RNA expression, before in-depth scRNA-seq (named SELECT-seq). We applied SELECT-seq to collect both TCR sequences and then transcriptomes from single cells of peripheral blood lymphocytes activated by a Mycobacterium tuberculosis (Mtb) lysate. TCR sequence analysis allowed us to preferentially select expanded conventional CD8+ T cells as well as invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells. The iNKT and MAIT cells have a highly similar transcriptional pattern, indicating that they carry out similar immunological functions and differ considerably from conventional CD8+ T cells. While there is no relationship between expression profiles and clonal expansion in iNKT or MAIT cells, highly expanded conventional CD8+ T cells down-regulate the interleukin 2 (IL-2) receptor alpha (IL2RA, or CD25) protein and show signs of senescence. This suggests inherent limits to clonal expansion that act to diversify the T cell response repertoire.

  View details for PubMedID 30992377

• Regional Variation in RBM20 Causes a Highly Penetrant Arrhythmogenic Cardiomyopathy. Circulation. Heart failure Parikh, V. N., Caleshu, C., Reuter, C., Lazzeroni, L. C., Ingles, J., Garcia, J., McCaleb, K., Adesiyun, T., Sedaghat-Hamedani, F., Kumar, S., Graw, S., Gigli, M., Stolfo, D., Dal Ferro, M., Ing, A. Y., Nussbaum, R., Funke, B., Wheeler, M. T., Hershberger, R. E., Cook, S., Steinmetz, L. M., Lakdawala, N. K., Taylor, M. R., Mestroni, L., Merlo, M., Sinagra, G., Semsarian, C., Meder, B., Judge, D. P., Ashley, E. 2019; 12 (3): e005371

  Abstract

  Background Variants in the cardiomyocyte-specific RNA splicing factor RBM20 have been linked to familial cardiomyopathy, but the causative genetic architecture and clinical consequences of this disease are incompletely defined. Methods and Results To define the genetic architecture of RBM20 cardiomyopathy, we first established a database of RBM20 variants associated with cardiomyopathy and compared these to variants observed in the general population with respect to their location in the RBM20 coding transcript. We identified 2 regions significantly enriched for cardiomyopathy-associated variants in exons 9 and 11. We then assembled a registry of 74 patients with RBM20 variants from 8 institutions across the world (44 index cases and 30 from cascade testing). This RBM20 patient registry revealed highly prevalent family history of sudden cardiac death (51%) and cardiomyopathy (72%) among index cases and a high prevalence of composite arrhythmias (including atrial fibrillation, nonsustained ventricular tachycardia, implantable cardiac defibrillator discharge, and sudden cardiac arrest, 43%). Patients harboring variants in cardiomyopathy-enriched regions identified by our variant database analysis were enriched for these findings. Further, these characteristics were more prevalent in the RBM20 registry than in large cohorts of patients with dilated cardiomyopathy and TTNtv cardiomyopathy and not significantly different from a cohort of patients with LMNA-associated cardiomyopathy. Conclusions Our data establish RBM20 cardiomyopathy as a highly penetrant and arrhythmogenic cardiomyopathy. These findings underline the importance of arrhythmia surveillance and family screening in this disease and represent the first step in defining the genetic architecture of RBM20 disease causality on a population level.

  View details for PubMedID 30871351

• Evolthon: A community endeavor to evolve lab evolution PLOS BIOLOGY Strauss, S., Schirman, D., Jona, G., Brooks, A. N., Kunjapur, A. M., Ba, A., Flint, A., Solt, A., Mershin, A., Dixit, A., Yona, A. H., Csorgo, B., Busby, B., Hennig, B. P., Pal, C., Schraivogel, D., Schultz, D., Wernick, D. G., Agashe, D., Levi, D., Zabezhinsky, D., Russ, D., Sass, E., Tamar, E., Herz, E., Levy, E. D., Church, G. M., Yelin, I., Nachman, I., Gerst, J. E., Georgeson, J. M., Adamala, K. P., Steinmetz, L. M., Ruebsam, M., Ralser, M., Klutstein, M., Desai, M. M., Walunjkar, N., Yin, N., Hefetz, N., Jakimo, N., Snitser, O., Adini, O., Kumar, P., Smith, R., Zeidan, R., Hazan, R., Rak, R., Kishony, R., Johnson, S., Nouriel, S., Vonesch, S. C., Foster, S., Dagan, T., Wein, T., Karydis, T., Wannier, T. M., Stiles, T., Olin-Sandoval, V., Mueller, W. F., Bar-On, Y. M., Dahan, O., Pilpel, Y. 2019; 17 (3): e3000182

  Abstract

  In experimental evolution, scientists evolve organisms in the lab, typically by challenging them to new environmental conditions. How best to evolve a desired trait? Should the challenge be applied abruptly, gradually, periodically, sporadically? Should one apply chemical mutagenesis, and do strains with high innate mutation rate evolve faster? What are ideal population sizes of evolving populations? There are endless strategies, beyond those that can be exposed by individual labs. We therefore arranged a community challenge, Evolthon, in which students and scientists from different labs were asked to evolve Escherichia coli or Saccharomyces cerevisiae for an abiotic stress-low temperature. About 30 participants from around the world explored diverse environmental and genetic regimes of evolution. After a period of evolution in each lab, all strains of each species were competed with one another. In yeast, the most successful strategies were those that used mating, underscoring the importance of sex in evolution. In bacteria, the fittest strain used a strategy based on exploration of different mutation rates. Different strategies displayed variable levels of performance and stability across additional challenges and conditions. This study therefore uncovers principles of effective experimental evolutionary regimens and might prove useful also for biotechnological developments of new strains and for understanding natural strategies in evolutionary arms races between species. Evolthon constitutes a model for community-based scientific exploration that encourages creativity and cooperation.

  View details for PubMedID 30925180

• Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science (New York, N.Y.) Zuo, E., Sun, Y., Wei, W., Yuan, T., Ying, W., Sun, H., Yuan, L., Steinmetz, L. M., Li, Y., Yang, H. 2019

  Abstract

  Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single nucleotide polymorphism in individuals. Here, we developed a method named GOTI (Genome-wide Off-target analysis by Two-cell embryo Injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole genome sequences of progeny cells of edited vs. non-edited blastomeres at E14.5 showed that off-target single nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. In contrast, cytosine base editing induced SNVs with over 20-fold higher frequencies, requiring a solution to address its fidelity.

  View details for PubMedID 30819928

• Sensitive high-throughput single-cell RNA-seq reveals within-clonal transcript correlations in yeast populations. Nature microbiology Nadal-Ribelles, M., Islam, S., Wei, W., Latorre, P., Nguyen, M., de Nadal, E., Posas, F., Steinmetz, L. M. 2019

  Abstract

  Single-cell RNA sequencing has revealed extensive cellular heterogeneity within many organisms, but few methods have been developed for microbial clonal populations. The yeast genome displays unusually dense transcript spacing, with interleaved and overlapping transcription from both strands, resulting in a minuscule but complex pool of RNA that is protected by a resilient cell wall. Here, we have developed a sensitive, scalable and inexpensive yeast single-cell RNA-seq (yscRNA-seq) method that digitally counts transcript start sites in a strand- and isoform-specific manner. YscRNA-seq detects the expression of low-abundance, noncoding RNAs and at least half of the protein-coding genome in each cell. In clonal cells, we observed a negative correlation for the expression of sense-antisense pairs, whereas paralogs and divergent transcripts co-expressed. By combining yscRNA-seq with index sorting, we uncovered a linear relationship between cell size and RNA content. Although we detected an average of ~3.5 molecules per gene, the number of expressed isoforms is restricted at the single-cell level. Remarkably, the expression of metabolic genes is highly variable, whereas their stochastic expression primes cells for increased fitness towards the corresponding environmental challenge. These findings suggest that functional transcript diversity acts as a mechanism that provides a selective advantage to individual cells within otherwise transcriptionally heterogeneous populations.

  View details for PubMedID 30718850

• Combined single-cell and spatial transcriptomics reveal the molecular, cellular and spatial bone marrow niche organization. Nature cell biology Baccin, C. n., Al-Sabah, J. n., Velten, L. n., Helbling, P. M., Grünschläger, F. n., Hernández-Malmierca, P. n., Nombela-Arrieta, C. n., Steinmetz, L. M., Trumpp, A. n., Haas, S. n. 2019

  Abstract

  The bone marrow constitutes the primary site for life-long blood production and skeletal regeneration. However, its cellular and spatial organization remains controversial. Here, we combine single-cell and spatially resolved transcriptomics to systematically map the molecular, cellular and spatial composition of distinct bone marrow niches. This allowed us to transcriptionally profile all major bone-marrow-resident cell types, determine their localization and clarify sources of pro-haematopoietic factors. Our data demonstrate that Cxcl12-abundant-reticular (CAR) cell subsets (Adipo-CAR and Osteo-CAR) differentially localize to sinusoidal and arteriolar surfaces, act locally as 'professional cytokine-secreting cells' and thereby establish peri-vascular micro-niches. Importantly, the three-dimensional bone-marrow organization can be accurately inferred from single-cell transcriptome data using the RNA-Magnet algorithm described here. Together, our study reveals the cellular and spatial organization of bone marrow niches and offers a systematic approach to dissect the complex organization of whole organs.

  View details for DOI 10.1038/s41556-019-0439-6

  View details for PubMedID 31871321

• Biological plasticity rescues target activity in CRISPR knock outs. Nature methods Smits, A. H., Ziebell, F. n., Joberty, G. n., Zinn, N. n., Mueller, W. F., Clauder-Münster, S. n., Eberhard, D. n., Fälth Savitski, M. n., Grandi, P. n., Jakob, P. n., Michon, A. M., Sun, H. n., Tessmer, K. n., Bürckstümmer, T. n., Bantscheff, M. n., Steinmetz, L. M., Drewes, G. n., Huber, W. n. 2019

  Abstract

  Gene knock outs (KOs) are efficiently engineered through CRISPR-Cas9-induced frameshift mutations. While the efficiency of DNA editing is readily verified by DNA sequencing, a systematic understanding of the efficiency of protein elimination has been lacking. Here we devised an experimental strategy combining RNA sequencing and triple-stage mass spectrometry to characterize 193 genetically verified deletions targeting 136 distinct genes generated by CRISPR-induced frameshifts in HAP1 cells. We observed residual protein expression for about one third of the quantified targets, at variable levels from low to original, and identified two causal mechanisms, translation reinitiation leading to N-terminally truncated target proteins or skipping of the edited exon leading to protein isoforms with internal sequence deletions. Detailed analysis of three truncated targets, BRD4, DNMT1 and NGLY1, revealed partial preservation of protein function. Our results imply that systematic characterization of residual protein expression or function in CRISPR-Cas9-generated KO lines is necessary for phenotype interpretation.

  View details for DOI 10.1038/s41592-019-0614-5

  View details for PubMedID 31659326

• Liver-specific deletion of Ngly1 causes abnormal nuclear morphology and lipid metabolism under food stress. Biochimica et biophysica acta. Molecular basis of disease Fujihira, H. n., Masahara-Negishi, Y. n., Akimoto, Y. n., Hirayama, H. n., Lee, H. C., Story, B. A., Mueller, W. F., Jakob, P. n., Clauder-Münster, S. n., Steinmetz, L. M., Radhakrishnan, S. K., Kawakami, H. n., Kamada, Y. n., Miyoshi, E. n., Yokomizo, T. n., Suzuki, T. n. 2019: 165588

  Abstract

  The cytoplasmic peptide:N-glycanase (Ngly1) is a de-N-glycosylating enzyme that cleaves N-glycans from misfolded glycoproteins and is involved in endoplasmic reticulum-associated degradation. The recent discovery of NGLY1-deficiency, which causes severe systemic symptoms, drew attention to the physiological function of Ngly1 in mammals. While several studies have been carried out to reveal the physiological necessity of Ngly1, the semi-lethal nature of Ngly1-deficient animals made it difficult to analyze its function in adults. In this study, we focus on the physiological function of Ngly1 in liver (hepatocyte)-specific Ngly1-deficient mice generated using the cre-loxP system. We found that hepatocyte-specific Ngly1-deficient mice showed abnormal hepatocyte nuclear size/morphology with aging but did not show other notable defects in unstressed conditions. This nuclear phenotype did not appear to be related to the function of the only gene currently reported to rescue Ngly1-deficient murine lethality so far, endo-β-N-acetylglucosaminidase. We also found that under a high fructose diet induced stress, the hepatocyte-specific Ngly1-deletion resulted in liver transaminases elevation and increased lipid droplet accumulation. We showed that the processing and localization of the transcription factor, nuclear factor erythroid 2-like 1 (Nfe2l1), was impaired in the Ngly1-deficient hepatocytes. Therefore, Nfe2l1, at least partially, contributes to the phenotypes observed in hepatocyte-specific Ngly1-deficient mice. Our results indicate that Ngly1 plays important roles in the adult liver impacting nuclear morphology and lipid metabolism. Hepatocyte-specific Ngly1-deficient mice could thus serve as a valuable animal model for assessing in vivo efficacy of drugs and/or treatment for NGLY1-deficiency.

  View details for DOI 10.1016/j.bbadis.2019.165588

  View details for PubMedID 31733337

• Opposing T cell responses in experimental autoimmune encephalomyelitis. Nature Saligrama, N. n., Zhao, F. n., Sikora, M. J., Serratelli, W. S., Fernandes, R. A., Louis, D. M., Yao, W. n., Ji, X. n., Idoyaga, J. n., Mahajan, V. B., Steinmetz, L. M., Chien, Y. H., Hauser, S. L., Oksenberg, J. R., Garcia, K. C., Davis, M. M. 2019

  Abstract

  Experimental autoimmune encephalomyelitis is a model for multiple sclerosis. Here we show that induction generates successive waves of clonally expanded CD4+, CD8+ and γδ+ T cells in the blood and central nervous system, similar to gluten-challenge studies of patients with coeliac disease. We also find major expansions of CD8+ T cells in patients with multiple sclerosis. In autoimmune encephalomyelitis, we find that most expanded CD4+ T cells are specific for the inducing myelin peptide MOG35-55. By contrast, surrogate peptides derived from a yeast peptide major histocompatibility complex library of some of the clonally expanded CD8+ T cells inhibit disease by suppressing the proliferation of MOG-specific CD4+ T cells. These results suggest that the induction of autoreactive CD4+ T cells triggers an opposing mobilization of regulatory CD8+ T cells.

  View details for DOI 10.1038/s41586-019-1467-x

  View details for PubMedID 31391585

• Genetic analysis reveals functions of atypical polyubiquitin chains. eLife Meza Gutierrez, F., Simsek, D., Mizrak, A., Deutschbauer, A., Braberg, H., Johnson, J., Xu, J., Shales, M., Nguyen, M., Tamse-Kuehn, R., Palm, C., Steinmetz, L. M., Krogan, N. J., Toczyski, D. P. 2018; 7

  Abstract

  Although polyubiquitin chains linked through all lysines of ubiquitin exist, specific functions are well-established only for lysine-48 and lysine-63 linkages in Saccharomyces cerevisiae. To uncover pathways regulated by distinct linkages, genetic interactions between a gene deletion library and a panel of lysine-to-arginine ubiquitin mutants were systematically identified. The K11R mutant had strong genetic interactions with threonine biosynthetic genes. Consistently, we found that K11R mutants import threonine poorly. The K11R mutant also exhibited a strong genetic interaction with a subunit of the anaphase-promoting complex (APC), suggesting a role in cell cycle regulation. K11-linkages are important for vertebrate APC function, but this was not previously described in yeast. We show that the yeast APC also modifies substrates with K11-linkages in vitro, and that those chains contribute to normal APC-substrate turnover in vivo. This study reveals comprehensive genetic interactomes of polyubiquitin chains and characterizes the role of K11-chains in two biological pathways.

  View details for PubMedID 30547882

• Conventional and Neo-antigenic Peptides Presented by beta Cells Are Targeted by Circulating Naive CD8+T Cells in Type 1 Diabetic and Healthy Donors CELL METABOLISM Gonzalez-Duque, S., Azoury, M., Colli, M. L., Afonso, G., Turatsinze, J., Nigi, L., Lalanne, A., Sebastiani, G., Carre, A., Pinto, S., Culina, S., Corcos, N., Bugliani, M., Marchetti, P., Armanet, M., Diedisheim, M., Kyewski, B., Steinmetz, L. M., Buus, S., You, S., Dubois-Laforgue, D., Larger, E., Beressi, J., Bruno, G., Dotta, F., Scharfmann, R., Eizirik, D. L., Verdier, Y., Vinh, J., Mallone, R. 2018; 28 (6): 946-+

  Abstract

  Although CD8+ T-cell-mediated autoimmune β cell destruction occurs in type 1 diabetes (T1D), the target epitopes processed and presented by β cells are unknown. To identify them, we combined peptidomics and transcriptomics strategies. Inflammatory cytokines increased peptide presentation in vitro, paralleling upregulation of human leukocyte antigen (HLA) class I expression. Peptide sources featured several insulin granule proteins and all known β cell antigens, barring islet-specific glucose-6-phosphatase catalytic subunit-related protein. Preproinsulin yielded HLA-A2-restricted epitopes previously described. Secretogranin V and its mRNA splice isoform SCG5-009, proconvertase-2, urocortin-3, the insulin gene enhancer protein ISL-1, and an islet amyloid polypeptide transpeptidation product emerged as antigens processed into HLA-A2-restricted epitopes, which, as those already described, were recognized by circulating naive CD8+ T cells in T1D and healthy donors and by pancreas-infiltrating cells in T1D donors. This peptidome opens new avenues to understand antigen processing by β cells and for the development of T cell biomarkers and tolerogenic vaccination strategies.

  View details for PubMedID 30078552

• High Frequency Actionable Pathogenic Exome Variants in an Average-Risk Cohort. Cold Spring Harbor molecular case studies Rego, S., Dagan-Rosenfeld, O., Zhou, W., Sailani, M. R., Limcaoco, P., Colbert, E., Avina, M., Wheeler, J., Craig, C., Salins, D., Rost, H. L., Dunn, J., McLaughlin, T., Steinmetz, L. M., Bernstein, J. A., Snyder, M. P. 2018

  Abstract

  Exome sequencing is increasingly utilized in both clinical and non-clinical settings, but little is known about its utility in healthy individuals. Most previous studies on this topic have examined a small subset of genes known to be implicated in human disease and/or have used automated pipelines to assess pathogenicity of known variants. In order to determine the frequency of both medically actionable and non-actionable but medically relevant exome findings in the general population we assessed the exomes of 70 participants who have been extensively characterized over the past several years as part of a longitudinal integrated multi-omics profiling study. We analyzed exomes by identifying rare likely pathogenic and pathogenic variants in genes associated with Mendelian disease in the Online Mendelian Inheritance in Man (OMIM) database. We then used American College of Medical Genetics (ACMG) guidelines for the classification of rare sequence variants. Additionally, we assessed pharmacogenetic variants. Twelve out of 70 (17%) participants had medically actionable findings in Mendelian disease genes. Five had phenotypes or family histories associated with their genetic variants. The frequency of actionable variants is higher than that reported in most previous studies and suggests added benefit from utilizing expanded gene lists and manual curation to assess actionable findings. A total of 63 participants (90%) had additional non-actionable findings, including 60 who were found to be carriers for recessive diseases and 21 who have increased Alzheimer's disease risk due to heterozygous or homozygous APOE e4 alleles (18 participants had both). Our results suggest that exome sequencing may have considerable more utility for health management in the general population than previously thought.

  View details for PubMedID 30487145

• Regional Variation in RBM20 Causes a Highly Penetrant Arrhythmogenic Cardiomyopathy. Parikh, V. N., Caleshu, C., Reuter, C., Lazzeroni, L., Ingles, J., Kumar, S., Garcia, J., McCaleb, K., Adesiyun, T., Dedaghat-Hamedani, F., Graw, S., Gigli, M., Stolfo, D., Dal Ferro, M., Ing, A., Nussbaum, R., Funke, B., Wheeler, M. T., Hershberger, R. E., Cook, S., Steinmetz, L., Lakdawala, N. K., Taylor, M. R., Mestroni, L., Merlo, M., Sinagra, G., Semsarian, C., Meder, B., Judge, D. P., Ashley, E. A. LIPPINCOTT WILLIAMS & WILKINS. 2018

  View details for Web of Science ID 000528619405020

• Gain of CTCF-Anchored Chromatin Loops Marks the Exit from Naive Pluripotency. Cell systems Pekowska, A., Klaus, B., Xiang, W., Severino, J., Daigle, N., Klein, F. A., Oles, M., Casellas, R., Ellenberg, J., Steinmetz, L. M., Bertone, P., Huber, W. 2018

  Abstract

  The genome of pluripotent stem cells adopts a unique three-dimensional architecture featuring weakly condensed heterochromatin and large nucleosome-free regions. Yet, it is unknown whether structural loops and contact domains display characteristics that distinguish embryonic stem cells (ESCs) from differentiated cell types. We used genome-wide chromosome conformation capture and super-resolution imaging to determine nuclear organization in mouse ESC and neural stem cell (NSC) derivatives. We found that loss of pluripotency is accompanied by widespread gain of structural loops. This general architectural change correlates with enhanced binding of CTCF and cohesins and more pronounced insulation of contacts across chromatin boundaries in lineage-committed cells. Reprogramming NSCs to pluripotency restores the unique features of ESC domain topology. Domains defined by the anchors of loops established upon differentiation are enriched for developmental genes. Chromatin loop formation is a pervasive structural alteration to the genome that accompanies exit from pluripotency and delineates the spatial segregation of developmentally regulated genes.

  View details for PubMedID 30414923

• Rpd3L HDAC links H3K4me3 to transcriptional repression memory NUCLEIC ACIDS RESEARCH Lee, B., Choi, A., Kim, J., Jun, Y., Woo, H., Ha, S., Yoon, C., Hwang, J., Steinmetz, L., Buratowski, S., Lee, S., Kim, H., Kim, T. 2018; 46 (16): 8261–74

  Abstract

  Transcriptional memory is critical for the faster reactivation of necessary genes upon environmental changes and requires that the genes were previously in an active state. However, whether transcriptional repression also displays 'memory' of the prior transcriptionally inactive state remains unknown. In this study, we show that transcriptional repression of ∼540 genes in yeast occurs much more rapidly if the genes have been previously repressed during carbon source shifts. This novel transcriptional response has been termed transcriptional repression memory (TREM). Interestingly, Rpd3L histone deacetylase (HDAC), targeted to active promoters induces TREM. Mutants for Rpd3L exhibit increased acetylation at active promoters and delay TREM significantly. Surprisingly, the interaction between H3K4me3 and Rpd3L via the Pho23 PHD finger is critical to promote histone deacetylation and TREM by Rpd3L. Therefore, we propose that an active mark, H3K4me3 enriched at active promoters, instructs Rpd3L HDAC to induce histone deacetylation and TREM.

  View details for PubMedID 29982589

  View details for PubMedCentralID PMC6144869

• NAD(P)HX repair deficiency causes central metabolic perturbations in yeast and human cells FEBS JOURNAL Becker-Kettern, J., Paczia, N., Conrotte, J., Zhu, C., Fiehn, O., Jung, P. P., Steinmetz, L. M., Linster, C. L. 2018; 285 (18): 3376–3401

  Abstract

  NADHX and NADPHX are hydrated and redox inactive forms of the NADH and NADPH cofactors, known to inhibit several dehydrogenases in vitro. A metabolite repair system that is conserved in all domains of life and that comprises the two enzymes NAD(P)HX dehydratase and NAD(P)HX epimerase, allows reconversion of both the S- and R-epimers of NADHX and NADPHX to the normal cofactors. An inherited deficiency in this system has recently been shown to cause severe neurometabolic disease in children. Although evidence for the presence of NAD(P)HX has been obtained in plant and human cells, little is known about the mechanism of formation of these derivatives in vivo and their potential effects on cell metabolism. Here, we show that NAD(P)HX dehydratase deficiency in yeast leads to an important, temperature-dependent NADHX accumulation in quiescent cells with a concomitant depletion of intracellular NAD+ and serine pools. We demonstrate that NADHX potently inhibits the first step of the serine synthesis pathway in yeast. Human cells deficient in the NAD(P)HX dehydratase also accumulated NADHX and showed decreased viability. In addition, those cells consumed more glucose and produced more lactate, potentially indicating impaired mitochondrial function. Our results provide first insights into how NADHX accumulation affects cellular functions and pave the way for a better understanding of the mechanism(s) underlying the rapid and severe neurodegeneration leading to early death in NADHX repair-deficient children.

  View details for PubMedID 30098110

• HEx: A heterologous expression platform for the discovery of fungal natural products SCIENCE ADVANCES Harvey, C. B., Tang, M., Schlecht, U., Horecka, J., Fischer, C. R., Lin, H., Li, J., Naughton, B., Cherry, J., Miranda, M., Li, Y., Chu, A. M., Hennessy, J. R., Vandova, G. A., Inglis, D., Aiyar, R. S., Steinmetz, L. M., Davis, R. W., Medema, M. H., Sattely, E., Khosla, C., St Onge, R. P., Tang, Y., Hillenmeyer, M. E. 2018; 4 (4): eaar5459

  Abstract

  For decades, fungi have been a source of U.S. Food and Drug Administration-approved natural products such as penicillin, cyclosporine, and the statins. Recent breakthroughs in DNA sequencing suggest that millions of fungal species exist on Earth, with each genome encoding pathways capable of generating as many as dozens of natural products. However, the majority of encoded molecules are difficult or impossible to access because the organisms are uncultivable or the genes are transcriptionally silent. To overcome this bottleneck in natural product discovery, we developed the HEx (Heterologous EXpression) synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and their encoded metabolites in Saccharomyces cerevisiae. We applied this platform to 41 fungal biosynthetic gene clusters from diverse fungal species from around the world, 22 of which produced detectable compounds. These included novel compounds with unexpected biosynthetic origins, particularly from poorly studied species. This result establishes the HEx platform for rapid discovery of natural products from any fungal species, even those that are uncultivable, and opens the door to discovery of the next generation of natural products.

  View details for PubMedID 29651464

• Multiplexed ChIP-Seq Using Direct Nucleosome Barcoding: A Tool for High-Throughput Chromatin Analysis. Methods in molecular biology (Clifton, N.J.) Chabbert, C. D., Adjalley, S. H., Steinmetz, L. M., Pelechano, V. n. 2018; 1689: 177–94

  Abstract

  Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) or microarray hybridization (ChIP-on-chip) are standard methods for the study of transcription factor binding sites and histone chemical modifications. However, these approaches only allow profiling of a single factor or protein modification at a time.In this chapter, we present Bar-ChIP, a higher throughput version of ChIP-Seq that relies on the direct ligation of molecular barcodes to chromatin fragments. Bar-ChIP enables the concurrent profiling of multiple DNA-protein interactions and is therefore amenable to experimental scale-up, without the need for any robotic instrumentation.

  View details for PubMedID 29027175

• Evolutionary-like selection on-a-chip: Using microfluidics to isolate the fittest sperm Chinnasamy, T., Kingsley, J., Durmus, N. G., Turek, P. J., Rosen, M. P., Behr, B., Steinmetz, L. M., Tuzel, E., Demirci, U. OXFORD UNIV PRESS. 2017: 187–88

  View details for Web of Science ID 000416390501201

• A new fate mapping system reveals context-dependent random or clonal expansion of microglia. Nature neuroscience Tay, T. L., Mai, D., Dautzenberg, J., Fernández-Klett, F., Lin, G., SAGAR, Datta, M., Drougard, A., Stempfl, T., Ardura-Fabregat, A., Staszewski, O., Margineanu, A., Sporbert, A., Steinmetz, L. M., Pospisilik, J. A., Jung, S., Priller, J., Grün, D., Ronneberger, O., Prinz, M. 2017; 20 (6): 793-803

  Abstract

  Microglia constitute a highly specialized network of tissue-resident immune cells that is important for the control of tissue homeostasis and the resolution of diseases of the CNS. Little is known about how their spatial distribution is established and maintained in vivo. Here we establish a new multicolor fluorescence fate mapping system to monitor microglial dynamics during steady state and disease. Our findings suggest that microglia establish a dense network with regional differences, and the high regional turnover rates found challenge the universal concept of microglial longevity. Microglial self-renewal under steady state conditions constitutes a stochastic process. During pathology this randomness shifts to selected clonal microglial expansion. In the resolution phase, excess disease-associated microglia are removed by a dual mechanism of cell egress and apoptosis to re-establish the stable microglial network. This study unravels the dynamic yet discrete self-organization of mature microglia in the healthy and diseased CNS.

  View details for DOI 10.1038/nn.4547

  View details for PubMedID 28414331

• Assembly of functionally integrated human forebrain spheroids NATURE Birey, F., Andersen, J., Makinson, C. D., Islam, S., Wei, W., Huber, N., Fan, H. C., Metzler, K. R., Panagiotakos, G., Thom, N., O'Rourke, N. A., Steinmetz, L. M., Bernstein, J. A., Hallmayer, J., Huguenard, J. R., Pasca, S. P. 2017; 545 (7652): 54-?

  Abstract

  The development of the nervous system involves a coordinated succession of events including the migration of GABAergic (γ-aminobutyric-acid-releasing) neurons from ventral to dorsal forebrain and their integration into cortical circuits. However, these interregional interactions have not yet been modelled with human cells. Here we generate three-dimensional spheroids from human pluripotent stem cells that resemble either the dorsal or ventral forebrain and contain cortical glutamatergic or GABAergic neurons. These subdomain-specific forebrain spheroids can be assembled in vitro to recapitulate the saltatory migration of interneurons observed in the fetal forebrain. Using this system, we find that in Timothy syndrome-a neurodevelopmental disorder that is caused by mutations in the CaV1.2 calcium channel-interneurons display abnormal migratory saltations. We also show that after migration, interneurons functionally integrate with glutamatergic neurons to form a microphysiological system. We anticipate that this approach will be useful for studying neural development and disease, and for deriving spheroids that resemble other brain regions to assemble circuits in vitro.

  View details for DOI 10.1038/nature22330

  View details for PubMedID 28445465

• Modulating Crossover Frequency and Interference for Obligate Crossovers in Saccharomyces cerevisiae Meiosis G3-GENES GENOMES GENETICS Chakraborty, P., Pankajam, A. V., Lin, G., Dutta, A., Nandanan, K. G., Tekkedil, M. M., Shinohara, A., Steinmetz, L. M., Thazath, N. K. 2017; 7 (5): 1511-1524

  Abstract

  Meiotic crossover frequencies show wide variation among organisms. But most organisms maintain at least one crossover per homolog pair (obligate crossover). In Saccharomyces cerevisiae, previous studies have shown crossover frequencies are reduced in the mismatch repair related mutant mlh3Δ and enhanced in a meiotic checkpoint mutant pch2Δ by up to twofold at specific chromosomal loci, but both mutants maintain high spore viability. We analyzed meiotic recombination events genome-wide in mlh3Δ, pch2Δ, and mlh3Δ pch2Δ mutants to test the effect of variation in crossover frequency on obligate crossovers. mlh3Δ showed ∼30% genome-wide reduction in crossovers (64 crossovers per meiosis) and loss of the obligate crossover, but nonexchange chromosomes were efficiently segregated. pch2Δ showed ∼50% genome-wide increase in crossover frequency (137 crossovers per meiosis), elevated noncrossovers as well as loss of chromosome size dependent double-strand break formation. Meiotic defects associated with pch2∆ did not cause significant increase in nonexchange chromosome frequency. Crossovers were restored to wild-type frequency in the double mutant mlh3Δ pch2Δ (100 crossovers per meiosis), but obligate crossovers were compromised. Genetic interference was reduced in mlh3Δ, pch2Δ, and mlh3Δ pch2Δ. Triple mutant analysis of mlh3Δ pch2Δ with other resolvase mutants showed that most of the crossovers in mlh3Δ pch2Δ are made through the Mus81-Mms4 pathway. These results are consistent with a requirement for increased crossover frequencies in the absence of genetic interference for obligate crossovers. In conclusion, these data suggest crossover frequencies and the strength of genetic interference in an organism are mutually optimized to ensure obligate crossovers.

  View details for DOI 10.1534/g3.117.040071

  View details for Web of Science ID 000400822200012

  View details for PubMedCentralID PMC5427503

• INO80 represses osmostress induced gene expression by resetting promoter proximal nucleosomes NUCLEIC ACIDS RESEARCH Klopf, E., Schmidt, H. A., Clauder-Muenster, S., Steinmetz, L. M., Schueller, C. 2017; 45 (7): 3752-3766

  View details for DOI 10.1093/nar/gkw1292

  View details for Web of Science ID 000399448400022

• Myt1l safeguards neuronal identity by actively repressing many non-neuronal fates NATURE Mall, M., Kareta, M. S., Chanda, S., Ahlenius, H., Perotti, N., Zhou, B., Grieder, S. D., Ge, X., Drake, S., Ang, C. E., Walker, B. M., Vierbuchen, T., Fuentes, D. R., Brennecke, P., Nitta, K. R., Jolma, A., Steinmetz, L. M., Taipale, J., Sudhof, T. C., Wernig, M. 2017; 544 (7649): 245-?

  Abstract

  Normal differentiation and induced reprogramming require the activation of target cell programs and silencing of donor cell programs. In reprogramming, the same factors are often used to reprogram many different donor cell types. As most developmental repressors, such as RE1-silencing transcription factor (REST) and Groucho (also known as TLE), are considered lineage-specific repressors, it remains unclear how identical combinations of transcription factors can silence so many different donor programs. Distinct lineage repressors would have to be induced in different donor cell types. Here, by studying the reprogramming of mouse fibroblasts to neurons, we found that the pan neuron-specific transcription factor Myt1-like (Myt1l) exerts its pro-neuronal function by direct repression of many different somatic lineage programs except the neuronal program. The repressive function of Myt1l is mediated via recruitment of a complex containing Sin3b by binding to a previously uncharacterized N-terminal domain. In agreement with its repressive function, the genomic binding sites of Myt1l are similar in neurons and fibroblasts and are preferentially in an open chromatin configuration. The Notch signalling pathway is repressed by Myt1l through silencing of several members, including Hes1. Acute knockdown of Myt1l in the developing mouse brain mimicked a Notch gain-of-function phenotype, suggesting that Myt1l allows newborn neurons to escape Notch activation during normal development. Depletion of Myt1l in primary postmitotic neurons de-repressed non-neuronal programs and impaired neuronal gene expression and function, indicating that many somatic lineage programs are actively and persistently repressed by Myt1l to maintain neuronal identity. It is now tempting to speculate that similar 'many-but-one' lineage repressors exist for other cell fates; such repressors, in combination with lineage-specific activators, would be prime candidates for use in reprogramming additional cell types.

  View details for DOI 10.1038/nature21722

  View details for PubMedID 28379941

• A method for high-throughput production of sequence-verified DNA libraries and strain collections. Molecular systems biology Smith, J. D., Schlecht, U., Xu, W., Suresh, S., Horecka, J., Proctor, M. J., Aiyar, R. S., Bennett, R. A., Chu, A., Li, Y. F., Roy, K., Davis, R. W., Steinmetz, L. M., Hyman, R. W., Levy, S. F., St Onge, R. P. 2017; 13 (2): 913-?

  Abstract

  The low costs of array-synthesized oligonucleotide libraries are empowering rapid advances in quantitative and synthetic biology. However, high synthesis error rates, uneven representation, and lack of access to individual oligonucleotides limit the true potential of these libraries. We have developed a cost-effective method called Recombinase Directed Indexing (REDI), which involves integration of a complex library into yeast, site-specific recombination to index library DNA, and next-generation sequencing to identify desired clones. We used REDI to generate a library of ~3,300 DNA probes that exhibited > 96% purity and remarkable uniformity (> 95% of probes within twofold of the median abundance). Additionally, we created a collection of ~9,000 individually accessible CRISPR interference yeast strains for > 99% of genes required for either fermentative or respiratory growth, demonstrating the utility of REDI for rapid and cost-effective creation of strain collections from oligonucleotide pools. Our approach is adaptable to any complex DNA library, and fundamentally changes how these libraries can be parsed, maintained, propagated, and characterized.

  View details for DOI 10.15252/msb.20167233

  View details for PubMedID 28193641

  View details for PubMedCentralID PMC5327727

• Inhibition of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor Cytotoxicity. ACS central science Tomlin, F. M., Gerling-Driessen, U. I., Liu, Y. C., Flynn, R. A., Vangala, J. R., Lentz, C. S., Clauder-Muenster, S. n., Jakob, P. n., Mueller, W. F., Ordoñez-Rueda, D. n., Paulsen, M. n., Matsui, N. n., Foley, D. n., Rafalko, A. n., Suzuki, T. n., Bogyo, M. n., Steinmetz, L. M., Radhakrishnan, S. K., Bertozzi, C. R. 2017; 3 (11): 1143–55

  Abstract

  Proteasome inhibitors are used to treat blood cancers such as multiple myeloma (MM) and mantle cell lymphoma. The efficacy of these drugs is frequently undermined by acquired resistance. One mechanism of proteasome inhibitor resistance may involve the transcription factor Nuclear Factor, Erythroid 2 Like 1 (NFE2L1, also referred to as Nrf1), which responds to proteasome insufficiency or pharmacological inhibition by upregulating proteasome subunit gene expression. This "bounce-back" response is achieved through a unique mechanism. Nrf1 is constitutively translocated into the ER lumen, N-glycosylated, and then targeted for proteasomal degradation via the ER-associated degradation (ERAD) pathway. Proteasome inhibition leads to accumulation of cytosolic Nrf1, which is then processed to form the active transcription factor. Here we show that the cytosolic enzyme N-glycanase 1 (NGLY1, the human PNGase) is essential for Nrf1 activation in response to proteasome inhibition. Chemical or genetic disruption of NGLY1 activity results in the accumulation of misprocessed Nrf1 that is largely excluded from the nucleus. Under these conditions, Nrf1 is inactive in regulating proteasome subunit gene expression in response to proteasome inhibition. Through a small molecule screen, we identified a cell-active NGLY1 inhibitor that disrupts the processing and function of Nrf1. The compound potentiates the cytotoxicity of carfilzomib, a clinically used proteasome inhibitor, against MM and T cell-derived acute lymphoblastic leukemia (T-ALL) cell lines. Thus, NGLY1 inhibition prevents Nrf1 activation and represents a new therapeutic approach for cancers that depend on proteasome homeostasis.

  View details for PubMedID 29202016

• The State of Systems Genetics in 2017. Cell systems Baliga, N. S., Björkegren, J. L., Boeke, J. D., Boutros, M. n., Crawford, N. P., Dudley, A. M., Farber, C. R., Jones, A. n., Levey, A. I., Lusis, A. J., Mak, H. C., Nadeau, J. H., Noyes, M. B., Petretto, E. n., Seyfried, N. T., Steinmetz, L. M., Vonesch, S. C. 2017; 4 (1): 7–15

  Abstract

  Cell Systems invited 16 experts to share their views on the field of systems genetics. In questions repeated in the headings, we asked them to define systems genetics, highlight its relevance to researchers outside the field, discuss what makes a strong systems genetics paper, and paint a picture of where the field is heading in the coming years. Their responses, ordered by the journal but otherwise unedited, make it clear that deciphering genotype to phenotype relationships is a central challenge of systems genetics and will require understanding how networks and higher-order properties of biological systems underlie complex traits. In addition, our experts illuminate the applications and relevance of systems genetics to human disease, the gut microbiome, development of tools that connect the global research community, sustainability, drug discovery, patient-specific disease and network models, and personalized treatments. Finally, a table of suggested reading provides a sample of influential work in the field.

  View details for PubMedID 28125793

• Genome Dynamics of HybridSaccharomyces cerevisiaeDuring Vegetative and Meiotic Divisions. G3 (Bethesda, Md.) Dutta, A. n., Lin, G. n., Pankajam, A. V., Chakraborty, P. n., Bhat, N. n., Steinmetz, L. M., Nishant, K. T. 2017; 7 (11): 3669–79

  Abstract

  Mutation and recombination are the major sources of genetic diversity in all organisms. In the baker's yeast, all mutation rate estimates are in homozygous background. We determined the extent of genetic change through mutation and loss of heterozygosity (LOH) in a heterozygousSaccharomyces cerevisiaegenome during successive vegetative and meiotic divisions. We measured genome-wide LOH and base mutation rates during vegetative and meiotic divisions in a hybrid (S288c/YJM789)S. cerevisiaestrain. The S288c/YJM789 hybrid showed nearly complete reduction in heterozygosity within 31 generations of meioses and improved spore viability. LOH in the meiotic lines was driven primarily by the mating of spores within the tetrad. The S288c/YJM789 hybrid lines propagated vegetatively for the same duration as the meiotic lines, showed variable LOH (from 2 to 3% and up to 35%). Two of the vegetative lines with extensive LOH showed frequent and large internal LOH tracts that suggest a high frequency of recombination repair. These results suggest significant LOH can occur in the S288c/YJM789 hybrid during vegetative propagation presumably due to return to growth events. The average base substitution rates for the vegetative lines (1.82 × 10-10per base per division) and the meiotic lines (1.22 × 10-10per base per division) are the first genome-wide mutation rate estimates for a hybrid yeast. This study therefore provides a novel context for the analysis of mutation rates (especially in the context of detecting LOH during vegetative divisions), compared to previous mutation accumulation studies in yeast that used homozygous backgrounds.

  View details for PubMedID 28916648

  View details for PubMedCentralID PMC5677154

• Modulation of mRNA and lncRNA expression dynamics by the Set2-Rpd3S pathway NATURE COMMUNICATIONS Kim, J. H., Lee, B. B., Oh, Y. M., Zhu, C., Steinmetz, L. M., Lee, Y., Kim, W. K., Lee, S. B., Buratowski, S., Kim, T. 2016; 7

  Abstract

  H3K36 methylation by Set2 targets Rpd3S histone deacetylase to transcribed regions of mRNA genes, repressing internal cryptic promoters and slowing elongation. Here we explore the function of this pathway by analysing transcription in yeast undergoing a series of carbon source shifts. Approximately 80 mRNA genes show increased induction upon SET2 deletion. A majority of these promoters have overlapping lncRNA transcription that targets H3K36me3 and deacetylation by Rpd3S to the mRNA promoter. We previously reported a similar mechanism for H3K4me2-mediated repression via recruitment of the Set3C histone deacetylase. Here we show that the distance between an mRNA and overlapping lncRNA promoter determines whether Set2-Rpd3S or Set3C represses. This analysis also reveals many previously unreported cryptic ncRNAs induced by specific carbon sources, showing that cryptic promoters can be environmentally regulated. Therefore, in addition to repression of cryptic transcription and modulation of elongation, H3K36 methylation maintains optimal expression dynamics of many mRNAs and ncRNAs.

  View details for DOI 10.1038/ncomms13534

  View details for PubMedID 27892458

• A privacy-preserving solution for compressed storage and selective retrieval of genomic data. Genome research Huang, Z., Ayday, E., Lin, H., Aiyar, R. S., Molyneaux, A., Xu, Z., Fellay, J., Steinmetz, L. M., Hubaux, J. 2016

  Abstract

  In clinical genomics, the continuous evolution of bioinformatic algorithms and sequencing platforms makes it beneficial to store patients' complete aligned genomic data in addition to variant calls relative to a reference sequence. Due to the large size of human genome sequence data files (varying from 30 GB to 200 GB depending on coverage), two major challenges facing genomics laboratories are the costs of storage and the efficiency of the initial data processing. In addition, privacy of genomic data is becoming an increasingly serious concern, yet no standard data storage solutions exist that enable compression, encryption, and selective retrieval. Here we present a privacy-preserving solution named SECRAM (Selective retrieval on Encrypted and Compressed Reference-oriented Alignment Map) for the secure storage of compressed aligned genomic data. Our solution enables selective retrieval of encrypted data and improves the efficiency of downstream analysis (e.g., variant calling). Compared with BAM, the de facto standard for storing aligned genomic data, SECRAM uses 18% less storage. Compared with CRAM, one of the most compressed nonencrypted formats (using 34% less storage than BAM), SECRAM maintains efficient compression and downstream data processing, while allowing for unprecedented levels of security in genomic data storage. Compared with previous work, the distinguishing features of SECRAM are that (1) it is position-based instead of read-based, and (2) it allows random querying of a subregion from a BAM-like file in an encrypted form. Our method thus offers a space-saving, privacy-preserving, and effective solution for the storage of clinical genomic data.

  View details for PubMedID 27789525

• A global genetic interaction network maps a wiring diagram of cellular function SCIENCE Costanzo, M., VanderSluis, B., Koch, E. N., Baryshnikova, A., Pons, C., Tan, G., Wang, W., Usaj, M., Hanchard, J., Lee, S. D., Pelechano, V., Styles, E. B., Billmann, M., van Leeuwen, J., van Dyk, N., Lin, Z., Kuzmin, E., Nelson, J., Piotrowski, J. S., Srikumar, T., Bahr, S., Chen, Y., Deshpande, R., Kurat, C. F., Li, S. C., Li, Z., Usaj, M. M., Okada, H., Pascoe, N., San Luis, B., Sharifpoor, S., Shuteriqi, E., Simpkins, S. W., Snider, J., Suresh, H. G., Tan, Y., Zhu, H., Malod-Dognin, N., Janjic, V., Przulj, N., Troyanskaya, O. G., Stagljar, I., Xia, T., Ohya, Y., Gingras, A., Raught, B., Boutros, M., Steinmetz, L. M., Moore, C. L., Rosebrock, A. P., Caudy, A. A., Myers, C. L., Andrews, B., Boone, C. 2016; 353 (6306): 1381-?

  Abstract

  We generated a global genetic interaction network for Saccharomyces cerevisiae, constructing more than 23 million double mutants, identifying about 550,000 negative and about 350,000 positive genetic interactions. This comprehensive network maps genetic interactions for essential gene pairs, highlighting essential genes as densely connected hubs. Genetic interaction profiles enabled assembly of a hierarchical model of cell function, including modules corresponding to protein complexes and pathways, biological processes, and cellular compartments. Negative interactions connected functionally related genes, mapped core bioprocesses, and identified pleiotropic genes, whereas positive interactions often mapped general regulatory connections among gene pairs, rather than shared functionality. The global network illustrates how coherent sets of genetic interactions connect protein complex and pathway modules to map a functional wiring diagram of the cell.

  View details for DOI 10.1126/science.aaf1420

  View details for PubMedID 27708008

• Principles for RNA metabolism and alternative transcription initiation within closely spaced promoters NATURE GENETICS Chen, Y., Pai, A. A., Herudek, J., Lubas, M., Meola, N., Jaervelin, A. I., Andersson, R., Pelechano, V., Steinmetz, L. M., Jensen, T. H., Sandelin, A. 2016; 48 (9): 984-?

  Abstract

  Mammalian transcriptomes are complex and formed by extensive promoter activity. In addition, gene promoters are largely divergent and initiate transcription of reverse-oriented promoter upstream transcripts (PROMPTs). Although PROMPTs are commonly terminated early, influenced by polyadenylation sites, promoters often cluster so that the divergent activity of one might impact another. Here we found that the distance between promoters strongly correlates with the expression, stability and length of their associated PROMPTs. Adjacent promoters driving divergent mRNA transcription support PROMPT formation, but owing to polyadenylation site constraints, these transcripts tend to spread into the neighboring mRNA on the same strand. This mechanism to derive new alternative mRNA transcription start sites (TSSs) is also evident at closely spaced promoters supporting convergent mRNA transcription. We suggest that basic building blocks of divergently transcribed core promoter pairs, in combination with the wealth of TSSs in mammalian genomes, provide a framework with which evolution shapes transcriptomes.

  View details for DOI 10.1038/ng.3616

  View details for Web of Science ID 000382398800008

  View details for PubMedID 27455346

  View details for PubMedCentralID PMC5008441

• Meiotic Interactors of a Mitotic Gene TAO3 Revealed by Functional Analysis of its Rare Variant G3-GENES GENOMES GENETICS Gupta, S., Radhakrishnan, A., Nitin, R., Raharja-Liu, P., Lin, G., Steinmetz, L. M., Gagneur, J., Sinha, H. 2016; 6 (8): 2255-2263

  Abstract

  Studying the molecular consequences of rare genetic variants has the potential to identify novel and hitherto uncharacterized pathways causally contributing to phenotypic variation. Here, we characterize the functional consequences of a rare coding variant of TAO3, previously reported to contribute significantly to sporulation efficiency variation in Saccharomyces cerevisiae During mitosis, the common TAO3 allele interacts with CBK1-a conserved NDR kinase. Both TAO3 and CBK1 are components of the RAM signaling network that regulates cell separation and polarization during mitosis. We demonstrate that the role of the rare allele TAO3(4477C) in meiosis is distinct from its role in mitosis by being independent of ACE2-a RAM network target gene. By quantitatively measuring cell morphological dynamics, and expressing the TAO3(4477C) allele conditionally during sporulation, we show that TAO3 has an early role in meiosis. This early role of TAO3 coincides with entry of cells into meiotic division. Time-resolved transcriptome analyses during early sporulation identified regulators of carbon and lipid metabolic pathways as candidate mediators. We show experimentally that, during sporulation, the TAO3(4477C) allele interacts genetically with ERT1 and PIP2, regulators of the tricarboxylic acid cycle and gluconeogenesis metabolic pathways, respectively. We thus uncover a meiotic functional role for TAO3, and identify ERT1 and PIP2 as novel regulators of sporulation efficiency. Our results demonstrate that studying the causal effects of genetic variation on the underlying molecular network has the potential to provide a more extensive understanding of the pathways driving a complex trait.

  View details for DOI 10.1534/g3.116.029900/-/DC1

  View details for Web of Science ID 000381282300002

  View details for PubMedID 27317780

  View details for PubMedCentralID PMC4978881

• Protein Abundance Control by Non-coding Antisense Transcription CELL REPORTS Huber, F., Bunina, D., Gupta, I., Khmelinskii, A., Meurer, M., Theer, P., Steinmetz, L. M., Knop, M. 2016; 15 (12): 2625-2636

  Abstract

  Stable unannotated transcripts (SUTs), some of which overlap protein-coding genes in antisense direction, are a class of non-coding RNAs. While case studies have reported important regulatory roles for several of such RNAs, their general impact on protein abundance regulation of the overlapping gene is not known. To test this, we employed seamless gene manipulation to repress antisense SUTs of 162 yeast genes by using a unidirectional transcriptional terminator and a GFP tag. We found that the mere presence of antisense SUTs was not sufficient to influence protein abundance, that observed effects of antisense SUTs correlated with sense transcript start site overlap, and that the effects were generally weak and led to reduced protein levels. Antisense regulated genes showed increased H3K4 di- and trimethylation and had slightly lower than expected noise levels. Our results suggest that the functionality of antisense RNAs has gene and condition-specific components.

  View details for DOI 10.1016/j.celrep.2016.05.043

  View details for Web of Science ID 000378255900007

  View details for PubMedID 27292640

  View details for PubMedCentralID PMC4920891

• Functional interplay between MSL1 and CDK7 controls RNA polymerase II Ser5 phosphorylation NATURE STRUCTURAL & MOLECULAR BIOLOGY Chlamydas, S., Holz, H., Samata, M., Chelmicki, T., Georgiev, P., Pelechano, V., Dundar, F., Dasmeh, P., Mittler, G., Cadete, F. T., Ramirez, F., Conrad, T., Wei, W., Raja, S., Manke, T., Luscombe, N. M., Steinmetz, L. M., Akhtar, A. 2016; 23 (6): 580-589

  Abstract

  Proper gene expression requires coordinated interplay among transcriptional coactivators, transcription factors and the general transcription machinery. We report here that MSL1, a central component of the dosage compensation complex in Drosophila melanogaster and Drosophila virilis, displays evolutionarily conserved sex-independent binding to promoters. Genetic and biochemical analyses reveal a functional interaction of MSL1 with CDK7, a subunit of the Cdk-activating kinase (CAK) complex of the general transcription factor TFIIH. Importantly, MSL1 depletion leads to decreased phosphorylation of Ser5 of RNA polymerase II. In addition, we demonstrate that MSL1 is a phosphoprotein, and transgenic flies expressing MSL1 phosphomutants show mislocalization of the histone acetyltransferase MOF and histone H4 K16 acetylation, thus ultimately causing male lethality due to a failure of dosage compensation. We propose that, by virtue of its interaction with components of the general transcription machinery, MSL1 exists in different phosphorylation states, thereby modulating transcription in flies.

  View details for DOI 10.1038/nsmb.3233

  View details for PubMedID 27183194

• Translational Capacity of a Cell Is Determined during Transcription Elongation via the Ccr4-Not Complex CELL REPORTS Gupta, I., Villanyi, Z., Kassem, S., Hughes, C., Panasenko, O. O., Steinmetz, L. M., Collart, M. A. 2016; 15 (8): 1782-1794

  Abstract

  The current understanding of gene expression considers transcription and translation to be independent processes. Challenging this notion, we found that translation efficiency is determined during transcription elongation through the imprinting of mRNAs with Not1, the central scaffold of the Ccr4-Not complex. We determined that another subunit of the complex, Not5, defines Not1 binding to specific mRNAs, particularly those produced from ribosomal protein genes. This imprinting mechanism specifically regulates ribosomal protein gene expression, which in turn determines the translational capacity of cells. We validate our model by SILAC and polysome profiling experiments. As a proof of concept, we demonstrate that enhanced translation compensates for transcriptional elongation stress. Taken together, our data indicate that in addition to defining mRNA stability, components of the Ccr4-Not imprinting complex regulate RNA translatability, thus ensuring global gene expression homeostasis.

  View details for DOI 10.1016/j.celrep.2016.04.055

  View details for Web of Science ID 000376654500014

  View details for PubMedID 27184853

  View details for PubMedCentralID PMC4880543

• The cellular growth rate controls overall mRNA turnover, and modulates either transcription or degradation rates of particular gene regulons NUCLEIC ACIDS RESEARCH Garcia-Martinez, J., Delgado-Ramos, L., Ayala, G., Pelechano, V., Medina, D. A., Carrasco, F., Gonzalez, R., Andres-Leon, E., Steinmetz, L., Warringer, J., Chavez, S., Perez-Ortin, J. E. 2016; 44 (8): 3643-3658

  Abstract

  We analyzed 80 different genomic experiments, and found a positive correlation between both RNA polymerase II transcription and mRNA degradation with growth rates in yeast. Thus, in spite of the marked variation in mRNA turnover, the total mRNA concentration remained approximately constant. Some genes, however, regulated their mRNA concentration by uncoupling mRNA stability from the transcription rate. Ribosome-related genes modulated their transcription rates to increase mRNA levels under fast growth. In contrast, mitochondria-related and stress-induced genes lowered mRNA levels by reducing mRNA stability or the transcription rate, respectively. We also detected these regulations within the heterogeneity of a wild-type cell population growing in optimal conditions. The transcriptomic analysis of sorted microcolonies confirmed that the growth rate dictates alternative expression programs by modulating transcription and mRNA decay.The regulation of overall mRNA turnover keeps a constant ratio between mRNA decay and the dilution of [mRNA] caused by cellular growth. This regulation minimizes the indiscriminate transmission of mRNAs from mother to daughter cells, and favors the response capacity of the latter to physiological signals and environmental changes. We also conclude that, by uncoupling mRNA synthesis from decay, cells control the mRNA abundance of those gene regulons that characterize fast and slow growth.

  View details for DOI 10.1093/nar/gkv1512

  View details for Web of Science ID 000376389000023

  View details for PubMedID 26717982

  View details for PubMedCentralID PMC4856968

• DChIPRep, an R/Bioconductor package for differential enrichment analysis in chromatin studies PEERJ Chabbert, C. D., Steinmetz, L. M., Klaus, B. 2016; 4

  Abstract

  The genome-wide study of epigenetic states requires the integrative analysis of histone modification ChIP-seq data. Here, we introduce an easy-to-use analytic framework to compare profiles of enrichment in histone modifications around classes of genomic elements, e.g. transcription start sites (TSS). Our framework is available via the user-friendly R/Bioconductor package DChIPRep. DChIPRep uses biological replicate information as well as chromatin Input data to allow for a rigorous assessment of differential enrichment. DChIPRep is available for download through the Bioconductor project at http://bioconductor.org/packages/DChIPRep. Contact. DChIPRep@gmail.com.

  View details for DOI 10.7717/peerj.1981

  View details for Web of Science ID 000375186000008

  View details for PubMedID 27168989

  View details for PubMedCentralID PMC4860309

• Landscape and Dynamics of Transcription Initiation in the Malaria Parasite Plasmodium falciparum CELL REPORTS Adjalley, S. H., Chabbert, C. D., Klaus, B., Pelechano, V., Steinmetz, L. M. 2016; 14 (10): 2463-2475

  Abstract

  A comprehensive map of transcription start sites (TSSs) across the highly AT-rich genome of P. falciparum would aid progress toward deciphering the molecular mechanisms that underlie the timely regulation of gene expression in this malaria parasite. Using high-throughput sequencing technologies, we generated a comprehensive atlas of transcription initiation events at single-nucleotide resolution during the parasite intra-erythrocytic developmental cycle. This detailed analysis of TSS usage enabled us to define architectural features of plasmodial promoters. We demonstrate that TSS selection and strength are constrained by local nucleotide composition. Furthermore, we provide evidence for coordinate and stage-specific TSS usage from distinct sites within the same transcription unit, thereby producing transcript isoforms, a subset of which are developmentally regulated. This work offers a framework for further investigations into the interactions between genomic sequences and regulatory factors governing the complex transcriptional program of this major human pathogen.

  View details for DOI 10.1016/j.celrep.2016.02.025

  View details for Web of Science ID 000371998700017

  View details for PubMedID 26947071

  View details for PubMedCentralID PMC4806524

• Integrating Cell Phone Imaging with Magnetic Levitation (i-LEV) for Label-Free Blood Analysis at the Point-of-Living. Small Baday, M., Calamak, S., Durmus, N. G., Davis, R. W., Steinmetz, L. M., Demirci, U. 2016; 12 (9): 1222-1229

  Abstract

  There is an emerging need for portable, robust, inexpensive, and easy-to-use disease diagnosis and prognosis monitoring platforms to share health information at the point-of-living, including clinical and home settings. Recent advances in digital health technologies have improved early diagnosis, drug treatment, and personalized medicine. Smartphones with high-resolution cameras and high data processing power enable intriguing biomedical applications when integrated with diagnostic devices. Further, these devices have immense potential to contribute to public health in resource-limited settings where there is a particular need for portable, rapid, label-free, easy-to-use, and affordable biomedical devices to diagnose and continuously monitor patients for precision medicine, especially those suffering from rare diseases, such as sickle cell anemia, thalassemia, and chronic fatigue syndrome. Here, a magnetic levitation-based diagnosis system is presented in which different cell types (i.e., white and red blood cells) are levitated in a magnetic gradient and separated due to their unique densities. Moreover, an easy-to-use, smartphone incorporated levitation system for cell analysis is introduced. Using our portable imaging magnetic levitation (i-LEV) system, it is shown that white and red blood cells can be identified and cell numbers can be quantified without using any labels. In addition, cells levitated in i-LEV can be distinguished at single-cell resolution, potentially enabling diagnosis and monitoring, as well as clinical and research applications.

  View details for DOI 10.1002/smll.201501845

  View details for PubMedID 26523938

  View details for PubMedCentralID PMC4775401

• Genome-wide quantification of 5'-phosphorylated mRNA degradation intermediates for analysis of ribosome dynamics. Nature protocols Pelechano, V., Wei, W., Steinmetz, L. M. 2016; 11 (2): 359-376

  Abstract

  Co-translational mRNA degradation is a widespread process in which 5'-3' exonucleolytic degradation follows the last translating ribosome, thus producing an in vivo ribosomal footprint that delimits the 5' position of the mRNA molecule within the ribosome. To study this degradation process and ribosome dynamics, we developed 5PSeq, which is a method that profiles the genome-wide abundance of mRNA degradation intermediates by virtue of their 5'-phosphorylated (5'P) ends. The approach involves targeted ligation of an oligonucleotide to the 5'P end of mRNA degradation intermediates, followed by depletion of rRNA molecules, reverse transcription of 5'P mRNAs and Illumina high-throughput sequencing. 5PSeq can identify translational pauses at rare codons that are often masked when using alternative methods. This approach can be applied to previously extracted RNA samples, and it is straightforward and does not require polyribosome purification or in vitro RNA footprinting. The protocol we describe here can be applied to Saccharomyces cerevisiae and potentially to other eukaryotic organisms. Three days are required to generate 5PSeq libraries.

  View details for DOI 10.1038/nprot.2016.026

  View details for PubMedID 26820793

• Comprehensive Identification of RNA-Binding Proteins by RNA Interactome Capture. Methods in molecular biology (Clifton, N.J.) Castello, A., Horos, R., Strein, C., Fischer, B., Eichelbaum, K., Steinmetz, L. M., Krijgsveld, J., Hentze, M. W. 2016; 1358: 131-139

  Abstract

  RNA associates with RNA-binding proteins (RBPs) from synthesis to decay, forming dynamic ribonucleoproteins (RNPs). In spite of the preeminent role of RBPs regulating RNA fate, the scope of cellular RBPs has remained largely unknown. We have recently developed a novel and comprehensive method to identify the repertoire of active RBPs of cultured cells, called RNA interactome capture. Using in vivo UV cross-linking on cultured cells, proteins are covalently bound to RNA if the contact between the two is direct ("zero distance"). Protein-RNA complexes are purified by poly(A) tail-dependent oligo(dT) capture and analyzed by quantitative mass spectrometry. Because UV irradiation is applied to living cells and purification is performed using highly stringent washes, RNA interactome capture identifies physiologic and direct protein-RNA interactions. Applied to HeLa cells, this protocol revealed the near-complete repertoire of RBPs, including hundreds of novel RNA binders. Apart from its RBP discovery capacity, quantitative and comparative RNA interactome capture can also be used to study the responses of the RBP repertoire to different physiological cues and processes, including metabolic stress, differentiation, development, or the response to drugs.

  View details for DOI 10.1007/978-1-4939-3067-8_8

  View details for PubMedID 26463381

• Sensing a revolution. Molecular systems biology Steinmetz, L. M., Jones, A. n. 2016; 12 (4): 867

  View details for PubMedID 27118815

  View details for PubMedCentralID PMC4848763

• SYGNALing a Red Light for Glioblastoma. Cell systems Brooks, A. N., Mueller, W. F., Steinmetz, L. M. 2016; 3 (2): 118–20

  Abstract

  A new multiomic network inference pipeline, SYGNAL, integrates patient data with mechanistically accurate transcriptional regulatory networks to predict drug combinations with synergistic anti-proliferative effects on glioblastoma multiforme.

  View details for PubMedID 27559923

• Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design. Genome biology Smith, J. D., Suresh, S., Schlecht, U., Wu, M., Wagih, O., Peltz, G., Davis, R. W., Steinmetz, L. M., Parts, L., St Onge, R. P. 2016; 17 (1): 45-?

  Abstract

  Genome-scale CRISPR interference (CRISPRi) has been used in human cell lines; however, the features of effective guide RNAs (gRNAs) in different organisms have not been well characterized. Here, we define rules that determine gRNA effectiveness for transcriptional repression in Saccharomyces cerevisiae.We create an inducible single plasmid CRISPRi system for gene repression in yeast, and use it to analyze fitness effects of gRNAs under 18 small molecule treatments. Our approach correctly identifies previously described chemical-genetic interactions, as well as a new mechanism of suppressing fluconazole toxicity by repression of the ERG25 gene. Assessment of multiple target loci across treatments using gRNA libraries allows us to determine generalizable features associated with gRNA efficacy. Guides that target regions with low nucleosome occupancy and high chromatin accessibility are clearly more effective. We also find that the best region to target gRNAs is between the transcription start site (TSS) and 200 bp upstream of the TSS. Finally, unlike nuclease-proficient Cas9 in human cells, the specificity of truncated gRNAs (18 nt of complementarity to the target) is not clearly superior to full-length gRNAs (20 nt of complementarity), as truncated gRNAs are generally less potent against both mismatched and perfectly matched targets.Our results establish a powerful functional and chemical genomics screening method and provide guidelines for designing effective gRNAs, which consider chromatin state and position relative to the target gene TSS. These findings will enable effective library design and genome-wide programmable gene repression in many genetic backgrounds.

  View details for DOI 10.1186/s13059-016-0900-9

  View details for PubMedID 26956608

  View details for PubMedCentralID PMC4784398

• Genome-Wide Identification of Alternative Polyadenylation Events Using 3'T-Fill. Methods in molecular biology (Clifton, N.J.) Wilkening, S., Pelechano, V., Steinmetz, L. M. 2016; 1358: 295-302

  Abstract

  Due to the increasing appreciation of the impact of alternative polyadenylation on cellular biology, our straightforward, scalable method is of interest to any researcher studying eukaryotic transcription. In addition to high quality gene expression measurements, it precisely maps poly(A) sites and thereby permits the distinction between differential 3'UTR isoforms. As sequencing through long homopolymer stretches is not possible on the Illumina platform, we developed a method that fills up the poly(A) stretch with dTTPs before the sequencing reaction starts.

  View details for DOI 10.1007/978-1-4939-3067-8_18

  View details for PubMedID 26463391

• Loss of the Yeast SR Protein Npl3 Alters Gene Expression Due to Transcription Readthrough PLOS GENETICS Holmes, R. K., Tuck, A. C., Zhu, C., Dunn-Davies, H. R., Kudla, G., Clauder-Munster, S., Granneman, S., Steinmetz, L. M., Guthrie, C., Tollervey, D. 2015; 11 (12)

  View details for DOI 10.1371/journal.pgen.1005735

  View details for Web of Science ID 000368518400064

• Chromatin Dynamics and the RNA Exosome Function in Concert to Regulate Transcriptional Homeostasis CELL REPORTS Rege, M., Subramanian, V., Zhu, C., Hsieh, T. S., Weiner, A., Friedman, N., Clauder-Muenster, S., Steinmetz, L. M., Rando, O. J., Boyer, L. A., Peterson, C. L. 2015; 13 (8): 1610-1622

  Abstract

  The histone variant H2A.Z is a hallmark of nucleosomes flanking promoters of protein-coding genes and is often found in nucleosomes that carry lysine 56-acetylated histone H3 (H3-K56Ac), a mark that promotes replication-independent nucleosome turnover. Here, we find that H3-K56Ac promotes RNA polymerase II occupancy at many protein-coding and noncoding loci, yet neither H3-K56Ac nor H2A.Z has a significant impact on steady-state mRNA levels in yeast. Instead, broad effects of H3-K56Ac or H2A.Z on RNA levels are revealed only in the absence of the nuclear RNA exosome. H2A.Z is also necessary for the expression of divergent, promoter-proximal noncoding RNAs (ncRNAs) in mouse embryonic stem cells. Finally, we show that H2A.Z functions with H3-K56Ac to facilitate formation of chromosome interaction domains (CIDs). Our study suggests that H2A.Z and H3-K56Ac work in concert with the RNA exosome to control mRNA and ncRNA expression, perhaps in part by regulating higher-order chromatin structures.

  View details for DOI 10.1016/j.celrep.2015.10.030

  View details for Web of Science ID 000365404900011

  View details for PubMedID 26586442

  View details for PubMedCentralID PMC4662874

• Inflammation-Induced Emergency Megakaryopoiesis Driven by Hematopoietic Stem Cell-like Megakaryocyte Progenitors CELL STEM CELL Haas, S., Hansson, J., Klimmeck, D., Loeffler, D., Velten, L., UcKelmann, H., Wurzer, S., Prendergast, A. M., Schnell, A., Hexel, K., Santarella-Mellwig, R., Blaszkiewicz, S., Kuck, A., Geiger, H., Milsom, M. D., Steinmetz, L. M., Schroeder, T., Trumpp, A., Krijgsveld, J., Essers, M. A. 2015; 17 (4): 422-434

  Abstract

  Infections are associated with extensive platelet consumption, representing a high risk for health. However, the mechanism coordinating the rapid regeneration of the platelet pool during such stress conditions remains unclear. Here, we report that the phenotypic hematopoietic stem cell (HSC) compartment contains stem-like megakaryocyte-committed progenitors (SL-MkPs), a cell population that shares many features with multipotent HSCs and serves as a lineage-restricted emergency pool for inflammatory insults. During homeostasis, SL-MkPs are maintained in a primed but quiescent state, thus contributing little to steady-state megakaryopoiesis. Even though lineage-specific megakaryocyte transcripts are expressed, protein synthesis is suppressed. In response to acute inflammation, SL-MkPs become activated, resulting in megakaryocyte protein production from pre-existing transcripts and a maturation of SL-MkPs and other megakaryocyte progenitors. This results in an efficient replenishment of platelets that are lost during inflammatory insult. Thus, our study reveals an emergency machinery that counteracts life-threatening platelet depletions during acute inflammation.

  View details for DOI 10.1016/j.stem.2015.07.007

  View details for Web of Science ID 000365693800009

  View details for PubMedID 26299573

• Genetic Control of Chromatin States in Humans Involves Local and Distal Chromosomal Interactions CELL Grubert, F., Zaugg, J. B., Kasowski, M., Ursu, O., Spacek, D. V., Martin, A. R., Greenside, P., Srivas, R., Phanstiel, D. H., Pekowska, A., Heidari, N., Euskirchen, G., Huber, W., Pritchard, J. K., Bustamante, C. D., Steinmetz, L. M., Kundaje, A., Snyder, M. 2015; 162 (5): 1051-1065

  Abstract

  Deciphering the impact of genetic variants on gene regulation is fundamental to understanding human disease. Although gene regulation often involves long-range interactions, it is unknown to what extent non-coding genetic variants influence distal molecular phenotypes. Here, we integrate chromatin profiling for three histone marks in lymphoblastoid cell lines (LCLs) from 75 sequenced individuals with LCL-specific Hi-C and ChIA-PET-based chromatin contact maps to uncover one of the largest collections of local and distal histone quantitative trait loci (hQTLs). Distal QTLs are enriched within topologically associated domains and exhibit largely concordant variation of chromatin state coordinated by proximal and distal non-coding genetic variants. Histone QTLs are enriched for common variants associated with autoimmune diseases and enable identification of putative target genes of disease-associated variants from genome-wide association studies. These analyses provide insights into how genetic variation can affect human disease phenotypes by coordinated changes in chromatin at interacting regulatory elements.

  View details for DOI 10.1016/j.cell.2015.07.048

  View details for Web of Science ID 000360589900015

  View details for PubMedCentralID PMC4556133

• Multitarget, quantitative nanoplasmonic electrical field-enhanced resonating device (NE2RD) for diagnostics. Proceedings of the National Academy of Sciences of the United States of America Inci, F., Filippini, C., Baday, M., Ozen, M. O., Calamak, S., Durmus, N. G., Wang, S., Hanhauser, E., Hobbs, K. S., Juillard, F., Kuang, P. P., Vetter, M. L., Carocci, M., Yamamoto, H. S., Takagi, Y., Yildiz, U. H., Akin, D., Wesemann, D. R., Singhal, A., Yang, P. L., Nibert, M. L., Fichorova, R. N., Lau, D. T., Henrich, T. J., Kaye, K. M., Schachter, S. C., Kuritzkes, D. R., Steinmetz, L. M., Gambhir, S. S., Davis, R. W., Demirci, U. 2015; 112 (32): E4354-63

  Abstract

  Recent advances in biosensing technologies present great potential for medical diagnostics, thus improving clinical decisions. However, creating a label-free general sensing platform capable of detecting multiple biotargets in various clinical specimens over a wide dynamic range, without lengthy sample-processing steps, remains a considerable challenge. In practice, these barriers prevent broad applications in clinics and at patients' homes. Here, we demonstrate the nanoplasmonic electrical field-enhanced resonating device (NE(2)RD), which addresses all these impediments on a single platform. The NE(2)RD employs an immunodetection assay to capture biotargets, and precisely measures spectral color changes by their wavelength and extinction intensity shifts in nanoparticles without prior sample labeling or preprocessing. We present through multiple examples, a label-free, quantitative, portable, multitarget platform by rapidly detecting various protein biomarkers, drugs, protein allergens, bacteria, eukaryotic cells, and distinct viruses. The linear dynamic range of NE(2)RD is five orders of magnitude broader than ELISA, with a sensitivity down to 400 fg/mL This range and sensitivity are achieved by self-assembling gold nanoparticles to generate hot spots on a 3D-oriented substrate for ultrasensitive measurements. We demonstrate that this precise platform handles multiple clinical samples such as whole blood, serum, and saliva without sample preprocessing under diverse conditions of temperature, pH, and ionic strength. The NE(2)RD's broad dynamic range, detection limit, and portability integrated with a disposable fluidic chip have broad applications, potentially enabling the transition toward precision medicine at the point-of-care or primary care settings and at patients' homes.

  View details for DOI 10.1073/pnas.1510824112

  View details for PubMedID 26195743

  View details for PubMedCentralID PMC4538635

• Multitarget, quantitative nanoplasmonic electrical field-enhanced resonating device ((NERD)-R-2) for diagnostics PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Inci, F., Filippini, C., Baday, M., Ozen, M. O., Calamak, S., Durmus, N. G., Wang, S., Hanhauser, E., Hobbs, K. S., Juillard, F., Kuang, P. P., Vetter, M. L., Carocci, M., Yamamoto, H. S., Takagi, Y., Yildiz, U. H., Akin, D., Wesemann, D. R., Singhal, A., Yang, P. L., Nibert, M. L., Fichorova, R. N., Lau, D. T., Henrich, T. J., Kaye, K. M., Schachter, S. C., Kuritzkes, D. R., Steinmetz, L. M., Gambhir, S. S., Davis, R. W., Demirci, U. 2015; 112 (32): E4354-E4363

  Abstract

  Recent advances in biosensing technologies present great potential for medical diagnostics, thus improving clinical decisions. However, creating a label-free general sensing platform capable of detecting multiple biotargets in various clinical specimens over a wide dynamic range, without lengthy sample-processing steps, remains a considerable challenge. In practice, these barriers prevent broad applications in clinics and at patients' homes. Here, we demonstrate the nanoplasmonic electrical field-enhanced resonating device (NE(2)RD), which addresses all these impediments on a single platform. The NE(2)RD employs an immunodetection assay to capture biotargets, and precisely measures spectral color changes by their wavelength and extinction intensity shifts in nanoparticles without prior sample labeling or preprocessing. We present through multiple examples, a label-free, quantitative, portable, multitarget platform by rapidly detecting various protein biomarkers, drugs, protein allergens, bacteria, eukaryotic cells, and distinct viruses. The linear dynamic range of NE(2)RD is five orders of magnitude broader than ELISA, with a sensitivity down to 400 fg/mL This range and sensitivity are achieved by self-assembling gold nanoparticles to generate hot spots on a 3D-oriented substrate for ultrasensitive measurements. We demonstrate that this precise platform handles multiple clinical samples such as whole blood, serum, and saliva without sample preprocessing under diverse conditions of temperature, pH, and ionic strength. The NE(2)RD's broad dynamic range, detection limit, and portability integrated with a disposable fluidic chip have broad applications, potentially enabling the transition toward precision medicine at the point-of-care or primary care settings and at patients' homes.

  View details for DOI 10.1073/pnas.1510824112

  View details for Web of Science ID 000359285100006

  View details for PubMedID 26195743

  View details for PubMedCentralID PMC4538635

• Magnetic levitation of single cells. Proceedings of the National Academy of Sciences of the United States of America Durmus, N. G., Tekin, H. C., Guven, S., Sridhar, K., Arslan Yildiz, A., Calibasi, G., Ghiran, I., Davis, R. W., Steinmetz, L. M., Demirci, U. 2015; 112 (28): E3661-8

  Abstract

  Several cellular events cause permanent or transient changes in inherent magnetic and density properties of cells. Characterizing these changes in cell populations is crucial to understand cellular heterogeneity in cancer, immune response, infectious diseases, drug resistance, and evolution. Although magnetic levitation has previously been used for macroscale objects, its use in life sciences has been hindered by the inability to levitate microscale objects and by the toxicity of metal salts previously applied for levitation. Here, we use magnetic levitation principles for biological characterization and monitoring of cells and cellular events. We demonstrate that each cell type (i.e., cancer, blood, bacteria, and yeast) has a characteristic levitation profile, which we distinguish at an unprecedented resolution of 1 × 10(-4) g⋅mL(-1). We have identified unique differences in levitation and density blueprints between breast, esophageal, colorectal, and nonsmall cell lung cancer cell lines, as well as heterogeneity within these seemingly homogenous cell populations. Furthermore, we demonstrate that changes in cellular density and levitation profiles can be monitored in real time at single-cell resolution, allowing quantification of heterogeneous temporal responses of each cell to environmental stressors. These data establish density as a powerful biomarker for investigating living systems and their responses. Thereby, our method enables rapid, density-based imaging and profiling of single cells with intriguing applications, such as label-free identification and monitoring of heterogeneous biological changes under various physiological conditions, including antibiotic or cancer treatment in personalized medicine.

  View details for DOI 10.1073/pnas.1509250112

  View details for PubMedID 26124131

  View details for PubMedCentralID PMC4507238

• The Nuclear PolyA-Binding Protein Nab2p Is Essential for mRNA Production CELL REPORTS Schmid, M., Olszewski, P., Pelechano, V., Gupta, I., Steinmetz, L. M., Jensen, T. H. 2015; 12 (1): 128-139

  Abstract

  Polyadenylation of mRNA is a key step in eukaryotic gene expression. However, despite the major impact of poly(A) tails on mRNA metabolism, the precise roles of poly(A)-binding proteins (PABPs) in nuclear mRNA biogenesis remain elusive. Here, we demonstrate that rapid nuclear depletion of the S. cerevisiae PABP Nab2p leads to a global loss of cellular mRNA, but not of RNA lacking poly(A) tails. Disappearance of mRNA is a nuclear event, but not due to decreased transcription. Instead, the absence of Nab2p results in robust nuclear mRNA decay by the ribonucleolytic RNA exosome in a polyadenylation-dependent process. We conclude that Nab2p is required to protect early mRNA and therefore constitutes a crucial nuclear mRNA biogenesis factor.

  View details for DOI 10.1016/j.celrep.2015.06.008

  View details for Web of Science ID 000357673300014

  View details for PubMedID 26119729

• Yeast as a system for modeling mitochondrial disease mechanisms and discovering therapies. Disease models & mechanisms Lasserre, J., Dautant, A., Aiyar, R. S., Kucharczyk, R., Glatigny, A., Tribouillard-Tanvier, D., Rytka, J., Blondel, M., Skoczen, N., Reynier, P., Pitayu, L., Rötig, A., Delahodde, A., Steinmetz, L. M., Dujardin, G., Procaccio, V., di Rago, J. 2015; 8 (6): 509-526

  Abstract

  Mitochondrial diseases are severe and largely untreatable. Owing to the many essential processes carried out by mitochondria and the complex cellular systems that support these processes, these diseases are diverse, pleiotropic, and challenging to study. Much of our current understanding of mitochondrial function and dysfunction comes from studies in the baker's yeast Saccharomyces cerevisiae. Because of its good fermenting capacity, S. cerevisiae can survive mutations that inactivate oxidative phosphorylation, has the ability to tolerate the complete loss of mitochondrial DNA (a property referred to as 'petite-positivity'), and is amenable to mitochondrial and nuclear genome manipulation. These attributes make it an excellent model system for studying and resolving the molecular basis of numerous mitochondrial diseases. Here, we review the invaluable insights this model organism has yielded about diseases caused by mitochondrial dysfunction, which ranges from primary defects in oxidative phosphorylation to metabolic disorders, as well as dysfunctions in maintaining the genome or in the dynamics of mitochondria. Owing to the high level of functional conservation between yeast and human mitochondrial genes, several yeast species have been instrumental in revealing the molecular mechanisms of pathogenic human mitochondrial gene mutations. Importantly, such insights have pointed to potential therapeutic targets, as have genetic and chemical screens using yeast.

  View details for DOI 10.1242/dmm.020438

  View details for PubMedID 26035862

  View details for PubMedCentralID PMC4457039

• Yeast as a system for modeling mitochondrial disease mechanisms and discovering therapies DISEASE MODELS & MECHANISMS Lasserre, J., Dautant, A., Aiyar, R. S., Kucharczyk, R., Glatigny, A., Tribouillard-Tanvier, D., Rytka, J., Blondel, M., Skoczen, N., Reynier, P., Pitayu, L., Roetig, A., Delahodde, A., Steinmetz, L. M., Dujardin, G., Procaccio, V., di Rago, J. 2015; 8 (6): 509-526

  Abstract

  Mitochondrial diseases are severe and largely untreatable. Owing to the many essential processes carried out by mitochondria and the complex cellular systems that support these processes, these diseases are diverse, pleiotropic, and challenging to study. Much of our current understanding of mitochondrial function and dysfunction comes from studies in the baker's yeast Saccharomyces cerevisiae. Because of its good fermenting capacity, S. cerevisiae can survive mutations that inactivate oxidative phosphorylation, has the ability to tolerate the complete loss of mitochondrial DNA (a property referred to as 'petite-positivity'), and is amenable to mitochondrial and nuclear genome manipulation. These attributes make it an excellent model system for studying and resolving the molecular basis of numerous mitochondrial diseases. Here, we review the invaluable insights this model organism has yielded about diseases caused by mitochondrial dysfunction, which ranges from primary defects in oxidative phosphorylation to metabolic disorders, as well as dysfunctions in maintaining the genome or in the dynamics of mitochondria. Owing to the high level of functional conservation between yeast and human mitochondrial genes, several yeast species have been instrumental in revealing the molecular mechanisms of pathogenic human mitochondrial gene mutations. Importantly, such insights have pointed to potential therapeutic targets, as have genetic and chemical screens using yeast.

  View details for DOI 10.1242/dmm.020438

  View details for Web of Science ID 000355559100001

  View details for PubMedCentralID PMC4457039

• Temporal expression profiling identifies pathways mediating effect of causal variant on phenotype. PLoS genetics Gupta, S., Radhakrishnan, A., Raharja-Liu, P., Lin, G., Steinmetz, L. M., Gagneur, J., Sinha, H. 2015; 11 (6)

  Abstract

  Even with identification of multiple causal genetic variants for common human diseases, understanding the molecular processes mediating the causal variants' effect on the disease remains a challenge. This understanding is crucial for the development of therapeutic strategies to prevent and treat disease. While static profiling of gene expression is primarily used to get insights into the biological bases of diseases, it makes differentiating the causative from the correlative effects difficult, as the dynamics of the underlying biological processes are not monitored. Using yeast as a model, we studied genome-wide gene expression dynamics in the presence of a causal variant as the sole genetic determinant, and performed allele-specific functional validation to delineate the causal effects of the genetic variant on the phenotype. Here, we characterized the precise genetic effects of a functional MKT1 allelic variant in sporulation efficiency variation. A mathematical model describing meiotic landmark events and conditional activation of MKT1 expression during sporulation specified an early meiotic role of this variant. By analyzing the early meiotic genome-wide transcriptional response, we demonstrate an MKT1-dependent role of novel modulators, namely, RTG1/3, regulators of mitochondrial retrograde signaling, and DAL82, regulator of nitrogen starvation, in additively effecting sporulation efficiency. In the presence of functional MKT1 allele, better respiration during early sporulation was observed, which was dependent on the mitochondrial retrograde regulator, RTG3. Furthermore, our approach showed that MKT1 contributes to sporulation independent of Puf3, an RNA-binding protein that steady-state transcription profiling studies have suggested to mediate MKT1-pleiotropic effects during mitotic growth. These results uncover interesting regulatory links between meiosis and mitochondrial retrograde signaling. In this study, we highlight the advantage of analyzing allele-specific transcriptional dynamics of mediating genes. Applications in higher eukaryotes can be valuable for inferring causal molecular pathways underlying complex dynamic processes, such as development, physiology and disease progression.

  View details for DOI 10.1371/journal.pgen.1005195

  View details for PubMedID 26039065

  View details for PubMedCentralID PMC4454590

• Variation in Crossover Frequencies Perturb Crossover Assurance Without Affecting Meiotic Chromosome Segregation in Saccharomyces cerevisiae GENETICS Krishnaprasad, G. N., Anand, M. T., Lin, G., Tekkedil, M. M., Steinmetz, L. M., Nishant, K. T. 2015; 199 (2): 399-412

  Abstract

  The segregation of homologous chromosomes during the Meiosis I division requires an obligate crossover per homolog pair (crossover assurance). In Saccharomyces cerevisiae and mammals, Msh4 and Msh5 proteins stabilize Holliday junctions and its progenitors to facilitate crossing over. S. cerevisiae msh4/5 hypomorphs that reduce crossover levels up to twofold at specific loci on chromosomes VII, VIII, and XV without affecting homolog segregation were identified recently. We use the msh4-R676W hypomorph to ask if the obligate crossover is insulated from variation in crossover frequencies, using a S. cerevisiae S288c/YJM789 hybrid to map recombination genome-wide. The msh4-R676W hypomorph made on average 64 crossovers per meiosis compared to 94 made in wild type and 49 in the msh4Δ mutant confirming the defect seen at individual loci on a genome-wide scale. Crossover reductions in msh4-R676W and msh4Δ were significant across chromosomes regardless of size, unlike previous observations made at specific loci. The msh4-R676W hypomorph showed reduced crossover interference. Although crossover reduction in msh4-R676W is modest, 42% of the four viable spore tetrads showed nonexchange chromosomes. These results, along with modeling of crossover distribution, suggest the significant reduction in crossovers across chromosomes and the loss of interference compromises the obligate crossover in the msh4 hypomorph. The high spore viability of the msh4 hypomorph is maintained by efficient segregation of the natural nonexchange chromosomes. Our results suggest that variation in crossover frequencies can compromise the obligate crossover and also support a mechanistic role for interference in obligate crossover formation.

  View details for DOI 10.1534/genetics.114.172320

  View details for Web of Science ID 000349459400009

  View details for PubMedID 25467183

  View details for PubMedCentralID PMC4317650

• Chromatin-dependent regulation of RNA polymerases II and III activity throughout the transcription cycle NUCLEIC ACIDS RESEARCH Jordan-Pla, A., Gupta, I., de Miguel-Jimenez, L., Steinmetz, L. M., Chavez, S., Pelechano, V., Perez-Ortin, J. E. 2015; 43 (2): 787-802

  Abstract

  The particular behaviour of eukaryotic RNA polymerases along different gene regions and amongst distinct gene functional groups is not totally understood. To cast light onto the alternative active or backtracking states of RNA polymerase II, we have quantitatively mapped active RNA polymerases at a high resolution following a new biotin-based genomic run-on (BioGRO) technique. Compared with conventional profiling with chromatin immunoprecipitation, the analysis of the BioGRO profiles in Saccharomyces cerevisiae shows that RNA polymerase II has unique activity profiles at both gene ends, which are highly dependent on positioned nucleosomes. This is the first demonstration of the in vivo influence of positioned nucleosomes on transcription elongation. The particular features at the 5' end and around the polyadenylation site indicate that this polymerase undergoes extensive specific-activity regulation in the initial and final transcription elongation phases. The genes encoding for ribosomal proteins show distinctive features at both ends. BioGRO also provides the first nascentome analysis for RNA polymerase III, which indicates that transcription of tRNA genes is poorly regulated at the individual copy level. The present study provides a novel perspective of the transcription cycle that incorporates inactivation/reactivation as an important aspect of RNA polymerase dynamics.

  View details for DOI 10.1093/nar/gku1349

  View details for Web of Science ID 000350209000017

  View details for PubMedID 25550430

  View details for PubMedCentralID PMC4333398

• The conserved histone deacetylase Rpd3 and its DNA binding subunit Ume6 control dynamic transcript architecture during mitotic growth and meiotic development NUCLEIC ACIDS RESEARCH Lardenois, A., Stuparevic, I., Liu, Y., Law, M. J., Becker, E., Smagulova, F., Waern, K., Guilleux, M., Horecka, J., Chu, A., Kervarrec, C., Strich, R., Snyder, M., Davis, R. W., Steinmetz, L. M., Primig, M. 2015; 43 (1): 115-128

  Abstract

  It was recently reported that the sizes of many mRNAs change when budding yeast cells exit mitosis and enter the meiotic differentiation pathway. These differences were attributed to length variations of their untranslated regions. The function of UTRs in protein translation is well established. However, the mechanism controlling the expression of distinct transcript isoforms during mitotic growth and meiotic development is unknown. In this study, we order developmentally regulated transcript isoforms according to their expression at specific stages during meiosis and gametogenesis, as compared to vegetative growth and starvation. We employ regulatory motif prediction, in vivo protein-DNA binding assays, genetic analyses and monitoring of epigenetic amino acid modification patterns to identify a novel role for Rpd3 and Ume6, two components of a histone deacetylase complex already known to repress early meiosis-specific genes in dividing cells, in mitotic repression of meiosis-specific transcript isoforms. Our findings classify developmental stage-specific early, middle and late meiotic transcript isoforms, and they point to a novel HDAC-dependent control mechanism for flexible transcript architecture during cell growth and differentiation. Since Rpd3 is highly conserved and ubiquitously expressed in many tissues, our results are likely relevant for development and disease in higher eukaryotes.

  View details for DOI 10.1093/nar/gku1185

  View details for Web of Science ID 000350207100017

  View details for PubMedID 25477386

  View details for PubMedCentralID PMC4288150

• Single-cell polyadenylation site mapping reveals 3' isoform choice variability. Molecular systems biology Velten, L., Anders, S., Pekowska, A., Järvelin, A. I., Huber, W., Pelechano, V., Steinmetz, L. M. 2015; 11 (6): 812-?

  Abstract

  Cell-to-cell variability in gene expression is important for many processes in biology, including embryonic development and stem cell homeostasis. While heterogeneity of gene expression levels has been extensively studied, less attention has been paid to mRNA polyadenylation isoform choice. 3' untranslated regions regulate mRNA fate, and their choice is tightly controlled during development, but how 3' isoform usage varies within genetically and developmentally homogeneous cell populations has not been explored. Here, we perform genome-wide quantification of polyadenylation site usage in single mouse embryonic and neural stem cells using a novel single-cell transcriptomic method, BATSeq. By applying BATBayes, a statistical framework for analyzing single-cell isoform data, we find that while the developmental state of the cell globally determines isoform usage, single cells from the same state differ in the choice of isoforms. Notably this variation exceeds random selection with equal preference in all cells, a finding that was confirmed by RNA FISH data. Variability in 3' isoform choice has potential implications on functional cell-to-cell heterogeneity as well as utility in resolving cell populations.

  View details for DOI 10.15252/msb.20156198

  View details for PubMedID 26040288

• Negative feedback buffers effects of regulatory variants MOLECULAR SYSTEMS BIOLOGY Bader, D. M., Wilkening, S., Lin, G., Tekkedil, M. M., Dietrich, K., Steinmetz, L. M., Gagneur, J. 2015; 11 (1)

  Abstract

  Mechanisms conferring robustness against regulatory variants have been controversial. Previous studies suggested widespread buffering of RNA misexpression on protein levels during translation. We do not find evidence that translational buffering is common. Instead, we find extensive buffering at the level of RNA expression, exerted through negative feedback regulation acting in trans, which reduces the effect of regulatory variants on gene expression. Our approach is based on a novel experimental design in which allelic differential expression in a yeast hybrid strain is compared to allelic differential expression in a pool of its spores. Allelic differential expression in the hybrid is due to cis-regulatory differences only. Instead, in the pool of spores allelic differential expression is not only due to cis-regulatory differences but also due to local trans effects that include negative feedback. We found that buffering through such local trans regulation is widespread, typically compensating for about 15% of cis-regulatory effects on individual genes. Negative feedback is stronger not only for essential genes, indicating its functional relevance, but also for genes with low to middle levels of expression, for which tight regulation matters most. We suggest that negative feedback is one mechanism of Waddington's canalization, facilitating the accumulation of genetic variants that might give selective advantage in different environments.

  View details for Web of Science ID 000349105100009

  View details for PubMedID 25634765

• A high-throughput ChIP-Seq for large-scale chromatin studies MOLECULAR SYSTEMS BIOLOGY Chabbert, C. D., Adjalley, S. H., Klaus, B., Fritsch, E. S., Gupta, I., Pelechano, V., Steinmetz, L. M. 2015; 11 (1)

  Abstract

  We present a modified approach of chromatin immuno-precipitation followed by sequencing (ChIP-Seq), which relies on the direct ligation of molecular barcodes to chromatin fragments, thereby permitting experimental scale-up. With Bar-ChIP now enabling the concurrent profiling of multiple DNA-protein interactions, we report the simultaneous generation of 90 ChIP-Seq datasets without any robotic instrumentation. We demonstrate that application of Bar-ChIP to a panel of Saccharomyces cerevisiae chromatin-associated mutants provides a rapid and accurate genome-wide overview of their chromatin status. Additionally, we validate the utility of this technology to derive novel biological insights by identifying a role for the Rpd3S complex in maintaining H3K14 hypo-acetylation in gene bodies. We also report an association between the presence of intragenic H3K4 tri-methylation and the emergence of cryptic transcription in a Set2 mutant. Finally, we uncover a crosstalk between H3K14 acetylation and H3K4 methylation in this mutant. These results show that Bar-ChIP enables biological discovery through rapid chromatin profiling at single-nucleosome resolution for various conditions and protein modifications at once.

  View details for Web of Science ID 000349105100007

  View details for PubMedID 25583149

• Expression of nuclear and mitochondrial genes encoding ATP synthase is synchronized by disassembly of a multisynthetase complex. Molecular cell Frechin, M., Enkler, L., Tetaud, E., Laporte, D., Senger, B., Blancard, C., Hammann, P., Bader, G., Clauder-Münster, S., Steinmetz, L. M., Martin, R. P., di Rago, J., Becker, H. D. 2014; 56 (6): 763-776

  Abstract

  In eukaryotic cells, oxidative phosphorylation involves multisubunit complexes of mixed genetic origin. Assembling these complexes requires an organelle-independent synchronizing system for the proper expression of nuclear and mitochondrial genes. Here we show that proper expression of the F1FO ATP synthase (complex V) depends on a cytosolic complex (AME) made of two aminoacyl-tRNA synthetases (cERS and cMRS) attached to an anchor protein, Arc1p. When yeast cells adapt to respiration the Snf1/4 glucose-sensing pathway inhibits ARC1 expression triggering simultaneous release of cERS and cMRS. Free cMRS and cERS relocate to the nucleus and mitochondria, respectively, to synchronize nuclear transcription and mitochondrial translation of ATP synthase genes. Strains releasing asynchronously the two aminoacyl-tRNA synthetases display aberrant expression of nuclear and mitochondrial genes encoding subunits of complex V resulting in severe defects of the oxidative phosphorylation mechanism. This work shows that the AME complex coordinates expression of enzymes that require intergenomic control.

  View details for DOI 10.1016/j.molcel.2014.10.015

  View details for PubMedID 25453761

• Roadblock Termination by Reb1p Restricts Cryptic and Readthrough Transcription MOLECULAR CELL Colin, J., Candelli, T., Porrua, O., Boulay, J., Zhu, C., Lacroute, F., Steinmetz, L. M., Libri, D. 2014; 56 (5): 667-680

  Abstract

  Widely transcribed compact genomes must cope with the major challenge of frequent overlapping or concurrent transcription events. Efficient and timely transcription termination is crucial to control pervasive transcription and prevent transcriptional interference. In yeast, transcription termination of RNA polymerase II (RNAPII) occurs via two possible pathways that both require recognition of termination signals on nascent RNA by specific factors. We describe here an additional mechanism of transcription termination for RNAPII and demonstrate its biological significance. We show that the transcriptional activator Reb1p bound to DNA is a roadblock for RNAPII, which pauses and is ubiquitinated, thus triggering termination. Reb1p-dependent termination generates a class of cryptic transcripts that are degraded in the nucleus by the exosome. We also observed transcriptional interference between neighboring genes in the absence of Reb1p. This work demonstrates the importance of roadblock termination for controlling pervasive transcription and preventing transcription through gene regulatory regions.

  View details for DOI 10.1016/j.molcel.2014.10.026

  View details for Web of Science ID 000346653300008

  View details for PubMedID 25479637

• Mitochondrial protein sorting as a therapeutic target for ATP synthase disorders NATURE COMMUNICATIONS Aiyar, R. S., Bohnert, M., Duvezin-Caubet, S., Voisset, C., Gagneur, J., Fritsch, E. S., Couplan, E., von der Malsburg, K., Funaya, C., Soubigou, F., Courtin, F., Suresh, S., Kucharczyk, R., Evrard, J., Antony, C., St Onge, R. P., Blondel, M., di Rago, J., van der Laan, M., Steinmetz, L. M. 2014; 5

  Abstract

  Mitochondrial diseases are systemic, prevalent and often fatal; yet treatments remain scarce. Identifying molecular intervention points that can be therapeutically targeted remains a major challenge, which we confronted via a screening assay we developed. Using yeast models of mitochondrial ATP synthase disorders, we screened a drug repurposing library, and applied genomic and biochemical techniques to identify pathways of interest. Here we demonstrate that modulating the sorting of nuclear-encoded proteins into mitochondria, mediated by the TIM23 complex, proves therapeutic in both yeast and patient-derived cells exhibiting ATP synthase deficiency. Targeting TIM23-dependent protein sorting improves an array of phenotypes associated with ATP synthase disorders, including biogenesis and activity of the oxidative phosphorylation machinery. Our study establishes mitochondrial protein sorting as an intervention point for ATP synthase disorders, and because of the central role of this pathway in mitochondrial biogenesis, it holds broad value for the treatment of mitochondrial diseases.

  View details for DOI 10.1038/ncomms6585

  View details for Web of Science ID 000347224100001

  View details for PubMedID 25519239

• Transcription mediated insulation and interference direct gene cluster expression switches ELIFE Tania Nguyen, T., Fischl, H., Howe, F. S., Woloszczuk, R., Barros, A. S., Xu, Z., Brown, D., Murray, S. C., Haenni, S., Halstead, J. M., O'Connor, L., Shipkovenska, G., Steinmetz, L. M., Mellor, J. 2014; 3

  Abstract

  In yeast, many tandemly arranged genes show peak expression in different phases of the metabolic cycle (YMC) or in different carbon sources, indicative of regulation by a bi-modal switch, but it is not clear how these switches are controlled. Using native elongating transcript analysis (NET-seq), we show that transcription itself is a component of bi-modal switches, facilitating reciprocal expression in gene clusters. HMS2, encoding a growth-regulated transcription factor, switches between sense- or antisense-dominant states that also coordinate up- and down-regulation of transcription at neighbouring genes. Engineering HMS2 reveals alternative mono-, di- or tri-cistronic and antisense transcription units (TUs), using different promoter and terminator combinations, that underlie state-switching. Promoters or terminators are excluded from functional TUs by read-through transcriptional interference, while antisense TUs insulate downstream genes from interference. We propose that the balance of transcriptional insulation and interference at gene clusters facilitates gene expression switches during intracellular and extracellular environmental change.

  View details for DOI 10.7554/eLife.03635

  View details for Web of Science ID 000345639000007

  View details for PubMedID 25407679

• Induced Mutations in Yeast Cell Populations Adapting to an Unforeseen Challenge PLOS ONE Moore, L. S., Wei, W., Stolovicki, E., Benbenishty, T., Wilkening, S., Steinmetz, L. M., Braun, E., David, L. 2014; 9 (10)

  Abstract

  The modern evolutionary synthesis assumes that mutations occur at random, independently of the environment in which they confer an advantage. However, there are indications that cells facing challenging conditions can adapt rapidly, utilizing processes beyond selection of pre-existing genetic variation. Here, we show that a strong regulatory challenge can induce mutations in many independent yeast cells, in the absence of general mutagenesis. Whole genome sequencing of cell lineages reveals a repertoire of independent mutations within a single lineage that arose only after the cells were exposed to the challenging environment, while other cells in the same lineage adapted without any mutation in their genomes. Thus, our experiments uncovered multiple alternative routes for heritable adaptation that were all induced in the same lineage during a short time period. Our results demonstrate the existence of adaptation mechanisms beyond random mutation, suggesting a tight connection between physiological and genetic processes.

  View details for DOI 10.1371/journal.pone.0111133

  View details for Web of Science ID 000343662800087

  View details for PubMedID 25340744

  View details for PubMedCentralID PMC4207790

• A Genome-Wide Map of Mitochondrial DNA Recombination in Yeast GENETICS Fritsch, E. S., Chabbert, C. D., Klaus, B., Steinmetz, L. M. 2014; 198 (2): 755-U428

  Abstract

  In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome maintenance.

  View details for DOI 10.1534/genetics.114.166637

  View details for Web of Science ID 000343885300033

  View details for PubMedID 25081569

  View details for PubMedCentralID PMC4196626

• Ultrasensitive proteome analysis using paramagnetic bead technology MOLECULAR SYSTEMS BIOLOGY Hughes, C. S., Foehr, S., Garfield, D. A., Furlong, E. E., Steinmetz, L. M., Krijgsveld, J. 2014; 10 (10)

  Abstract

  In order to obtain a systems-level understanding of a complex biological system, detailed proteome information is essential. Despite great progress in proteomics technologies, thorough interrogation of the proteome from quantity-limited biological samples is hampered by inefficiencies during processing. To address these challenges, here we introduce a novel protocol using paramagnetic beads, termed Single-Pot Solid-Phase-enhanced Sample Preparation (SP3). SP3 provides a rapid and unbiased means of proteomic sample preparation in a single tube that facilitates ultrasensitive analysis by outperforming existing protocols in terms of efficiency, scalability, speed, throughput, and flexibility. To illustrate these benefits, characterization of 1,000 HeLa cells and single Drosophila embryos is used to establish that SP3 provides an enhanced platform for profiling proteomes derived from sub-microgram amounts of material. These data present a first view of developmental stage-specific proteome dynamics in Drosophila at a single-embryo resolution, permitting characterization of inter-individual expression variation. Together, the findings of this work position SP3 as a superior protocol that facilitates exciting new directions in multiple areas of proteomics ranging from developmental biology to clinical applications.

  View details for DOI 10.15252/msb.20145625

  View details for Web of Science ID 000344595300003

  View details for PubMedID 25358341

• The Not5 Subunit of the Ccr4-Not Complex Connects Transcription and Translation PLOS GENETICS Villanyi, Z., Ribaud, V., Kassem, S., Panasenko, O. O., Pahi, Z., Gupta, I., Steinmetz, L., Boros, I., Collart, M. A. 2014; 10 (10)

  Abstract

  Recent studies have suggested that a sub-complex of RNA polymerase II composed of Rpb4 and Rpb7 couples the nuclear and cytoplasmic stages of gene expression by associating with newly made mRNAs in the nucleus, and contributing to their translation and degradation in the cytoplasm. Here we show by yeast two hybrid and co-immunoprecipitation experiments, followed by ribosome fractionation and fluorescent microscopy, that a subunit of the Ccr4-Not complex, Not5, is essential in the nucleus for the cytoplasmic functions of Rpb4. Not5 interacts with Rpb4; it is required for the presence of Rpb4 in polysomes, for interaction of Rpb4 with the translation initiation factor eIF3 and for association of Rpb4 with mRNAs. We find that Rpb7 presence in the cytoplasm and polysomes is much less significant than that of Rpb4, and that it does not depend upon Not5. Hence Not5-dependence unlinks the cytoplasmic functions of Rpb4 and Rpb7. We additionally determine with RNA immunoprecipitation and native gel analysis that Not5 is needed in the cytoplasm for the co-translational assembly of RNA polymerase II. This stems from the importance of Not5 for the association of the R2TP Hsp90 co-chaperone with polysomes translating RPB1 mRNA to protect newly synthesized Rpb1 from aggregation. Hence taken together our results show that Not5 interconnects translation and transcription.

  View details for DOI 10.1371/journal.pgen.1004569

  View details for Web of Science ID 000344650700002

  View details for PubMedID 25340856

• Heritability and genetic basis of protein level variation in an outbred population GENOME RESEARCH Parts, L., Liu, Y., Tekkedil, M. M., Steinmetz, L. M., Caudy, A. A., Fraser, A. G., Boone, C., Andrews, B. J., Rosebrock, A. P. 2014; 24 (8): 1363-1370

  Abstract

  The genetic basis of heritable traits has been studied for decades. Although recent mapping efforts have elucidated genetic determinants of transcript levels, mapping of protein abundance has lagged. Here, we analyze levels of 4084 GFP-tagged yeast proteins in the progeny of a cross between a laboratory and a wild strain using flow cytometry and high-content microscopy. The genotype of trans variants contributed little to protein level variation between individual cells but explained >50% of the variance in the population's average protein abundance for half of the GFP fusions tested. To map trans-acting factors responsible, we performed flow sorting and bulk segregant analysis of 25 proteins, finding a median of five protein quantitative trait loci (pQTLs) per GFP fusion. Further, we find that cis-acting variants predominate; the genotype of a gene and its surrounding region had a large effect on protein level six times more frequently than the rest of the genome combined. We present evidence for both shared and independent genetic control of transcript and protein abundance: More than half of the expression QTLs (eQTLs) contribute to changes in protein levels of regulated genes, but several pQTLs do not affect their cognate transcript levels. Allele replacements of genes known to underlie trans eQTL hotspots confirmed the correlation of effects on mRNA and protein levels. This study represents the first genome-scale measurement of genetic contribution to protein levels in single cells and populations, identifies more than a hundred trans pQTLs, and validates the propagation of effects associated with transcript variation to protein abundance.

  View details for DOI 10.1101/gr.170506.113

  View details for Web of Science ID 000339860200011

  View details for PubMedID 24823668

  View details for PubMedCentralID PMC4120089

• Genome-wide identification of transcript start and end sites by transcript isoform sequencing NATURE PROTOCOLS Pelechano, V., Wei, W., Jakob, P., Steinmetz, L. M. 2014; 9 (7): 1740-1759

  Abstract

  Hundreds of transcript isoforms with varying boundaries and alternative regulatory signals are transcribed from the genome, even in a genetically homogeneous population of cells. To study this transcriptional heterogeneity, we developed transcript isoform sequencing (TIF-seq), a method that allows the genome-wide profiling of full-length transcript isoforms defined by their exact 5' and 3' boundaries. TIF-seq entails the generation of full-length cDNA libraries, followed by their circularization and the sequencing of the junction fragments spanning the 5' and 3' transcript ends. By determining the respective co-occurrence of start and end sites of individual transcript molecules, TIF-seq can distinguish variations that conventional approaches for mapping single ends cannot, such as short abortive transcripts, bicistronic messages and overlapping transcripts that differ in lengths. The TIF-seq protocol we describe here can be applied to any eukaryotic organism (e.g., yeast, human), and it requires 6-10 d for generating TIF-seq libraries, 10 d for sequencing and 2-3 d for analysis.

  View details for DOI 10.1038/nprot.2014.121

  View details for Web of Science ID 000338777400015

  View details for PubMedCentralID PMC4111111

• Yeast Growth Plasticity Is Regulated by Environment-Specific Multi-QTL Interactions G3-GENES GENOMES GENETICS Bhatia, A., Yadav, A., Zhu, C., Gagneur, J., Radhakrishnan, A., Steinmetz, L. M., Bhanot, G., Sinha, H. 2014; 4 (5): 769-777

  Abstract

  For a unicellular, nonmotile organism like Saccharomyces cerevisiae, carbon sources act as nutrients and as signaling molecules; consequently, these sources affect various fitness parameters, including growth. It is therefore advantageous for yeast strains to adapt their growth to carbon source variation. The ability of a given genotype to manifest different phenotypes in varying environments is known as phenotypic plasticity. To identify quantitative trait loci (QTL) that drive plasticity in growth, two growth parameters (growth rate and biomass) were measured for a set of meiotic recombinants of two genetically divergent yeast strains grown in different carbon sources. To identify QTL contributing to plasticity across pairs of environments, gene-environment interaction mapping was performed, which identified several QTL that have a differential effect across environments, some of which act antagonistically across pairs of environments. Multi-QTL analysis identified loci interacting with previously known growth affecting QTL as well as novel two-QTL interactions that affect growth. A QTL that had no significant independent effect was found to alter growth rate and biomass for several carbon sources through two-QTL interactions. Our study demonstrates that environment-specific epistatic interactions contribute to the growth plasticity in yeast. We propose that a targeted scan for epistatic interactions, such as the one described here, can help unravel mechanisms regulating phenotypic plasticity.

  View details for DOI 10.1534/g3.113.009142

  View details for Web of Science ID 000336483900001

  View details for PubMedID 24474169

  View details for PubMedCentralID PMC4025475

• Role of histone modifications and early termination in pervasive transcription and antisense-mediated gene silencing in yeast. Nucleic acids research Castelnuovo, M., Zaugg, J. B., Guffanti, E., Maffioletti, A., Camblong, J., Xu, Z., Clauder-Münster, S., Steinmetz, L. M., Luscombe, N. M., Stutz, F. 2014; 42 (7): 4348-4362

  Abstract

  Most genomes, including yeast Saccharomyces cerevisiae, are pervasively transcribed producing numerous non-coding RNAs, many of which are unstable and eliminated by nuclear or cytoplasmic surveillance pathways. We previously showed that accumulation of PHO84 antisense RNA (asRNA), in cells lacking the nuclear exosome component Rrp6, is paralleled by repression of sense transcription in a process dependent on the Hda1 histone deacetylase (HDAC) and the H3K4 histone methyl transferase Set1. Here we investigate this process genome-wide and measure the whole transcriptome of various histone modification mutants in a Δrrp6 strain using tiling arrays. We confirm widespread occurrence of potentially antisense-dependent gene regulation and identify three functionally distinct classes of genes that accumulate asRNAs in the absence of Rrp6. These classes differ in whether the genes are silenced by the asRNA and whether the silencing is HDACs and histone methyl transferase-dependent. Among the distinguishing features of asRNAs with regulatory potential, we identify weak early termination by Nrd1/Nab3/Sen1, extension of the asRNA into the open reading frame promoter and dependence of the silencing capacity on Set1 and the HDACs Hda1 and Rpd3 particularly at promoters undergoing extensive chromatin remodelling. Finally, depending on the efficiency of Nrd1/Nab3/Sen1 early termination, asRNA levels are modulated and their capability of silencing is changed.

  View details for DOI 10.1093/nar/gku100

  View details for PubMedID 24497191

  View details for PubMedCentralID PMC3985671

• An Evaluation of High-Throughput Approaches to QTL Mapping in Saccharomyces cerevisiae. Genetics Wilkening, S., Lin, G., Fritsch, E. S., Tekkedil, M. M., Anders, S., Kuehn, R., Nguyen, M., Aiyar, R. S., Proctor, M., Sakhanenko, N. A., Galas, D. J., Gagneur, J., Deutschbauer, A., Steinmetz, L. M. 2014; 196 (3): 853-865

  Abstract

  Dissecting the molecular basis of quantitative traits is a significant challenge and is essential for understanding complex diseases. Even in model organisms, precisely determining causative genes and their interactions has remained elusive, due in part to difficulty in narrowing intervals to single genes and in detecting epistasis or linked quantitative trait loci. These difficulties are exacerbated by limitations in experimental design, such as low numbers of analyzed individuals or of polymorphisms between parental genomes. We address these challenges by applying three independent high-throughput approaches for QTL mapping to map the genetic variants underlying 11 phenotypes in two genetically distant Saccharomyces cerevisiae strains, namely (1) individual analysis of >700 meiotic segregants, (2) bulk segregant analysis, and (3) reciprocal hemizygosity scanning, a new genome-wide method that we developed. We reveal differences in the performance of each approach and, by combining them, identify eight polymorphic genes that affect eight different phenotypes: colony shape, flocculation, growth on two nonfermentable carbon sources, and resistance to two drugs, salt, and high temperature. Our results demonstrate the power of individual segregant analysis to dissect QTL and address the underestimated contribution of interactions between variants. We also reveal confounding factors like mutations and aneuploidy in pooled approaches, providing valuable lessons for future designs of complex trait mapping studies.

  View details for DOI 10.1534/genetics.113.160291

  View details for PubMedID 24374355

  View details for PubMedCentralID PMC3948811

• Control of Cdc28 CDK1 by a Stress-Induced lncRNA MOLECULAR CELL Nadal-Ribelles, M., Sole, C., Xu, Z., Steinmetz, L. M., de Nadal, E., Posas, F. 2014; 53 (4): 549-561

  View details for DOI 10.1016/j.molcel.2014.01.006

  View details for Web of Science ID 000331660200005

• Alternative polyadenylation diversifies post-transcriptional regulation by selective RNA-protein interactions MOLECULAR SYSTEMS BIOLOGY Gupta, I., Clauder-Muenster, S., Klaus, B., Jaervelin, A. I., Aiyar, R. S., Benes, V., Wilkening, S., Huber, W., Pelechano, V., Steinmetz, L. M. 2014; 10 (2)

  Abstract

  Recent research has uncovered extensive variability in the boundaries of transcript isoforms, yet the functional consequences of this variation remain largely unexplored. Here, we systematically discriminate between the molecular phenotypes of overlapping coding and non-coding transcriptional events from each genic locus using a novel genome-wide, nucleotide-resolution technique to quantify the half-lives of 3' transcript isoforms in yeast. Our results reveal widespread differences in stability among isoforms for hundreds of genes in a single condition, and that variation of even a single nucleotide in the 3' untranslated region (UTR) can affect transcript stability. While previous instances of negative associations between 3' UTR length and transcript stability have been reported, here, we find that shorter isoforms are not necessarily more stable. We demonstrate the role of RNA-protein interactions in conditioning isoform-specific stability, showing that PUF3 binds and destabilizes specific polyadenylation isoforms. Our findings indicate that although the functional elements of a gene are encoded in DNA sequence, the selective incorporation of these elements into RNA through transcript boundary variation allows a single gene to have diverse functional consequences.

  View details for DOI 10.1002/msb.135068

  View details for Web of Science ID 000332203200002

• High-density tiling microarray analysis of the full transcriptional activity of yeast. Methods in molecular biology (Clifton, N.J.) David, L., Clauder-Münster, S., Steinmetz, L. M. 2014; 1205: 257-273

  Abstract

  Understanding the relationship between DNA sequence variation and phenotypic variation in complex or quantitative traits is one of the major challenges in modern biology. We are witnessing a deluge of DNA sequence information and association studies of genetic polymorphisms with phenotypes of interest in families and populations. In addition, it has become clear that large portions of eukaryotic genomes beyond protein-coding genes are transcribed, generating numerous noncoding RNA (ncRNA) molecules whose functions remain mostly unknown.DNA oligonucleotide microarrays constitute a powerful technology for studying the expression of genes in different organisms. The Saccharomyces cerevisiae tiling array presents a significant advance over previous array-based platforms. It has a high density of overlapping probes that start on average every 8 bp along each strand of the genome, enabling precise definition of transcript structure. Furthermore, the array includes probes specific for the polymorphic positions of another, distantly related yeast strain, allowing accurate measurement of allele-specific expression in a hybrid of the two strains. This technology thus allows high-resolution, quantitative, strand- and allele-specific measurements of transcription from a full eukaryotic genome. In this chapter, we describe the methods for extracting RNA, synthesizing first-strand cDNA, fragmenting, and labeling of samples for hybridization to the tiling array. Combining genome-wide information on variation in DNA sequence with variation in transcript structure and levels promises to increase our understanding of the genotype-to-phenotype relationship.

  View details for DOI 10.1007/978-1-4939-1363-3_16

  View details for PubMedID 25213250

• The Role of Ctk1 Kinase in Termination of Small Non-Coding RNAs PLOS ONE Lenstra, T. L., Tudek, A., Clauder, S., Xu, Z., Pachis, S. T., van Leenen, D., Kemmeren, P., Steinmetz, L. M., Libri, D., Holstege, F. C. 2013; 8 (12)

  Abstract

  Transcription termination in Saccharomyces cerevisiae can be performed by at least two distinct pathways and is influenced by the phosphorylation status of the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II). Late termination of mRNAs is performed by the CPF/CF complex, the recruitment of which is dependent on CTD-Ser2 phosphorylation (Ser2P). Early termination of shorter cryptic unstable transcripts (CUTs) and small nucleolar/nuclear RNAs (sno/snRNAs) is performed by the Nrd1-Nab3-Sen1 (NNS) complex that binds phosphorylated CTD-Ser5 (Ser5P) via the CTD-interacting domain (CID) of Nrd1p. In this study, mutants of the different termination pathways were compared by genome-wide expression analysis. Surprisingly, the expression changes observed upon loss of the CTD-Ser2 kinase Ctk1p are more similar to those derived from alterations in the Ser5P-dependent NNS pathway, than from loss of CTD-Ser2P binding factors. Tiling array analysis of ctk1Δ cells reveals readthrough at snoRNAs, at many cryptic unstable transcripts (CUTs) and stable uncharacterized transcripts (SUTs), but only at some mRNAs. Despite the suggested predominant role in termination of mRNAs, we observed that a CTK1 deletion or a Pol II CTD mutant lacking all Ser2 positions does not result in a global mRNA termination defect. Rather, termination defects in these strains are widely observed at NNS-dependent genes. These results indicate that Ctk1p and Ser2 CTD phosphorylation have a wide impact in termination of small non-coding RNAs but only affect a subset of mRNA coding genes.

  View details for DOI 10.1371/journal.pone.0080495

  View details for Web of Science ID 000327949300022

  View details for PubMedID 24324601

• Gene regulation by antisense transcription. Nature reviews. Genetics Pelechano, V., Steinmetz, L. M. 2013; 14 (12): 880-893

  Abstract

  Antisense transcription, which was initially considered by many as transcriptional noise, is increasingly being recognized as an important regulator of gene expression. It is widespread among all kingdoms of life and has been shown to influence - either through the act of transcription or through the non-coding RNA that is produced - almost all stages of gene expression, from transcription and translation to RNA degradation. Antisense transcription can function as a fast evolving regulatory switch and a modular scaffold for protein complexes, and it can 'rewire' regulatory networks. The genomic arrangement of antisense RNAs opposite sense genes indicates that they might be part of self-regulatory circuits that allow genes to regulate their own expression.

  View details for DOI 10.1038/nrg3594

  View details for PubMedID 24217315

• Extensive Variation in Chromatin States Across Humans SCIENCE Kasowski, M., Kyriazopoulou-Panagiotopoulou, S., Grubert, F., Zaugg, J. B., Kundaje, A., Liu, Y., Boyle, A. P., Zhang, Q. C., Zakharia, F., Spacek, D. V., Li, J., Xie, D., Olarerin-George, A., Steinmetz, L. M., Hogenesch, J. B., Kellis, M., Batzoglou, S., Snyder, M. 2013; 342 (6159): 750-752

  Abstract

  The majority of disease-associated variants lie outside protein-coding regions, suggesting a link between variation in regulatory regions and disease predisposition. We studied differences in chromatin states using five histone modifications, cohesin, and CTCF in lymphoblastoid lines from 19 individuals of diverse ancestry. We found extensive signal variation in regulatory regions, which often switch between active and repressed states across individuals. Enhancer activity is particularly diverse among individuals, whereas gene expression remains relatively stable. Chromatin variability shows genetic inheritance in trios, correlates with genetic variation and population divergence, and is associated with disruptions of transcription factor binding motifs. Overall, our results provide insights into chromatin variation among humans.

  View details for DOI 10.1126/science.1242510

  View details for PubMedID 24136358

• Drift and conservation of differential exon usage across tissues in primate species. Proceedings of the National Academy of Sciences of the United States of America Reyes, A., Anders, S., Weatheritt, R. J., Gibson, T. J., Steinmetz, L. M., Huber, W. 2013; 110 (38): 15377-15382

  Abstract

  Alternative usage of exons provides genomes with plasticity to produce different transcripts from the same gene, modulating the function, localization, and life cycle of gene products. It affects most human genes. For a limited number of cases, alternative functions and tissue-specific roles are known. However, recent high-throughput sequencing studies have suggested that much alternative isoform usage across tissues is nonconserved, raising the question of the extent of its functional importance. We address this question in a genome-wide manner by analyzing the transcriptomes of five tissues for six primate species, focusing on exons that are 1:1 orthologous in all six species. Our results support a model in which differential usage of exons has two major modes: First, most of the exons show only weak differences, which are dominated by interspecies variability and may reflect neutral drift and noisy splicing. These cases dominate the genome-wide view and explain why conservation appears to be so limited. Second, however, a sizeable minority of exons show strong differences between tissues, which are mostly conserved. We identified a core set of 3,800 exons from 1,643 genes that show conservation of strongly tissue-dependent usage patterns from human at least to macaque. This set is enriched for exons encoding protein-disordered regions and untranslated regions. Our findings support the theory that isoform regulation is an important target of evolution in primates, and our method provides a powerful tool for discovering potentially functional tissue-dependent isoforms.

  View details for DOI 10.1073/pnas.1307202110

  View details for PubMedID 24003148

  View details for PubMedCentralID PMC3780897

• Polyadenylation site-induced decay of upstream transcripts enforces promoter directionality NATURE STRUCTURAL & MOLECULAR BIOLOGY Ntini, E., Jaervelin, A. I., Bornholdt, J., Chen, Y., Boyd, M., Jorgensen, M., Andersson, R., Hoof, I., Schein, A., Andersen, P. R., Andersen, P. K., Preker, P., Valen, E., Zhao, X., Pelechano, V., Steinmetz, L. M., Sandelin, A., Jensen, T. H. 2013; 20 (8): 923-?

  Abstract

  Active human promoters produce promoter-upstream transcripts (PROMPTs). Why these RNAs are coupled to decay, whereas their neighboring promoter-downstream mRNAs are not, is unknown. Here high-throughput sequencing demonstrates that PROMPTs generally initiate in the antisense direction closely upstream of the transcription start sites (TSSs) of their associated genes. PROMPT TSSs share features with mRNA-producing TSSs, including stalled RNA polymerase II (RNAPII) and the production of small TSS-associated RNAs. Notably, motif analyses around PROMPT 3' ends reveal polyadenylation (pA)-like signals. Mutagenesis studies demonstrate that PROMPT pA signals are functional but linked to RNA degradation. Moreover, pA signals are under-represented in promoter-downstream versus promoter-upstream regions, thus allowing for more efficient RNAPII progress in the sense direction from gene promoters. We conclude that asymmetric sequence distribution around human gene promoters serves to provide a directional RNA output from an otherwise bidirectional transcription process.

  View details for DOI 10.1038/nsmb.2640

  View details for Web of Science ID 000322715300004

  View details for PubMedID 23851456

• Multiple Genomic Changes Associated with Reorganization of Gene Regulation and Adaptation in Yeast MOLECULAR BIOLOGY AND EVOLUTION David, L., Ben-Harosh, Y., Stolovicki, E., Moore, L. S., Michelle Nguyen, M., Tamse, R., Dean, J., Mancera, E., Steinmetz, L. M., Braun, E. 2013; 30 (7): 1514-1526

  Abstract

  Frequently during evolution, new phenotypes evolved due to novelty in gene regulation, such as that caused by genome rewiring. This has been demonstrated by comparing common regulatory sequences among species and by identifying single regulatory mutations that are associated with new phenotypes. However, while a single mutation changes a single element, gene regulation is accomplished by a regulatory network involving multiple interactive elements. Therefore, to better understand regulatory evolution, we have studied how mutations contributed to the adaptation of cells to a regulatory challenge. We created a synthetic genome rewiring in yeast cells, challenged their gene regulation, and studied their adaptation. HIS3, an essential enzyme for histidine biosynthesis, was placed exclusively under a GAL promoter, which is induced by galactose and strongly repressed in glucose. Such rewired cells were faced with significant regulatory challenges in a repressive glucose medium. We identified several independent mutations in elements of the GAL system associated with the rapid adaptation of cells, such as the repressor GAL80 and the binding sites of the activator GAL4. Consistent with the extraordinarily high rate of cell adaptation, new regulation emerged during adaptation via multiple trajectories, including those involving mutations in elements of the GAL system. The new regulation of HIS3 tuned its expression according to histidine requirements with or without these significant mutations, indicating that additional factors participated in this regulation and that the regulatory network could reorganize in multiple ways to accommodate different mutations. This study, therefore, stresses network plasticity as an important property for regulatory adaptation and evolution.

  View details for DOI 10.1093/molbev/mst071

  View details for Web of Science ID 000321056200004

  View details for PubMedID 23589456

• System-wide identification of RNA-binding proteins by interactome capture NATURE PROTOCOLS Castello, A., Horos, R., Strein, C., Fischer, B., Eichelbaum, K., Steinmetz, L. M., Krijgsveld, J., Hentze, M. W. 2013; 8 (3): 491-500

  Abstract

  Owing to their preeminent biological functions, the repertoire of expressed RNA-binding proteins (RBPs) and their activity states are highly informative about cellular systems. We have developed a novel and unbiased technique, called interactome capture, for identifying the active RBPs of cultured cells. By making use of in vivo UV cross-linking of RBPs to polyadenylated RNAs, covalently bound proteins are captured with oligo(dT) magnetic beads. After stringent washes, the mRNA interactome is determined by quantitative mass spectrometry (MS). The protocol takes 3 working days for analysis of single proteins by western blotting, and about 2 weeks for the determination of complete cellular mRNA interactomes by MS. The most important advantage of interactome capture over other in vitro and in silico approaches is that only RBPs bound to RNA in a physiological environment are identified. When applied to HeLa cells, interactome capture revealed hundreds of novel RBPs. Interactome capture can also be broadly used to compare different biological states, including metabolic stress, cell cycle, differentiation, development or the response to drugs.

  View details for DOI 10.1038/nprot.2013.020

  View details for Web of Science ID 000317110600005

  View details for PubMedID 23411631

• An efficient method for genome-wide polyadenylation site mapping and RNA quantification. Nucleic acids research Wilkening, S., Pelechano, V., Järvelin, A. I., Tekkedil, M. M., Anders, S., Benes, V., Steinmetz, L. M. 2013; 41 (5)

  Abstract

  The use of alternative poly(A) sites is common and affects the post-transcriptional fate of mRNA, including its stability, subcellular localization and translation. Here, we present a method to identify poly(A) sites in a genome-wide and strand-specific manner. This method, termed 3'T-fill, initially fills in the poly(A) stretch with unlabeled dTTPs, allowing sequencing to start directly after the poly(A) tail into the 3'-untranslated regions (UTR). Our comparative analysis demonstrates that it outperforms existing protocols in quality and throughput and accurately quantifies RNA levels as only one read is produced from each transcript. We use this method to characterize the diversity of polyadenylation in Saccharomyces cerevisiae, showing that alternative RNA molecules are present even in a genetically identical cell population. Finally, we observe that overlap of convergent 3'-UTRs is frequent but sharply limited by coding regions, suggesting factors that restrict compression of the yeast genome.

  View details for DOI 10.1093/nar/gks1249

  View details for PubMedID 23295673

• Genotyping 1000 yeast strains by next-generation sequencing BMC GENOMICS Wilkening, S., Tekkedil, M. M., Lin, G., Fritsch, E. S., Wei, W., Gagneur, J., Lazinski, D. W., Camilli, A., Steinmetz, L. M. 2013; 14

  Abstract

  The throughput of next-generation sequencing machines has increased dramatically over the last few years; yet the cost and time for library preparation have not changed proportionally, thus representing the main bottleneck for sequencing large numbers of samples. Here we present an economical, high-throughput library preparation method for the Illumina platform, comprising a 96-well based method for DNA isolation for yeast cells, a low-cost DNA shearing alternative, and adapter ligation using heat inactivation of enzymes instead of bead cleanups.Up to 384 whole-genome libraries can be prepared from yeast cells in one week using this method, for less than 15 euros per sample. We demonstrate the robustness of this protocol by sequencing over 1000 yeast genomes at ~30x coverage. The sequence information from 768 yeast segregants derived from two divergent S. cerevisiae strains was used to generate a meiotic recombination map at unprecedented resolution. Comparisons to other datasets indicate a high conservation of recombination at a chromosome-wide scale, but differences at the local scale. Additionally, we detected a high degree of aneuploidy (3.6%) by examining the sequencing coverage in these segregants. Differences in allele frequency allowed us to attribute instances of aneuploidy to gains of chromosomes during meiosis or mitosis, both of which showed a strong tendency to missegregate specific chromosomes.Here we present a high throughput workflow to sequence genomes of large number of yeast strains at a low price. We have used this workflow to obtain recombination and aneuploidy data from hundreds of segregants, which can serve as a foundation for future studies of linkage, recombination, and chromosomal aberrations in yeast and higher eukaryotes.

  View details for DOI 10.1186/1471-2164-14-90

  View details for Web of Science ID 000315034700001

  View details for PubMedID 23394869

• Silencing of genes and alleles by RNAi in Anopheles gambiae. Methods in molecular biology (Clifton, N.J.) Lamacchia, M., Clayton, J. R., Wang-Sattler, R., Steinmetz, L. M., Levashina, E. A., Blandin, S. A. 2013; 923: 161-176

  Abstract

  Anopheles gambiae mosquitoes are the major vectors of human malaria parasites. However, mosquitoes are not passive hosts for parasites, actively limiting their development in vivo. Our current understanding of the mosquito antiparasitic response is mostly based on the phenotypic analysis of gene knockdowns obtained by RNA interference (RNAi), through the injection or transfection of long dsRNAs in adult mosquitoes or cultured cells, respectively. Recently, RNAi has been extended to silence specifically one allele of a given gene in a heterozygous context, thus allowing to compare the contribution of different alleles to a phenotype in the same genetic background.

  View details for PubMedID 22990777

• Natural sequence variants of yeast environmental sensors confer cell-to-cell expression variability. Molecular systems biology Fehrmann, S., Bottin-Duplus, H., Leonidou, A., Mollereau, E., Barthelaix, A., Wei, W., Steinmetz, L. M., Yvert, G. 2013; 9: 695-?

  Abstract

  Living systems may have evolved probabilistic bet hedging strategies that generate cell-to-cell phenotypic diversity in anticipation of environmental catastrophes, as opposed to adaptation via a deterministic response to environmental changes. Evolution of bet hedging assumes that genotypes segregating in natural populations modulate the level of intraclonal diversity, which so far has largely remained hypothetical. Using a fluorescent Pmet17-GFP reporter, we mapped four genetic loci conferring to a wild yeast strain an elevated cell-to-cell variability in the expression of MET17, a gene regulated by the methionine pathway. A frameshift mutation in the Erc1p transmembrane transporter, probably resulting from a release of laboratory strains from negative selection, reduced Pmet17-GFP expression variability. At a second locus, cis-regulatory polymorphisms increased mean expression of the Mup1p methionine permease, causing increased expression variability in trans. These results demonstrate that an expression quantitative trait locus (eQTL) can simultaneously have a deterministic effect in cis and a probabilistic effect in trans. Our observations indicate that the evolution of transmembrane transporter genes can tune intraclonal variation and may therefore be implicated in both reactive and anticipatory strategies of adaptation.

  View details for DOI 10.1038/msb.2013.53

  View details for PubMedID 24104478

• RNA Polymerase II Collision Interrupts Convergent Transcription MOLECULAR CELL Hobson, D. J., Wei, W., Steinmetz, L. M., Svejstrup, J. Q. 2012; 48 (3): 365-374

  Abstract

  Antisense noncoding transcripts, genes-within-genes, and convergent gene pairs are prevalent among eukaryotes. The existence of such transcription units raises the question of what happens when RNA polymerase II (RNAPII) molecules collide head-to-head. Here we use a combination of biochemical and genetic approaches in yeast to show that polymerases transcribing opposite DNA strands cannot bypass each other. RNAPII stops but does not dissociate upon head-to-head collision in vitro, suggesting that opposing polymerases represent insurmountable obstacles for each other. Head-to-head collision in vivo also results in RNAPII stopping, and removal of collided RNAPII from the DNA template can be achieved via ubiquitylation-directed proteolysis. Indeed, in cells lacking efficient RNAPII polyubiquitylation, the half-life of collided polymerases increases, so that they can be detected between convergent genes. These results provide insight into fundamental mechanisms of gene traffic control and point to an unexplored effect of antisense transcription on gene regulation via polymerase collision.

  View details for DOI 10.1016/j.molce1.2012.08.027

  View details for Web of Science ID 000311260900006

  View details for PubMedID 23041286

  View details for PubMedCentralID PMC3504299

• Extensive Degradation of RNA Precursors by the Exosome in Wild-Type Cells MOLECULAR CELL Gudipati, R. K., Xu, Z., Lebreton, A., Seraphin, B., Steinmetz, L. M., Jacquier, A., Libri, D. 2012; 48 (3): 409-421

  Abstract

  The exosome is a complex involved in the maturation of rRNA and sn-snoRNA, in the degradation of short-lived noncoding RNAs, and in the quality control of RNAs produced in mutants. It contains two catalytic subunits, Rrp6p and Dis3p, whose specific functions are not fully understood. We analyzed the transcriptome of combinations of Rrp6p and Dis3p catalytic mutants by high-resolution tiling arrays. We show that Dis3p and Rrp6p have both overlapping and specific roles in degrading distinct classes of substrates. We found that transcripts derived from more than half of intron-containing genes are degraded before splicing. Surprisingly, we also show that the exosome degrades large amounts of tRNA precursors despite the absence of processing defects. These results underscore the notion that large amounts of RNAs produced in wild-type cells are discarded before entering functional pathways and suggest that kinetic competition with degradation proofreads the efficiency and accuracy of processing.

  View details for DOI 10.1016/j.molce1.2012.08.018

  View details for Web of Science ID 000311260900010

  View details for PubMedID 23000176

• Gene Loops Enhance Transcriptional Directionality SCIENCE Tan-Wong, S. M., Zaugg, J. B., Camblong, J., Xu, Z., Zhang, D. W., Mischo, H. E., Ansari, A. Z., Luscombe, N. M., Steinmetz, L. M., Proudfoot, N. J. 2012; 338 (6107): 671-675

  Abstract

  Eukaryotic genomes are extensively transcribed, forming both messenger RNAs (mRNAs) and noncoding RNAs (ncRNAs). ncRNAs made by RNA polymerase II often initiate from bidirectional promoters (nucleosome-depleted chromatin) that synthesize mRNA and ncRNA in opposite directions. We demonstrate that, by adopting a gene-loop conformation, actively transcribed mRNA encoding genes restrict divergent transcription of ncRNAs. Because gene-loop formation depends on a protein factor (Ssu72) that coassociates with both the promoter and the terminator, the inactivation of Ssu72 leads to increased synthesis of promoter-associated divergent ncRNAs, referred to as Ssu72-restricted transcripts (SRTs). Similarly, inactivation of individual gene loops by gene mutation enhances SRT synthesis. We demonstrate that gene-loop conformation enforces transcriptional directionality on otherwise bidirectional promoters.

  View details for DOI 10.1126/science.1224350

  View details for Web of Science ID 000310516000054

  View details for PubMedID 23019609

  View details for PubMedCentralID PMC3563069

• easyRNASeq: a bioconductor package for processing RNA-Seq data BIOINFORMATICS Delhomme, N., Padioleau, I., Furlong, E. E., Steinmetz, L. M. 2012; 28 (19): 2532-2533

  Abstract

  RNA sequencing is becoming a standard for expression profiling experiments and many tools have been developed in the past few years to analyze RNA-Seq data. Numerous 'Bioconductor' packages are available for next-generation sequencing data loading in R, e.g. ShortRead and Rsamtools as well as to perform differential gene expression analyses, e.g. DESeq and edgeR. However, the processing tasks lying in between these require the precise interplay of many Bioconductor packages, e.g. Biostrings, IRanges or external solutions are to be sought.We developed 'easyRNASeq', an R package that simplifies the processing of RNA sequencing data, hiding the complex interplay of the required packages behind a single functionality.The package is implemented in R (as of version 2.15) and is available from Bioconductor (as of version 2.10) at the URL: http://bioconductor.org/packages/release/bioc/html/easyRNASeq.html, where installation and usage instructions can be found.delhomme@embl.de.

  View details for DOI 10.1093/bioinformatics/bts477

  View details for Web of Science ID 000309687500022

  View details for PubMedID 22847932

• Set3 HDAC Mediates Effects of Overlapping Noncoding Transcription on Gene Induction Kinetics CELL Kim, T., Sandra, Z. X., Clauder-Muenster, S., Steinmetz, L. M., Buratowski, S. 2012; 150 (6): 1158-1169

  Abstract

  The Set3 histone deacetylase complex (Set3C) binds histone H3 dimethylated at lysine 4 (H3K4me2) to mediate deacetylation of histones in 5'-transcribed regions. To discern how Set3C affects gene expression, genome-wide transcription was analyzed in yeast undergoing a series of carbon source shifts. Deleting SET3 primarily caused changes during transition periods, as genes were induced or repressed. Surprisingly, a majority of Set3-affected genes are overlapped by noncoding RNA (ncRNA) transcription. Many Set3-repressed genes have H3K4me2 instead of me3 over promoter regions, due to either reduced H3K4me3 or ncRNA transcription from distal or antisense promoters. Set3C also represses internal cryptic promoters, but in different regions of genes than the Set2/Rpd3S pathway. Finally, Set3C stimulates some genes by repressing an overlapping antagonistic antisense transcript. These results show that overlapping noncoding transcription can fine-tune gene expression, not via the ncRNA but by depositing H3K4me2 to recruit the Set3C deacetylase.

  View details for DOI 10.1016/j.cell.2012.08.016

  View details for Web of Science ID 000308928100010

  View details for PubMedID 22959268

• Genetic Modifiers of Chromatin Acetylation Antagonize the Reprogramming of Epi-Polymorphisms PLOS GENETICS Abraham, A., Nagarajan, M., Veyrieras, J., Bottin, H., Steinmetz, L. M., Yvert, G. 2012; 8 (9)

  Abstract

  Natural populations are known to differ not only in DNA but also in their chromatin-associated epigenetic marks. When such inter-individual epigenomic differences (or "epi-polymorphisms") are observed, their stability is usually not known: they may or may not be reprogrammed over time or upon environmental changes. In addition, their origin may be purely epigenetic, or they may result from regulatory variation encoded in the DNA. Studying epi-polymorphisms requires, therefore, an assessment of their nature and stability. Here we estimate the stability of yeast epi-polymorphisms of chromatin acetylation, and we provide a genome-by-epigenome map of their genetic control. A transient epi-drug treatment was able to reprogram acetylation variation at more than one thousand nucleosomes, whereas a similar amount of variation persisted, distinguishing "labile" from "persistent" epi-polymorphisms. Hundreds of genetic loci underlied acetylation variation at 2,418 nucleosomes either locally (in cis) or distantly (in trans), and this genetic control overlapped only partially with the genetic control of gene expression. Trans-acting regulators were not necessarily associated with genes coding for chromatin modifying enzymes. Strikingly, "labile" and "persistent" epi-polymorphisms were associated with poor and strong genetic control, respectively, showing that genetic modifiers contribute to persistence. These results estimate the amount of natural epigenomic variation that can be lost after transient environmental exposures, and they reveal the complex genetic architecture of the DNA-encoded determinism of chromatin epi-polymorphisms. Our observations provide a basis for the development of population epigenetics.

  View details for DOI 10.1371/journal.pgen.1002958

  View details for Web of Science ID 000309817900033

  View details for PubMedID 23028365

• Experimental Relocation of the Mitochondrial ATP9 Gene to the Nucleus Reveals Forces Underlying Mitochondrial Genome Evolution PLOS GENETICS Bietenhader, M., Martos, A., Tetaud, E., Aiyar, R. S., Sellem, C. H., Kucharczyk, R., Clauder-Muenster, S., Giraud, M., Godard, F., Salin, B., Sagot, I., Gagneur, J., Dequard-Chablat, M., Contamine, V., Hermann-Le Denmat, S., Sainsard-Chanet, A., Steinmetz, L. M., di Rago, J. 2012; 8 (8)

  View details for DOI 10.1371/journal.pgen.1002876

  View details for Web of Science ID 000308529300034

• Rrp6p Controls mRNA Poly(A) Tail Length and Its Decoration with Poly(A) Binding Proteins MOLECULAR CELL Schmid, M., Poulsen, M. B., Olszewski, P., Pelechano, V., Saguez, C., Gupta, I., Steinmetz, L. M., Moore, C., Jensen, T. H. 2012; 47 (2): 267-280

  Abstract

  Poly(A) (pA) tail binding proteins (PABPs) control mRNA polyadenylation, stability, and translation. In a purified system, S. cerevisiae PABPs, Pab1p and Nab2p, are individually sufficient to provide normal pA tail length. However, it is unknown how this occurs in more complex environments. Here we find that the nuclear exosome subunit Rrp6p counteracts the in vitro and in vivo extension of mature pA tails by the noncanonical pA polymerase Trf4p. Moreover, PABP loading onto nascent pA tails is controlled by Rrp6p; while Pab1p is the major PABP, Nab2p only associates in the absence of Rrp6p. This is because Rrp6p can interact with Nab2p and displace it from pA tails, potentially leading to RNA turnover, as evidenced for certain pre-mRNAs. We suggest that a nuclear mRNP surveillance step involves targeting of Rrp6p by Nab2p-bound pA-tailed RNPs and that pre-mRNA abundance is regulated at this level.

  View details for DOI 10.1016/j.molcel.2012.05.005

  View details for Web of Science ID 000307084000013

  View details for PubMedID 22683267

• Insights into RNA Biology from an Atlas of Mammalian mRNA-Binding Proteins CELL Castello, A., Fischer, B., Eichelbaum, K., Horos, R., Beckmann, B. M., Strein, C., Davey, N. E., Humphreys, D. T., Preiss, T., Steinmetz, L. M., Krijgsveld, J., Hentze, M. W. 2012; 149 (6): 1393-1406

  Abstract

  RNA-binding proteins (RBPs) determine RNA fate from synthesis to decay. Employing two complementary protocols for covalent UV crosslinking of RBPs to RNA, we describe a systematic, unbiased, and comprehensive approach, termed "interactome capture," to define the mRNA interactome of proliferating human HeLa cells. We identify 860 proteins that qualify as RBPs by biochemical and statistical criteria, adding more than 300 RBPs to those previously known and shedding light on RBPs in disease, RNA-binding enzymes of intermediary metabolism, RNA-binding kinases, and RNA-binding architectures. Unexpectedly, we find that many proteins of the HeLa mRNA interactome are highly intrinsically disordered and enriched in short repetitive amino acid motifs. Interactome capture is broadly applicable to study mRNA interactome composition and dynamics in varied biological settings.

  View details for DOI 10.1016/j.cell.2012.04.031

  View details for Web of Science ID 000305119600021

  View details for PubMedID 22658674

• Experimental relocation of the mitochondrial ATP9 gene to the nucleus reveals forces underlying mitochondrial genome evolution. PLoS genetics Bietenhader, M., Martos, A., Tetaud, E., Aiyar, R. S., Sellem, C. H., Kucharczyk, R., Clauder-Münster, S., Giraud, M., Godard, F., Salin, B., Sagot, I., Gagneur, J., Déquard-Chablat, M., Contamine, V., Hermann-Le Denmat, S., Sainsard-Chanet, A., Steinmetz, L. M., di Rago, J. 2012; 8 (8)

  Abstract

  Only a few genes remain in the mitochondrial genome retained by every eukaryotic organism that carry out essential functions and are implicated in severe diseases. Experimentally relocating these few genes to the nucleus therefore has both therapeutic and evolutionary implications. Numerous unproductive attempts have been made to do so, with a total of only 5 successes across all organisms. We have taken a novel approach to relocating mitochondrial genes that utilizes naturally nuclear versions from other organisms. We demonstrate this approach on subunit 9/c of ATP synthase, successfully relocating this gene for the first time in any organism by expressing the ATP9 genes from Podospora anserina in Saccharomyces cerevisiae. This study substantiates the role of protein structure in mitochondrial gene transfer: expression of chimeric constructs reveals that the P. anserina proteins can be correctly imported into mitochondria due to reduced hydrophobicity of the first transmembrane segment. Nuclear expression of ATP9, while permitting almost fully functional oxidative phosphorylation, perturbs many cellular properties, including cellular morphology, and activates the heat shock response. Altogether, our study establishes a novel strategy for allotopic expression of mitochondrial genes, demonstrates the complex adaptations required to relocate ATP9, and indicates a reason that this gene was only transferred to the nucleus during the evolution of multicellular organisms.

  View details for DOI 10.1371/journal.pgen.1002876

  View details for PubMedID 22916027

• Genome-wide H4 K16 acetylation by SAS-I is deposited independently of transcription and histone exchange NUCLEIC ACIDS RESEARCH Heise, F., Chung, H., Weber, J. M., Xu, Z., Klein-Hitpass, L., Steinmetz, L. M., Vingron, M., Ehrenhofer-Murray, A. E. 2012; 40 (1): 65-74

  Abstract

  The MYST HAT Sas2 is part of the SAS-I complex that acetylates histone H4 lysine 16 (H4 K16Ac) and blocks the propagation of heterochromatin at the telomeres of Saccharomyces cerevisiae. In this study, we investigated Sas2-mediated H4 K16Ac on a genome-wide scale. Interestingly, H4 K16Ac loss in sas2Δ cells outside of the telomeric regions showed a distinctive pattern in that there was a pronounced decrease of H4 K16Ac within the majority of open reading frames (ORFs), but little change in intergenic regions. Furthermore, regions of low histone H3 exchange and low H3 K56 acetylation showed the most pronounced loss of H4 K16Ac in sas2Δ, indicating that Sas2 deposited this modification on chromatin independently of histone exchange. In agreement with the effect of Sas2 within ORFs, sas2Δ caused resistance to 6-azauracil, indicating a positive effect on transcription elongation in the absence of H4 K16Ac. In summary, our data suggest that Sas2-dependent H4 K16Ac is deposited into chromatin independently of transcription and histone exchange, and that it has an inhibitory effect on the ability of PolII to travel through the body of the gene.

  View details for DOI 10.1093/nar/gkr649

  View details for Web of Science ID 000298733500015

  View details for PubMedID 21908408

• Genome-wide polyadenylation site mapping. Methods in enzymology Pelechano, V., Wilkening, S., Järvelin, A. I., Tekkedil, M. M., Steinmetz, L. M. 2012; 513: 271-296

  Abstract

  Alternative polyadenylation site usage gives rise to variation in 3' ends of transcripts in diverse organisms ranging from yeast to human. Accurate mapping of polyadenylation sites of transcripts is of major biological importance, since the length of the 3'UTR can have a strong influence on transcript stability, localization, and translation. However, reads generated using total mRNA sequencing mostly lack the very 3' end of transcripts. Here, we present a method that allows simultaneous analysis of alternative 3' ends and transcriptome dynamics at high throughput. By using transcripts produced in vitro, the high precision of end mapping during the protocol can be controlled. This method is illustrated here for budding yeast. However, this method can be applied to any natural or artificially polyadenylated RNA.

  View details for DOI 10.1016/B978-0-12-391938-0.00012-4

  View details for PubMedID 22929774

• Accumulation of noncoding RNA due to an RNase P defect in Saccharomyces cerevisiae. RNA (New York, N.Y.) Marvin, M. C., Clauder-Münster, S., Walker, S. C., Sarkeshik, A., Yates, J. R., Steinmetz, L. M., Engelke, D. R. 2011; 17 (8): 1441-1450

  Abstract

  Ribonuclease P (RNase P) is an essential endoribonuclease that catalyzes the cleavage of the 5' leader of pre-tRNAs. In addition, a growing number of non-tRNA substrates have been identified in various organisms. RNase P varies in composition, as bacterial RNase P contains a catalytic RNA core and one protein subunit, while eukaryotic nuclear RNase P retains the catalytic RNA but has at least nine protein subunits. The additional eukaryotic protein subunits most likely provide additional functionality to RNase P, with one possibility being additional RNA recognition capabilities. To investigate the possible range of additional RNase P substrates in vivo, a strand-specific, high-density microarray was used to analyze what RNA accumulates with a mutation in the catalytic RNA subunit of nuclear RNase P in Saccharomyces cerevisiae. A wide variety of noncoding RNAs were shown to accumulate, suggesting that nuclear RNase P participates in the turnover of normally unstable nuclear RNAs. In some cases, the accumulated noncoding RNAs were shown to be antisense to transcripts that commensurately decreased in abundance. Pre-mRNAs containing introns also accumulated broadly, consistent with either compromised splicing or failure to efficiently turn over pre-mRNAs that do not enter the splicing pathway. Taken together with the high complexity of the nuclear RNase P holoenzyme and its relatively nonspecific capacity to bind and cleave mixed sequence RNAs, these data suggest that nuclear RNase P facilitates turnover of nuclear RNAs in addition to its role in pre-tRNA biogenesis.

  View details for DOI 10.1261/rna.2737511

  View details for PubMedID 21665995

• A yeast-based assay identifies drugs active against human mitochondrial disorders PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Couplan, E., Aiyar, R. S., Kucharczyk, R., Kabala, A., Ezkurdia, N., Gagneur, J., St Onge, R. P., Salin, B., Soubigou, F., Le Cann, M., Steinmetz, L. M., di Rago, J., Blondel, M. 2011; 108 (29): 11989-11994

  Abstract

  Due to the lack of relevant animal models, development of effective treatments for human mitochondrial diseases has been limited. Here we establish a rapid, yeast-based assay to screen for drugs active against human inherited mitochondrial diseases affecting ATP synthase, in particular NARP (neuropathy, ataxia, and retinitis pigmentosa) syndrome. This method is based on the conservation of mitochondrial function from yeast to human, on the unique ability of yeast to survive without production of ATP by oxidative phosphorylation, and on the amenability of the yeast mitochondrial genome to site-directed mutagenesis. Our method identifies chlorhexidine by screening a chemical library and oleate through a candidate approach. We show that these molecules rescue a number of phenotypes resulting from mutations affecting ATP synthase in yeast. These compounds are also active on human cybrid cells derived from NARP patients. These results validate our method as an effective high-throughput screening approach to identify drugs active in the treatment of human ATP synthase disorders and suggest that this type of method could be applied to other mitochondrial diseases.

  View details for DOI 10.1073/pnas.1101478108

  View details for Web of Science ID 000292876900057

  View details for PubMedID 21715656

  View details for PubMedCentralID PMC3141935

• Functional consequences of bidirectional promoters TRENDS IN GENETICS Wei, W., Pelechano, V., Jaervelin, A. I., Steinmetz, L. M. 2011; 27 (7): 267-276

  Abstract

  Several studies have shown that promoters of protein-coding genes are origins of pervasive non-coding RNA transcription and can initiate transcription in both directions. However, only recently have researchers begun to elucidate the functional implications of this bidirectionality and non-coding RNA production. Increasing evidence indicates that non-coding transcription at promoters influences the expression of protein-coding genes, revealing a new layer of transcriptional regulation. This regulation acts at multiple levels, from modifying local chromatin to enabling regional signal spreading and more distal regulation. Moreover, the bidirectional activity of a promoter is regulated at multiple points during transcription, giving rise to diverse types of transcripts.

  View details for DOI 10.1016/j.tig.2011.04.002

  View details for Web of Science ID 000292413300003

  View details for PubMedID 21601935

• Antisense expression increases gene expression variability and locus interdependency. Molecular systems biology Xu, Z., Wei, W., Gagneur, J., Clauder-Münster, S., Smolik, M., Huber, W., Steinmetz, L. M. 2011; 7: 468-?

  Abstract

  Genome-wide transcription profiling has revealed extensive expression of non-coding RNAs antisense to genes, yet their functions, if any, remain to be understood. In this study, we perform a systematic analysis of sense-antisense expression in response to genetic and environmental changes in yeast. We find that antisense expression is associated with genes of larger expression variability. This is characterized by more 'switching off' at low levels of expression for genes with antisense compared to genes without, yet similar expression at maximal induction. By disrupting antisense transcription, we demonstrate that antisense expression confers an on-off switch on gene regulation for the SUR7 gene. Consistent with this, genes that must respond in a switch-like manner, such as stress-response and environment-specific genes, are enriched for antisense expression. In addition, our data provide evidence that antisense expression initiated from bidirectional promoters enables the spreading of regulatory signals from one locus to neighbouring genes. These results indicate a general regulatory effect of antisense expression on sense genes and emphasize the importance of antisense-initiating regions downstream of genes in models of gene regulation.

  View details for DOI 10.1038/msb.2011.1

  View details for PubMedID 21326235

• Antisense expression increases gene expression variability and locus interdependency MOLECULAR SYSTEMS BIOLOGY Xu, Z., Wei, W., Gagneur, J., Clauder-Muenster, S., Smolik, M., Huber, W., Steinmetz, L. M. 2011; 7

  View details for DOI 10.1038/msb.2011.1

  View details for Web of Science ID 000288276200004

• Execution of the meiotic noncoding RNA expression program and the onset of gametogenesis in yeast require the conserved exosome subunit Rrp6 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lardenois, A., Liu, Y., Walther, T., Chalmel, F., Evrard, B., Granovskaia, M., Chu, A., Davis, R. W., Steinmetz, L. M., Primig, M. 2011; 108 (3): 1058-1063

  Abstract

  Budding yeast noncoding RNAs (ncRNAs) are pervasively transcribed during mitosis, and some regulate mitotic protein-coding genes. However, little is known about ncRNA expression during meiotic development. Using high-resolution profiling we identified an extensive meiotic ncRNA expression program interlaced with the protein-coding transcriptome via sense/antisense transcript pairs, bidirectional promoters, and ncRNAs that overlap the regulatory regions of genes. Meiotic unannotated transcripts (MUTs) are mitotic targets of the conserved exosome component Rrp6, which itself is degraded after the onset of meiosis when MUTs and other ncRNAs accumulate in successive waves. Diploid cells lacking Rrp6 fail to initiate premeiotic DNA replication normally and cannot undergo efficient meiotic development. The present study demonstrates a unique function for budding yeast Rrp6 in degrading distinct classes of meiotically induced ncRNAs during vegetative growth and the onset of meiosis and thus points to a critical role of differential ncRNA expression in the execution of a conserved developmental program.

  View details for DOI 10.1073/pnas.1016459108

  View details for Web of Science ID 000286310300034

  View details for PubMedID 21149693

  View details for PubMedCentralID PMC3024698

• Yeast Sen1 Helicase Protects the Genome from Transcription-Associated Instability MOLECULAR CELL Mischo, H. E., Gomez-Gonzalez, B., Grzechnik, P., Rondon, A. G., Wei, W., Steinmetz, L., Aguilera, A., Proudfoot, N. J. 2011; 41 (1): 21-32

  Abstract

  Sen1 of S. cerevisiae is a known component of the NRD complex implicated in transcription termination of nonpolyadenylated as well as some polyadenylated RNA polymerase II transcripts. We now show that Sen1 helicase possesses a wider function by restricting the occurrence of RNA:DNA hybrids that may naturally form during transcription, when nascent RNA hybridizes to DNA prior to its packaging into RNA protein complexes. These hybrids displace the nontranscribed strand and create R loop structures. Loss of Sen1 results in transient R loop accumulation and so elicits transcription-associated recombination. SEN1 genetically interacts with DNA repair genes, suggesting that R loop resolution requires proteins involved in homologous recombination. Based on these findings, we propose that R loop formation is a frequent event during transcription and a key function of Sen1 is to prevent their accumulation and associated genome instability.

  View details for DOI 10.1016/j.molcel.2010.12.007

  View details for Web of Science ID 000286692400006

  View details for PubMedID 21211720

• Genome-wide transcriptome analysis in yeast using high-density tiling arrays. Methods in molecular biology (Clifton, N.J.) David, L., Clauder-Münster, S., Steinmetz, L. M. 2011; 759: 107-123

  Abstract

  In the last decade, it became clear that transcription goes far beyond that of protein-coding genes. Most RNA molecules are transcribed from intergenic regions or introns and exhibit much variability in size, expression level, secondary structure, and evolutionary conservation. While for several types of non-coding RNAs some cellular functions have been reported, like for micro-RNAs and small nucleolar RNAs, for most others no indications of function or regulation have so far been found. Therefore, the RNA population inside a cell is diverse and cryptic and, thus, demands powerful methods to study its composition, abundance, and structure. DNA oligonucleotide microarrays have proven to be of great utility to study transcription of genes in various organisms. Recently, due to advancement in microarray technology, tiling microarrays that extend transcription measurement to genomic regions beyond protein-coding genes were designed for several species. The Saccharomyces cerevisiae yeast tiling array contains overlapping probes across the full genomic sequence, with consecutive probes starting every 8 bp on average on each strand, enabling strand-specific measurement of transcription from a full eukaryotic genome. Here, we describe the methods used to extract yeast RNA, convert it into first-strand cDNA, fragment, and label it for hybridization to the tiling array. This protocol will enable researchers not only to study which genes are expressed and to what levels, but also to identify non-coding RNAs and to study the structure of transcripts including their untranslated regions, alternative start, stop, and processing sites. This information will allow understanding their roles inside cells.

  View details for DOI 10.1007/978-1-61779-173-4_7

  View details for PubMedID 21863484

• Genome-wide survey of post-meiotic segregation during yeast recombination GENOME BIOLOGY Mancera, E., Bourgon, R., Huber, W., Steinmetz, L. M. 2011; 12 (4)

  Abstract

  When mismatches in heteroduplex DNA formed during meiotic recombination are left unrepaired, post-meiotic segregation of the two mismatched alleles occurs during the ensuing round of mitosis. This gives rise to somatic mosaicism in multicellular organisms and leads to unexpected allelic combinations among progeny. Despite its implications for inheritance, post-meiotic segregation has been studied at only a few loci.By genotyping tens of thousands of genetic markers in yeast segregants and their clonal progeny, we analyzed post-meiotic segregation at a genome-wide scale. We show that post-meiotic segregation occurs in close to 10% of recombination events. Although the overall number of markers affected in a single meiosis is small, the rate of post-meiotic segregation is more than five orders of magnitude larger than the base substitution mutation rate. Post-meiotic segregation took place with equal relative frequency in crossovers and non-crossovers, and usually at the edges of gene conversion tracts. Furthermore, post-meiotic segregation tended to occur in markers that are isolated from other heterozygosities and preferentially at polymorphism types that are relatively uncommon in the yeast species.Overall, our survey reveals the genome-wide characteristics of post-meiotic segregation. The results show that post-meiotic segregation is widespread in meiotic recombination and could be a significant determinant of allelic inheritance and allele frequencies at the population level.

  View details for DOI 10.1186/gb-2011-12-4-r36

  View details for Web of Science ID 000292871100004

  View details for PubMedID 21481229

• The Baker's Yeast Diploid Genome Is Remarkably Stable in Vegetative Growth and Meiosis PLOS GENETICS Nishant, K. T., Wei, W., Mancera, E., Argueso, J. L., Schlattl, A., Delhomme, N., Ma, X., Bustamante, C. D., Korbel, J. O., Gu, Z., Steinmetz, L. M., Alani, E. 2010; 6 (9)

  Abstract

  Accurate estimates of mutation rates provide critical information to analyze genome evolution and organism fitness. We used whole-genome DNA sequencing, pulse-field gel electrophoresis, and comparative genome hybridization to determine mutation rates in diploid vegetative and meiotic mutation accumulation lines of Saccharomyces cerevisiae. The vegetative lines underwent only mitotic divisions while the meiotic lines underwent a meiotic cycle every ∼20 vegetative divisions. Similar base substitution rates were estimated for both lines. Given our experimental design, these measures indicated that the meiotic mutation rate is within the range of being equal to zero to being 55-fold higher than the vegetative rate. Mutations detected in vegetative lines were all heterozygous while those in meiotic lines were homozygous. A quantitative analysis of intra-tetrad mating events in the meiotic lines showed that inter-spore mating is primarily responsible for rapidly fixing mutations to homozygosity as well as for removing mutations. We did not observe 1-2 nt insertion/deletion (in-del) mutations in any of the sequenced lines and only one structural variant in a non-telomeric location was found. However, a large number of structural variations in subtelomeric sequences were seen in both vegetative and meiotic lines that did not affect viability. Our results indicate that the diploid yeast nuclear genome is remarkably stable during the vegetative and meiotic cell cycles and support the hypothesis that peripheral regions of chromosomes are more dynamic than gene-rich central sections where structural rearrangements could be deleterious. This work also provides an improved estimate for the mutational load carried by diploid organisms.

  View details for DOI 10.1371/journal.pgen.1001109

  View details for Web of Science ID 000282369200047

  View details for PubMedID 20838597

  View details for PubMedCentralID PMC2936533

• Antagonistic Changes in Sensitivity to Antifungal Drugs by Mutations of an Important ABC Transporter Gene in a Fungal Pathogen PLOS ONE Guan, W., Jiang, H., Guo, X., Mancera, E., Xu, L., Li, Y., Steinmetz, L., Li, Y., Gu, Z. 2010; 5 (6)

  Abstract

  Fungal pathogens can be lethal, especially among immunocompromised populations, such as patients with AIDS and recipients of tissue transplantation or chemotherapy. Prolonged usage of antifungal reagents can lead to drug resistance and treatment failure. Understanding mechanisms that underlie drug resistance by pathogenic microorganisms is thus vital for dealing with this emerging issue. In this study, we show that dramatic sequence changes in PDR5, an ABC (ATP-binding cassette) efflux transporter protein gene in an opportunistic fungal pathogen, caused the organism to become hypersensitive to azole, a widely used antifungal drug. Surprisingly, the same mutations conferred growth advantages to the organism on polyenes, which are also commonly used antimycotics. Our results indicate that Pdr5p might be important for ergosterol homeostasis. The observed remarkable sequence divergence in the PDR5 gene in yeast strain YJM789 may represent an interesting case of adaptive loss of gene function with significant clinical implications.

  View details for DOI 10.1371/journal.pone.0011309

  View details for Web of Science ID 000279140800010

  View details for PubMedID 20593017

• Genetic analysis of variation in transcription factor binding in yeast NATURE Zheng, W., Zhao, H., Mancera, E., Steinmetz, L. M., Snyder, M. 2010; 464 (7292): 1187-U106

  Abstract

  Variation in transcriptional regulation is thought to be a major cause of phenotypic diversity. Although widespread differences in gene expression among individuals of a species have been observed, studies to examine the variability of transcription factor binding on a global scale have not been performed, and thus the extent and underlying genetic basis of transcription factor binding diversity is unknown. By mapping differences in transcription factor binding among individuals, here we present the genetic basis of such variation on a genome-wide scale. Whole-genome Ste12-binding profiles were determined using chromatin immunoprecipitation coupled with DNA sequencing in pheromone-treated cells of 43 segregants of a cross between two highly diverged yeast strains and their parental lines. We identified extensive Ste12-binding variation among individuals, and mapped underlying cis- and trans-acting loci responsible for such variation. We showed that most transcription factor binding variation is cis-linked, and that many variations are associated with polymorphisms residing in the binding motifs of Ste12 as well as those of several proposed Ste12 cofactors. We also identified two trans-factors, AMN1 and FLO8, that modulate Ste12 binding to promoters of more than ten genes under alpha-factor treatment. Neither of these two genes was previously known to regulate Ste12, and we suggest that they may be mediators of gene activity and phenotypic diversity. Ste12 binding strongly correlates with gene expression for more than 200 genes, indicating that binding variation is functional. Many of the variable-bound genes are involved in cell wall organization and biogenesis. Overall, these studies identified genetic regulators of molecular diversity among individuals and provide new insights into mechanisms of gene regulation.

  View details for DOI 10.1038/nature08934

  View details for Web of Science ID 000276891100036

  View details for PubMedID 20237471

  View details for PubMedCentralID PMC2941147

• Natural Single-Nucleosome Epi-Polymorphisms in Yeast PLOS GENETICS Nagarajan, M., Veyrieras, J., de Dieuleveult, M., Bottin, H., Fehrmann, S., Abraham, A., Croze, S., Steinmetz, L. M., Gidrol, X., Yvert, G. 2010; 6 (4)

  Abstract

  Epigenomes commonly refer to the sequence of presence/absence of specific epigenetic marks along eukaryotic chromatin. Complete histone-borne epigenomes have now been described at single-nucleosome resolution from various organisms, tissues, developmental stages, or diseases, yet their intra-species natural variation has never been investigated. We describe here that the epigenomic sequence of histone H3 acetylation at Lysine 14 (H3K14ac) differs greatly between two unrelated strains of the yeast Saccharomyces cerevisiae. Using single-nucleosome chromatin immunoprecipitation and mapping, we interrogated 58,694 nucleosomes and found that 5,442 of them differed in their level of H3K14 acetylation, at a false discovery rate (FDR) of 0.0001. These Single Nucleosome Epi-Polymorphisms (SNEPs) were enriched at regulatory sites and conserved non-coding DNA sequences. Surprisingly, higher acetylation in one strain did not imply higher expression of the relevant gene. However, SNEPs were enriched in genes of high transcriptional variability and one SNEP was associated with the strength of gene activation upon stimulation. Our observations suggest a high level of inter-individual epigenomic variation in natural populations, with essential questions on the origin of this diversity and its relevance to gene x environment interactions.

  View details for DOI 10.1371/journal.pgen.1000913

  View details for Web of Science ID 000277354200030

  View details for PubMedID 20421933

• High-resolution transcription atlas of the mitotic cell cycle in budding yeast GENOME BIOLOGY Granovskaia, M. V., Jensen, L. J., Ritchie, M. E., Toedling, J., Ning, Y., Bork, P., Huber, W., Steinmetz, L. M. 2010; 11 (3)

  Abstract

  Extensive transcription of non-coding RNAs has been detected in eukaryotic genomes and is thought to constitute an additional layer in the regulation of gene expression. Despite this role, their transcription through the cell cycle has not been studied; genome-wide approaches have only focused on protein-coding genes. To explore the complex transcriptome architecture underlying the budding yeast cell cycle, we used 8 bp tiling arrays to generate a 5 minute-resolution, strand-specific expression atlas of the whole genome.We discovered 523 antisense transcripts, of which 80 cycle or are located opposite periodically expressed mRNAs, 135 unannotated intergenic non-coding RNAs, of which 11 cycle, and 109 cell-cycle-regulated protein-coding genes that had not previously been shown to cycle. We detected periodic expression coupling of sense and antisense transcript pairs, including antisense transcripts opposite of key cell-cycle regulators, like FAR1 and TAF2.Our dataset presents the most comprehensive resource to date on gene expression during the budding yeast cell cycle. It reveals periodic expression of both protein-coding and non-coding RNA and profiles the expression of non-annotated RNAs throughout the cell cycle for the first time. This data enables hypothesis-driven mechanistic studies concerning the functions of non-coding RNAs.

  View details for DOI 10.1186/gb-2010-11-3-r24

  View details for Web of Science ID 000277309100007

  View details for PubMedID 20193063

• Dissecting the Genetic Basis of Resistance to Malaria Parasites in Anopheles gambiae SCIENCE Blandin, S. A., Wang-Sattler, R., Lamacchia, M., Gagneur, J., Lycett, G., Ning, Y., Levashina, E. A., Steinmetz, L. M. 2009; 326 (5949): 147-150

  Abstract

  The ability of Anopheles gambiae mosquitoes to transmit Plasmodium parasites is highly variable between individuals. However, the genetic basis of this variability has remained unknown. We combined genome-wide mapping and reciprocal allele-specific RNA interference (rasRNAi) to identify the genomic locus that confers resistance to malaria parasites and demonstrated that polymorphisms in a single gene encoding the antiparasitic thioester-containing protein 1 (TEP1) explain a substantial part of the variability in parasite killing. The link between TEP1 alleles and resistance to malaria may offer new tools for controlling malaria transmission. The successful application of rasRNAi in Anopheles suggests that it could also be applied to other organisms where RNAi is feasible to dissect complex phenotypes to the level of individual quantitative trait alleles.

  View details for DOI 10.1126/science.1175241

  View details for Web of Science ID 000270355600057

  View details for PubMedID 19797663

  View details for PubMedCentralID PMC2959166

• Trans-acting antisense RNAs mediate transcriptional gene cosuppression in S. cerevisiae GENES & DEVELOPMENT Camblong, J., Beyrouthy, N., Guffanti, E., Schlaepfer, G., Steinmetz, L. M., Stutz, F. 2009; 23 (13): 1534-1545

  Abstract

  Homology-dependent gene silencing, a phenomenon described as cosuppression in plants, depends on siRNAs. We provide evidence that in Saccharomyces cerevisiae, which is missing the RNAi machinery, protein coding gene cosuppression exists. Indeed, introduction of an additional copy of PHO84 on a plasmid or within the genome results in the cosilencing of both the transgene and the endogenous gene. This repression is transcriptional and position-independent and requires trans-acting antisense RNAs. Antisense RNAs induce transcriptional gene silencing both in cis and in trans, and the two pathways differ by the implication of the Hda1/2/3 complex. We also show that trans-silencing is influenced by the Set1 histone methyltransferase, which promotes antisense RNA production. Finally we show that although antisense-mediated cis-silencing occurs in other genes, trans-silencing so far depends on features specific to PHO84. All together our data highlight the importance of noncoding RNAs in mediating RNAi-independent transcriptional gene silencing.

  View details for DOI 10.1101/gad.522509

  View details for Web of Science ID 000267954800008

  View details for PubMedID 19571181

• Genome-wide allele- and strand-specific expression profiling MOLECULAR SYSTEMS BIOLOGY Gagneur, J., Sinha, H., Perocchi, F., Bourgon, R., Huber, W., Steinmetz, L. M. 2009; 5

  Abstract

  Recent reports have shown that most of the genome is transcribed and that transcription frequently occurs concurrently on both DNA strands. In diploid genomes, the expression level of each allele conditions the degree to which sequence polymorphisms affect the phenotype. It is thus essential to quantify expression in an allele- and strand-specific manner. Using a custom-designed tiling array and a new computational approach, we piloted measuring allele- and strand-specific expression in yeast. Confident quantitative estimates of allele-specific expression were obtained for about half of the coding and non-coding transcripts of a heterozygous yeast strain, of which 371 transcripts (13%) showed significant allelic differential expression greater than 1.5-fold. The data revealed complex allelic differential expression on opposite strands. Furthermore, combining allele-specific expression with linkage mapping enabled identifying allelic variants that act in cis and in trans to regulate allelic expression in the heterozygous strain. Our results provide the first high-resolution analysis of differential expression on all four strands of an eukaryotic genome.

  View details for DOI 10.1038/msb.2009.31

  View details for Web of Science ID 000267629300008

  View details for PubMedID 19536197

• Array-based genotyping in S. cerevisiae using semi-supervised clustering BIOINFORMATICS Bourgon, R., Mancera, E., Brozzi, A., Steinmetz, L. M., Huber, W. 2009; 25 (8): 1056-1062

  Abstract

  Microarrays provide an accurate and cost-effective method for genotyping large numbers of individuals at high resolution. The resulting data permit the identification of loci at which genetic variation is associated with quantitative traits, or fine mapping of meiotic recombination, which is a key determinant of genetic diversity among individuals. Several issues inherent to short oligonucleotide arrays -- cross-hybridization, or variability in probe response to target -- have the potential to produce genotyping errors. There is a need for improved statistical methods for array-based genotyping.We developed ssGenotyping (ssG), a multivariate, semi-supervised approach for using microarrays to genotype haploid individuals at thousands of polymorphic sites. Using a meiotic recombination dataset, we show that ssG is more accurate than existing supervised classification methods, and that it produces denser marker coverage. The ssG algorithm is able to fit probe-specific affinity differences and to detect and filter spurious signal, permitting high-confidence genotyping at nucleotide resolution. We also demonstrate that oligonucleotide probe response depends significantly on genomic background, even when the probe's specific target sequence is unchanged. As a result, supervised classifiers trained on reference strains may not generalize well to diverged strains; ssG's semi-supervised approach, on the other hand, adapts automatically.

  View details for DOI 10.1093/bioinformatics/btp104

  View details for Web of Science ID 000265094400011

  View details for PubMedID 19237444

• Widespread bidirectional promoters are the major source of cryptic transcripts in yeast NATURE Neil, H., Malabat, C., d'Aubenton-Carafa, Y., Xu, Z., Steinmetz, L. M., Jacquier, A. 2009; 457 (7232): 1038-U8

  Abstract

  Pervasive and hidden transcription is widespread in eukaryotes, but its global level, the mechanisms from which it originates and its functional significance are unclear. Cryptic unstable transcripts (CUTs) were recently described as a principal class of RNA polymerase II transcripts in Saccharomyces cerevisiae. These transcripts are targeted for degradation immediately after synthesis by the action of the Nrd1-exosome-TRAMP complexes. Although CUT degradation mechanisms have been analysed in detail, the genome-wide distribution at the nucleotide resolution and the prevalence of CUTs are unknown. Here we report the first high-resolution genomic map of CUTs in yeast, revealing a class of potentially functional CUTs and the intrinsic bidirectional nature of eukaryotic promoters. An RNA fraction highly enriched in CUTs was analysed by a 3' Long-SAGE (serial analysis of gene expression) approach adapted to deep sequencing. The resulting detailed genomic map of CUTs revealed that they derive from extremely widespread and very well defined transcription units and do not result from unspecific transcriptional noise. Moreover, the transcription of CUTs predominantly arises within nucleosome-free regions, most of which correspond to promoter regions of bona fide genes. Some of the CUTs start upstream from messenger RNAs and overlap their 5' end. Our study of glycolysis genes, as well as recent results from the literature, indicate that such concurrent transcription is potentially associated with regulatory mechanisms. Our data reveal numerous new CUTs with such a potential regulatory role. However, most of the identified CUTs corresponded to transcripts divergent from the promoter regions of genes, indicating that they represent by-products of divergent transcription occurring at many and possibly most promoters. Eukaryotic promoter regions are thus intrinsically bidirectional, a fundamental property that escaped previous analyses because in most cases divergent transcription generates short-lived unstable transcripts present at very low steady-state levels.

  View details for DOI 10.1038/nature07747

  View details for Web of Science ID 000263425400048

  View details for PubMedID 19169244

• Identification of mitochondrial disease genes through integrative analysis of multiple datasets METHODS Aiyar, R. S., Gagneur, J., Steinmetz, L. M. 2008; 46 (4): 248-255

  Abstract

  Determining the genetic factors in a disease is crucial to elucidating its molecular basis. This task is challenging due to a lack of information on gene function. The integration of large-scale functional genomics data has proven to be an effective strategy to prioritize candidate disease genes. Mitochondrial disorders are a prevalent and heterogeneous class of diseases that are particularly amenable to this approach. Here we explain the application of integrative approaches to the identification of mitochondrial disease genes. We first examine various datasets that can be used to evaluate the involvement of each gene in mitochondrial function. The data integration methodology is then described, accompanied by examples of common implementations. Finally, we discuss how gene networks are constructed using integrative techniques and applied to candidate gene prioritization. Relevant public data resources are indicated. This report highlights the success and potential of data integration as well as its applicability to the search for mitochondrial disease genes.

  View details for DOI 10.1016/j.ymeth.2008.10.002

  View details for Web of Science ID 000261647200003

  View details for PubMedID 18930150

• Sequential Elimination of Major-Effect Contributors Identifies Additional Quantitative Trait Loci Conditioning High-Temperature Growth in Yeast GENETICS Sinha, H., David, L., Pascon, R. C., Clauder-Muenster, S., Krishnakumar, S., Nguyen, M., Shi, G., Dean, J., Davis, R. W., Oefner, P. J., McCusker, J. H., Steinmetz, L. A. 2008; 180 (3): 1661-1670

  Abstract

  Several quantitative trait loci (QTL) mapping strategies can successfully identify major-effect loci, but often have poor success detecting loci with minor effects, potentially due to the confounding effects of major loci, epistasis, and limited sample sizes. To overcome such difficulties, we used a targeted backcross mapping strategy that genetically eliminated the effect of a previously identified major QTL underlying high-temperature growth (Htg) in yeast. This strategy facilitated the mapping of three novel QTL contributing to Htg of a clinically derived yeast strain. One QTL, which is linked to the previously identified major-effect QTL, was dissected, and NCS2 was identified as the causative gene. The interaction of the NCS2 QTL with the first major-effect QTL was background dependent, revealing a complex QTL architecture spanning these two linked loci. Such complex architecture suggests that more genes than can be predicted are likely to contribute to quantitative traits. The targeted backcrossing approach overcomes the difficulties posed by sample size, genetic linkage, and epistatic effects and facilitates identification of additional alleles with smaller contributions to complex traits.

  View details for DOI 10.1534/genetics.108.092932

  View details for Web of Science ID 000261036200033

  View details for PubMedID 18780730

  View details for PubMedCentralID PMC2581965

• Systematic screens for human disease genes, from yeast to human and back MOLECULAR BIOSYSTEMS Perocchi, F., Mancera, E., Steinmetz, L. M. 2008; 4 (1): 18-29

  Abstract

  Systematic screens for human disease genes have emerged in recent years, due to the wealth of information provided by genome sequences and large scale datasets. Here we review how integration of genomic data in yeast and human is helping to elucidate the genetic basis of mitochondrial diseases. The identification of nearly all yeast mitochondrial proteins and many of their functional interactions provides insight into the role of mitochondria in cellular processes. This information enables prioritization of the candidate genes underlying mitochondrial disorders. In an iterative fashion, the link between predicted human candidate genes and their disease phenotypes can be experimentally tested back in yeast.

  View details for DOI 10.1039/b709494a

  View details for Web of Science ID 000251673300002

  View details for PubMedID 18075670

• Mosaic Genome Architecture of the Anopheles gambiae Species Complex PLOS ONE Wang-Sattler, R., Blandin, S., Ning, Y., Blass, C., Dolo, G., Toure, Y. T., della Torre, A., Lanzaro, G. C., Steinmetz, L. M., Kafatos, F. C., Zheng, L. 2007; 2 (11)

  Abstract

  Attempts over the last three decades to reconstruct the phylogenetic history of the Anopheles gambiae species complex have been important for developing better strategies to control malaria transmission.We used fingerprint genotyping data from 414 field-collected female mosquitoes at 42 microsatellite loci to infer the evolutionary relationships of four species in the A. gambiae complex, the two major malaria vectors A. gambiae sensu stricto (A. gambiae s.s.) and A. arabiensis, as well as two minor vectors, A. merus and A. melas.We identify six taxonomic units, including a clear separation of West and East Africa A. gambiae s.s. S molecular forms. We show that the phylogenetic relationships vary widely between different genomic regions, thus demonstrating the mosaic nature of the genome of these species. The two major malaria vectors are closely related and closer to A. merus than to A. melas at the genome-wide level, which is also true if only autosomes are considered. However, within the Xag inversion region of the X chromosome, the M and two S molecular forms are most similar to A. merus. Near the X centromere, outside the Xag region, the two S forms are highly dissimilar to the other taxa. Furthermore, our data suggest that the centromeric region of chromosome 3 is a strong discriminator between the major and minor malaria vectors.Although further studies are needed to elucidate the basis of the phylogenetic variation among the different regions of the genome, the preponderance of sympatric admixtures among taxa strongly favor introgression of different genomic regions between species, rather than lineage sorting of ancestral polymorphism, as a possible mechanism.

  View details for DOI 10.1371/journal.pone.0001249

  View details for Web of Science ID 000207459300030

  View details for PubMedID 18043756

• Antisense artifacts in transcriptome microarray experiments are resolved by actinomycin D NUCLEIC ACIDS RESEARCH Perocchi, F., Xu, Z., Clauder-Muenster, S., Steinmetz, L. M. 2007; 35 (19)

  Abstract

  Recent transcription profiling studies have revealed an unanticipatedly large proportion of antisense transcription across eukaryotic and bacterial genomes. However, the extent and significance of antisense transcripts is controversial partly because experimental artifacts are suspected. Here, we present a method to generate clean genome-wide transcriptome profiles, using actinomycin D (ActD) during reverse transcription. We show that antisense artifacts appear to be triggered by spurious synthesis of second-strand cDNA during reverse transcription reactions. Strand-specific hybridization signals obtained from Saccharomyces cerevisiae tiling arrays were compared between samples prepared with and without ActD. Use of ActD removed about half of the detectable antisense transcripts, consistent with their being artifacts, while sense expression levels and about 200 antisense transcripts were not affected. Our findings thus facilitate a more accurate assessment of the extent and position of antisense transcription, towards a better understanding of its role in cells.

  View details for DOI 10.1093/nar/gkm683

  View details for Web of Science ID 000250827000034

  View details for PubMedID 17897965

• Assessing systems properties of yeast mitochondria through an interaction map of the organelle PLOS GENETICS Perocchi, F., Jensen, L. J., Gagneur, J., Ahting, U., von Mering, C., Bork, P., Prokisch, H., Steinmetz, L. M. 2006; 2 (10): 1612-1624

  Abstract

  Mitochondria carry out specialized functions; compartmentalized, yet integrated into the metabolic and signaling processes of the cell. Although many mitochondrial proteins have been identified, understanding their functional interrelationships has been a challenge. Here we construct a comprehensive network of the mitochondrial system. We integrated genome-wide datasets to generate an accurate and inclusive mitochondrial parts list. Together with benchmarked measures of protein interactions, a network of mitochondria was constructed in their cellular context, including extra-mitochondrial proteins. This network also integrates data from different organisms to expand the known mitochondrial biology beyond the information in the existing databases. Our network brings together annotated and predicted functions into a single framework. This enabled, for the entire system, a survey of mutant phenotypes, gene regulation, evolution, and disease susceptibility. Furthermore, we experimentally validated the localization of several candidate proteins and derived novel functional contexts for hundreds of uncharacterized proteins. Our network thus advances the understanding of the mitochondrial system in yeast and identifies properties of genes underlying human mitochondrial disorders.

  View details for DOI 10.1371/journal.pgen.0020170

  View details for Web of Science ID 000241922200011

  View details for PubMedID 17054397

• Transcript mapping with high-density oligonucleotide tiling arrays BIOINFORMATICS Huber, W., Toedling, J., Steinmetz, L. M. 2006; 22 (16): 1963-1970

  Abstract

  High-density DNA tiling microarrays are a powerful tool for the characterization of complete transcriptomes. The two major analytical challenges are the segmentation of the hybridization signal along genomic coordinates to accurately determine transcript boundaries and the adjustment of the sequence-dependent response of the oligonucleotide probes to achieve quantitative comparability of the signal between different probes.We describe a dynamic programming algorithm for finding a globally optimal fit of a piecewise constant expression profile along genomic coordinates. We developed a probe-specific background correction and scaling method that employs empirical probe response parameters determined from reference hybridizations with no need for paired mismatch probes. This combined analysis approach allows the accurate determination of dynamical changes in transcription architectures from hybridization data and will help to study the biological significance of complex transcriptional phenomena in eukaryotic genomes.R package tilingArray at http://www.bioconductor.org.

  View details for DOI 10.1093/bioinformatics/btl289

  View details for Web of Science ID 000239900200006

  View details for PubMedID 16787969

• Capturing cellular machines by systematic screens of protein complexes TRENDS IN MICROBIOLOGY Gagneur, J., David, L., Steinmetz, L. M. 2006; 14 (8): 336-339

  Abstract

  Two recent studies have provided the most complete screen for protein complexes in yeast to date, in which partners were identified for approximately half of the proteome. A comparison shows that these two datasets are complementary. In addition, one of the analyses points to a modular organization of the cellular protein network. These data will prove useful in defining principles and trends that arise when combining large-scale datasets of different natures, and in deriving properties of protein machines in cellular systems.

  View details for DOI 10.1016/j.tim.2006.06.002

  View details for Web of Science ID 000239886600003

  View details for PubMedID 16782340

• Complex genetic interactions in a quantitative trait locus PLOS GENETICS Sinha, H., Nicholson, B. P., Steinmetz, L. M., McCusker, J. H. 2006; 2 (2): 140-147

  Abstract

  Whether in natural populations or between two unrelated members of a species, most phenotypic variation is quantitative. To analyze such quantitative traits, one must first map the underlying quantitative trait loci. Next, and far more difficult, one must identify the quantitative trait genes (QTGs), characterize QTG interactions, and identify the phenotypically relevant polymorphisms to determine how QTGs contribute to phenotype. In this work, we analyzed three Saccharomyces cerevisiae high-temperature growth (Htg) QTGs (MKT1, END3, and RHO2). We observed a high level of genetic interactions among QTGs and strain background. Interestingly, while the MKT1 and END3 coding polymorphisms contribute to phenotype, it is the RHO2 3'UTR polymorphisms that are phenotypically relevant. Reciprocal hemizygosity analysis of the Htg QTGs in hybrids between S288c and ten unrelated S. cerevisiae strains reveals that the contributions of the Htg QTGs are not conserved in nine other hybrids, which has implications for QTG identification by marker-trait association. Our findings demonstrate the variety and complexity of QTG contributions to phenotype, the impact of genetic background, and the value of quantitative genetic studies in S. cerevisiae.

  View details for DOI 10.1371/journal.pgen.0020013

  View details for Web of Science ID 000239494300006

  View details for PubMedID 16462944

• Re-analysis of data and its integration FEBS LETTERS Jensen, L. J., Steinmetz, L. M. 2005; 579 (8): 1802-1807

  Abstract

  To understand a biological process it is clear that a single approach will not be sufficient, just like a single measurement on a protein--such as its expression level--does not describe protein function. Using reference sets of proteins as benchmarks different approaches can be scaled and integrated. Here, we demonstrate the power of data re-analysis and integration by applying it in a case study to data from deletion phenotype screens and mRNA expression profiling.

  View details for DOI 10.1016/j.febslet.2005.02.006

  View details for Web of Science ID 000227657800007

  View details for PubMedID 15763555

• Elevated evolutionary rates in the laboratory strain of Saccharomyces cerevisiae PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gu, Z. L., David, L., Petrov, D., Jones, T., Davis, R. W., Steinmetz, L. M. 2005; 102 (4): 1092-1097

  Abstract

  By using the maximum likelihood method, we made a genome-wide comparison of the evolutionary rates in the lineages leading to the laboratory strain (S288c) and a wild strain (YJM789) of Saccharomyces cerevisiae and found that genes in the laboratory strain tend to evolve faster than in the wild strain. The pattern of elevated evolution suggests that relaxation of selection intensity is the dominant underlying reason, which is consistent with recurrent bottlenecks in the S. cerevisiae laboratory strain population. Supporting this conclusion are the following observations: (i) the increases in nonsynonymous evolutionary rate occur for genes in all functional categories; (ii) most of the synonymous evolutionary rate increases in S288c occur in genes with strong codon usage bias; (iii) genes under stronger negative selection have a larger increase in nonsynonymous evolutionary rate; and (iv) more genes with adaptive evolution were detected in the laboratory strain, but they do not account for the majority of the increased evolution. The present discoveries suggest that experimental and possible industrial manipulations of the laboratory strain of yeast could have had a strong effect on the genetic makeup of this model organism. Furthermore, they imply an evolution of laboratory model organisms away from their wild counterparts, questioning the relevancy of the models especially when extensive laboratory cultivation has occurred. In addition, these results shed light on the evolution of livestock and crop species that have been under human domestication for years.

  View details for DOI 10.1073/pnas.0409159102

  View details for Web of Science ID 000226617900026

  View details for PubMedID 15647350

  View details for PubMedCentralID PMC545845

• Integrative analysis of the mitochondrial proteome in yeast PLOS BIOLOGY Prokisch, H., Scharfe, C., Camp, D. G., Xiao, W. Z., David, L., Andreoli, C., Monroe, M. E., Moore, R. J., Gritsenko, M. A., Kozany, C., Hixson, K. K., Mottaz, H. M., Zischka, H., Ueffing, M., Herman, Z. S., Davis, R. W., Meitinger, T., Oefner, P. J., Smith, R. D., Steinmetz, L. M. 2004; 2 (6): 795-804

  Abstract

  In this study yeast mitochondria were used as a model system to apply, evaluate, and integrate different genomic approaches to define the proteins of an organelle. Liquid chromatography mass spectrometry applied to purified mitochondria identified 546 proteins. By expression analysis and comparison to other proteome studies, we demonstrate that the proteomic approach identifies primarily highly abundant proteins. By expanding our evaluation to other types of genomic approaches, including systematic deletion phenotype screening, expression profiling, subcellular localization studies, protein interaction analyses, and computational predictions, we show that an integration of approaches moves beyond the limitations of any single approach. We report the success of each approach by benchmarking it against a reference set of known mitochondrial proteins, and predict approximately 700 proteins associated with the mitochondrial organelle from the integration of 22 datasets. We show that a combination of complementary approaches like deletion phenotype screening and mass spectrometry can identify over 75% of the known mitochondrial proteome. These findings have implications for choosing optimal genome-wide approaches for the study of other cellular systems, including organelles and pathways in various species. Furthermore, our systematic identification of genes involved in mitochondrial function and biogenesis in yeast expands the candidate genes available for mapping Mendelian and complex mitochondrial disorders in humans.

  View details for DOI 10.1371/journal.pbio.0020160

  View details for Web of Science ID 000222380400019

  View details for PubMedID 15208715

  View details for PubMedCentralID PMC423137

• Maximizing the potential of functional genomics NATURE REVIEWS GENETICS Steinmetz, L. M., Davis, R. W. 2004; 5 (3): 190-201

  View details for DOI 10.1038/nrg1293

  View details for Web of Science ID 000189334500014

  View details for PubMedID 14970821

• Role of duplicate genes in genetic robustness against null mutations NATURE Gu, Z. L., Steinmetz, L. M., Gu, X., Scharfe, C., Davis, R. W., Li, W. H. 2003; 421 (6918): 63-66

  Abstract

  Deleting a gene in an organism often has little phenotypic effect, owing to two mechanisms of compensation. The first is the existence of duplicate genes: that is, the loss of function in one copy can be compensated by the other copy or copies. The second mechanism of compensation stems from alternative metabolic pathways, regulatory networks, and so on. The relative importance of the two mechanisms has not been investigated except for a limited study, which suggested that the role of duplicate genes in compensation is negligible. The availability of fitness data for a nearly complete set of single-gene-deletion mutants of the Saccharomyces cerevisiae genome has enabled us to carry out a genome-wide evaluation of the role of duplicate genes in genetic robustness against null mutations. Here we show that there is a significantly higher probability of functional compensation for a duplicate gene than for a singleton, a high correlation between the frequency of compensation and the sequence similarity of two duplicates, and a higher probability of a severe fitness effect when the duplicate copy that is more highly expressed is deleted. We estimate that in S. cerevisiae at least a quarter of those gene deletions that have no phenotype are compensated by duplicate genes.

  View details for DOI 10.1038/nature01198

  View details for Web of Science ID 000180165500037

  View details for PubMedID 12511954

• Gene function on a genomic scale JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES Steinmetz, L. M., Deutschbauer, A. M. 2002; 782 (1-2): 151-163

  Abstract

  The ability to obtain experimental measurements for thousands of genes has revolutionized our view of biological systems. While traditional studies of gene function evaluated many different properties for a single gene, genomic approaches can measure a single property for thousands of genes. Over the last years, genomic approaches have been developed to measure many different properties, including gene expression, deletion phenotype, and protein characteristics. The promise of integrating these datasets has made it attractive to test whether genomic approaches can determine gene function with accuracy comparable to single gene approaches.

  View details for Web of Science ID 000179785100013

  View details for PubMedID 12458004

• Systematic screen for human disease genes in yeast NATURE GENETICS Steinmetz, L. M., Scharfe, C., Deutschbauer, A. M., Mokranjac, D., Herman, Z. S., Jones, T., Chu, A. M., Giaever, G., Prokisch, H., Oefner, P. J., Davis, R. W. 2002; 31 (4): 400-404

  Abstract

  High similarity between yeast and human mitochondria allows functional genomic study of Saccharomyces cerevisiae to be used to identify human genes involved in disease. So far, 102 heritable disorders have been attributed to defects in a quarter of the known nuclear-encoded mitochondrial proteins in humans. Many mitochondrial diseases remain unexplained, however, in part because only 40-60% of the presumed 700-1,000 proteins involved in mitochondrial function and biogenesis have been identified. Here we apply a systematic functional screen using the pre-existing whole-genome pool of yeast deletion mutants to identify mitochondrial proteins. Three million measurements of strain fitness identified 466 genes whose deletions impaired mitochondrial respiration, of which 265 were new. Our approach gave higher selection than other systematic approaches, including fivefold greater selection than gene expression analysis. To apply these advantages to human disorders involving mitochondria, human orthologs were identified and linked to heritable diseases using genomic map positions.

  View details for DOI 10.1038/ng929

  View details for Web of Science ID 000177147100016

  View details for PubMedID 12134146

• Evolutionary rate in the protein interaction network SCIENCE Fraser, H. B., Hirsh, A. E., Steinmetz, L. M., Scharfe, C., Feldman, M. W. 2002; 296 (5568): 750-752

  Abstract

  High-throughput screens have begun to reveal the protein interaction network that underpins most cellular functions in the yeast Saccharomyces cerevisiae. How the organization of this network affects the evolution of the proteins that compose it is a fundamental question in molecular evolution. We show that the connectivity of well-conserved proteins in the network is negatively correlated with their rate of evolution. Proteins with more interactors evolve more slowly not because they are more important to the organism, but because a greater proportion of the protein is directly involved in its function. At sites important for interaction between proteins, evolutionary changes may occur largely by coevolution, in which substitutions in one protein result in selection pressure for reciprocal changes in interacting partners. We confirm one predicted outcome of this process-namely, that interacting proteins evolve at similar rates.

  View details for Web of Science ID 000175281700060

  View details for PubMedID 11976460

• Dissecting the architecture of a quantitative trait locus in yeast NATURE Steinmetz, L. M., Sinha, H., Richards, D. R., Spiegelman, J. I., Oefner, P. J., McCusker, J. H., Davis, R. W. 2002; 416 (6878): 326-330

  Abstract

  Most phenotypic diversity in natural populations is characterized by differences in degree rather than in kind. Identification of the actual genes underlying these quantitative traits has proved difficult. As a result, little is known about their genetic architecture. The failures are thought to be due to the different contributions of many underlying genes to the phenotype and the ability of different combinations of genes and environmental factors to produce similar phenotypes. This study combined genome-wide mapping and a new genetic technique named reciprocal-hemizygosity analysis to achieve the complete dissection of a quantitative trait locus (QTL) in Saccharomyces cerevisiae. A QTL architecture was uncovered that was more complex than expected. Functional linkages both in cis and in trans were found between three tightly linked quantitative trait genes that are neither necessary nor sufficient in isolation. This arrangement of alleles explains heterosis (hybrid vigour), the increased fitness of the heterozygote compared with homozygotes. It also demonstrates a deficiency in current approaches to QTL dissection with implications extending to traits in other organisms, including human genetic diseases.

  View details for Web of Science ID 000174482200044

  View details for PubMedID 11907579

• Transcriptional regulation and function during the human cell cycle NATURE GENETICS Cho, R. J., Huang, M. X., Campbell, M. J., Dong, H. L., Steinmetz, L., Sapinoso, L., Hampton, G., Elledge, S. J., Davis, R. W., Lockhart, D. J. 2001; 27 (1): 48-54

  Abstract

  We report here the transcriptional profiling of the cell cycle on a genome-wide scale in human fibroblasts. We identified approximately 700 genes that display transcriptional fluctuation with a periodicity consistent with that of the cell cycle. Systematic analysis of these genes revealed functional organization within groups of coregulated transcripts. A diverse set of cytoskeletal reorganization genes exhibit cell-cycle-dependent regulation, indicating that biological pathways are redirected for the execution of cell division. Many genes involved in cell motility and remodeling of the extracellular matrix are expressed predominantly in M phase, indicating a mechanism for balancing proliferative and invasive cellular behavior. Transcripts upregulated during S phase displayed extensive overlap with genes induced by DNA damage; cell-cycle-regulated transcripts may therefore constitute coherent programs used in response to external stimuli. Our data also provide clues to biological function for hundreds of previously uncharacterized human genes.

  View details for Web of Science ID 000166187900014

  View details for PubMedID 11137997

• Combining genome sequences and new technologies for dissecting the genetics of complex phenotypes TRENDS IN PLANT SCIENCE Steinmetz, L. M., Mindrinos, M., Oefner, P. J. 2000; 5 (9): 397-401

  View details for Web of Science ID 000089442900019

  View details for PubMedID 10973096

• High-density arrays and insights into genome function BIOTECHNOLOGY & GENETIC ENGINEERING REVIEWS, VOL 17 Steinmetz, L. M., Davis, R. W. 2000; 17: 109-146

  View details for Web of Science ID 000167911600005

  View details for PubMedID 11255664

• A genome-wide transcriptional analysis of the mitotic cell cycle MOLECULAR CELL Cho, R. J., Campbell, M. J., Winzeler, E. A., Steinmetz, L., Conway, A., Wodicka, L., Wolfsberg, T. G., Gabrielian, A. E., Landsman, D., Lockhart, D. J., Davis, R. W. 1998; 2 (1): 65-73

  Abstract

  Progression through the eukaryotic cell cycle is known to be both regulated and accompanied by periodic fluctuation in the expression levels of numerous genes. We report here the genome-wide characterization of mRNA transcript levels during the cell cycle of the budding yeast S. cerevisiae. Cell cycle-dependent periodicity was found for 416 of the 6220 monitored transcripts. More than 25% of the 416 genes were found directly adjacent to other genes in the genome that displayed induction in the same cell cycle phase, suggesting a mechanism for local chromosomal organization in global mRNA regulation. More than 60% of the characterized genes that displayed mRNA fluctuation have already been implicated in cell cycle period-specific biological roles. Because more than 20% of human proteins display significant homology to yeast proteins, these results also link a range of human genes to cell cycle period-specific biological functions.

  View details for Web of Science ID 000075174500007

  View details for PubMedID 9702192