Casey Gifford

All Publications

• Considerations for reporting variants in novel candidate genes identified during clinical genomic testing. Genetics in medicine : official journal of the American College of Medical Genetics Chong, J. X., Berger, S. I., Baxter, S., Smith, E., Xiao, C., Calame, D. G., Hawley, M. H., Rivera-Munoz, E. A., DiTroia, S., Bamshad, M. J., Rehm, H. L. 2024: 101199

  Abstract

  Since the first novel gene discovery for a Mendelian condition was made via exome sequencing (ES), the rapid increase in the number of genes known to underlie Mendelian conditions coupled with the adoption of exome (and more recently, genome) sequencing by diagnostic testing labs has changed the landscape of genomic testing for rare disease. Specifically, many individuals suspected to have a Mendelian condition are now routinely offered clinical ES. This commonly results in a precise genetic diagnosis but frequently overlooks the identification of novel candidate genes. Such candidates are also less likely to be identified in the absence of large-scale gene discovery research programs. Accordingly, clinical laboratories have both the opportunity, and some might argue a responsibility, to contribute to novel gene discovery which should in turn increase the diagnostic yield for many conditions. However, clinical diagnostic laboratories must necessarily balance priorities for throughput, turnaround time, cost efficiency, clinician preferences, and regulatory constraints, and often do not have the infrastructure or resources to effectively participate in either clinical translational or basic genome science research efforts. For these and other reasons, many laboratories have historically refrained from broadly sharing potentially pathogenic variants in novel genes via networks like Matchmaker Exchange, much less reporting such results to ordering providers. Efforts to report such results are further complicated by a lack of guidelines for clinical reporting and interpretation of variants in novel candidate genes. Nevertheless, there are myriad benefits for many stakeholders, including patients/families, clinicians, researchers, if clinical laboratories systematically and routinely identify, share, and report novel candidate genes. To facilitate this change in practice, we developed criteria for triaging, sharing, and reporting novel candidate genes that are most likely to be promptly validated as underlying a Mendelian condition and translated to use in clinical settings.

  View details for DOI 10.1016/j.gim.2024.101199

  View details for PubMedID 38944749

• A lethal mitonuclear incompatibility in complex I of natural hybrids. Nature Moran, B. M., Payne, C. Y., Powell, D. L., Iverson, E. N., Donny, A. E., Banerjee, S. M., Langdon, Q. K., Gunn, T. R., Rodriguez-Soto, R. A., Madero, A., Baczenas, J. J., Kleczko, K. M., Liu, F., Matney, R., Singhal, K., Leib, R. D., Hernandez-Perez, O., Corbett-Detig, R., Frydman, J., Gifford, C., Schartl, M., Havird, J. C., Schumer, M. 2024

  Abstract

  The evolution of reproductive barriers is the first step in the formation of new species and can help us understand the diversification of life on Earth. These reproductive barriers often take the form of hybrid incompatibilities, in which alleles derived from two different species no longer interact properly in hybrids1-3. Theory predicts that hybrid incompatibilities may be more likely to arise at rapidly evolving genes4-6 and that incompatibilities involving multiple genes should be common7,8, but there has been sparse empirical data to evaluate these predictions. Here we describe a mitonuclear incompatibility involving three genes whose protein products are in physical contact within respiratory complex I of naturally hybridizing swordtail fish species. Individuals homozygous for mismatched protein combinations do not complete embryonic development or die as juveniles, whereas those heterozygous for the incompatibility have reduced complex I function and unbalanced representation of parental alleles in the mitochondrial proteome. We find that the effects of different genetic interactions on survival are non-additive, highlighting subtle complexity in the genetic architecture of hybrid incompatibilities. Finally, we document the evolutionary history of the genes involved, showing signals of accelerated evolution and evidence that an incompatibility has been transferred between species via hybridization.

  View details for DOI 10.1038/s41586-023-06895-8

  View details for PubMedID 38200310

  View details for PubMedCentralID 6502311

• Single Cell Multimodal Analyses Reveal Epigenomic and Transcriptomic Basis for Birth Defects in Maternal Diabetes. Nature cardiovascular research Nishino, T., Ranade, S. S., Pelonero, A., van Soldt, B. J., Ye, L., Alexanian, M., Koback, F., Huang, Y., Sadagopan, N., Lam, A., Zholudeva, L. V., Li, F., Padmanabhan, A., Thomas, R., van Bemmel, J. G., Gifford, C. A., Costa, M. W., Srivastava, D. 2023; 2 (12): 1190-1203

  Abstract

  Maternal diabetes mellitus is among the most frequent environmental contributors to congenital birth defects, including heart defects and craniofacial anomalies, yet the cell types affected and mechanisms of disruption are largely unknown. Using multi-modal single cell analyses, here we show that maternal diabetes affects the epigenomic landscape of specific subsets of cardiac and craniofacial progenitors during embryogenesis. A previously unrecognized cardiac progenitor subpopulation expressing the homeodomain-containing protein ALX3 showed prominent chromatin accessibility changes and acquired a more posterior identity. Similarly, a subpopulation of neural crest-derived cells in the second pharyngeal arch, which contributes to craniofacial structures, displayed abnormalities in the epigenetic landscape and axial patterning defects. Chromatin accessibility changes in both populations were associated with increased retinoic acid signaling, known to establish anterior-posterior identity. This work highlights how an environmental insult can have highly selective epigenomic consequences on discrete cell types leading to developmental patterning defects.

  View details for DOI 10.1038/s44161-023-00367-y

  View details for PubMedID 39183978

  View details for PubMedCentralID PMC11343316

• Single-cell multimodal analyses reveal epigenomic and transcriptomic basis for birth defects in maternal diabetes NATURE CARDIOVASCULAR RESEARCH Nishino, T., Ranade, S. S., Pelonero, A., van Soldt, B. J., Ye, L., Alexanian, M., Koback, F., Huang, Y., Wallace, L., Sadagopan, N., Lam, A., Zholudeva, L. V., Li, F., Padmanabhan, A., Thomas, R., van Bemmel, J. G., Gifford, C. A., Costa, M. W., Srivastava, D. 2023; 2 (12): 1190-+

  View details for DOI 10.1038/s44161-023-00367-y

  View details for Web of Science ID 001124837200001

• The multi-lineage transcription factor ISL1 controls cardiomyocyte cell fate through interaction with NKX2.5. Stem cell reports Maven, B. E., Gifford, C. A., Weilert, M., Gonzalez-Teran, B., Hüttenhain, R., Pelonero, A., Ivey, K. N., Samse-Knapp, K., Kwong, W., Gordon, D., McGregor, M., Nishino, T., Okorie, E., Rossman, S., Costa, M. W., Krogan, N. J., Zeitlinger, J., Srivastava, D. 2023

  Abstract

  Congenital heart disease often arises from perturbations of transcription factors (TFs) that guide cardiac development. ISLET1 (ISL1) is a TF that influences early cardiac cell fate, as well as differentiation of other cell types including motor neuron progenitors (MNPs) and pancreatic islet cells. While lineage specificity of ISL1 function is likely achieved through combinatorial interactions, its essential cardiac interacting partners are unknown. By assaying ISL1 genomic occupancy in human induced pluripotent stem cell-derived cardiac progenitors (CPs) or MNPs and leveraging the deep learning approach BPNet, we identified motifs of other TFs that predicted ISL1 occupancy in each lineage, with NKX2.5 and GATA motifs being most closely associated to ISL1 in CPs. Experimentally, nearly two-thirds of ISL1-bound loci were co-occupied by NKX2.5 and/or GATA4. Removal of NKX2.5 from CPs led to widespread ISL1 redistribution, and overexpression of NKX2.5 in MNPs led to ISL1 occupancy of CP-specific loci. These results reveal how ISL1 guides lineage choices through a combinatorial code that dictates genomic occupancy and transcription.

  View details for DOI 10.1016/j.stemcr.2023.09.014

  View details for PubMedID 37863045

• Functional Human iPSC-Derived Thymic Epithelial Progenitor Cells Reconstitute T Cell Development and Function in an In Vivo Model of Thymic Aplasia Slepicka, P., Hubka, K. M., Hanh Dan Nguyen, Mohammed, A., Wang, J., Gifford, C., Sebastiano, V., Weinacht, K. G. AMER SOC HEMATOLOGY. 2022: 7340-7341

  View details for DOI 10.1182/blood-2022-171133

  View details for Web of Science ID 000893230300154

• De novo and inherited variants in coding and regulatory regions in genetic cardiomyopathies. Human genomics Vadgama, N., Ameen, M., Sundaram, L., Gaddam, S., Genomics England Research Consortium, Gifford, C., Nasir, J., Karakikes, I. 2022; 16 (1): 55

  Abstract

  BACKGROUND: Cardiomyopathies are a leading cause of progressive heart failure and sudden cardiac death; however, their genetic aetiology remains poorly understood. We hypothesised that variants in noncoding regulatory regions and oligogenic inheritance mechanisms may help close the diagnostic gap.METHODS: We first analysed whole-genome sequencing data of 143 parent-offspring trios from Genomics England 100,000 Genomes Project. We used gene panel testing and a phenotype-based, variant prioritisation framework called Exomiser to identify candidate genes in trios. To assess the contribution of noncoding DNVs to cardiomyopathies, we intersected DNVs with open chromatin sequences from single-cell ATAC-seq data of cardiomyocytes. We also performed a case-control analysis in an exome-negative cohort, including 843 probands and 19,467 controls, to assess the association between noncoding variants in known cardiomyopathy genes and disease.RESULTS: In the trio analysis, a definite or probable genetic diagnosis was identified in 21 probands according to the American College of Medical Genetics guidelines. We identified novel DNVs in diagnostic-grade genes (RYR2, TNNT2, PTPN11, MYH7, LZR1, NKX2-5), and five cases harbouring a combination of prioritised variants, suggesting that oligogenic inheritance and genetic modifiers contribute to cardiomyopathies. Phenotype-based ranking of candidate genes identified in noncoding DNV analysis revealed JPH2 as the top candidate. Moreover, a case-control analysis revealed an enrichment of rare noncoding variants in regulatory elements of cardiomyopathy genes (p=.035, OR=1.43, 95% Cl=1.095-1.767) versus controls. Of the 25 variants associated with disease (p< 0.5), 23 are novel and nine are predicted to disrupt transcription factor binding motifs.CONCLUSION: Our results highlight complex genetic mechanisms in cardiomyopathies and reveal novel genes for future investigations.

  View details for DOI 10.1186/s40246-022-00420-0

  View details for PubMedID 36357925

• 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

• Transcription factor protein interactomes reveal genetic determinants in heart disease. Cell Gonzalez-Teran, B., Pittman, M., Felix, F., Thomas, R., Richmond-Buccola, D., Hüttenhain, R., Choudhary, K., Moroni, E., Costa, M. W., Huang, Y., Padmanabhan, A., Alexanian, M., Lee, C. Y., Maven, B. E., Samse-Knapp, K., Morton, S. U., McGregor, M., Gifford, C. A., Seidman, J. G., Seidman, C. E., Gelb, B. D., Colombo, G., Conklin, B. R., Black, B. L., Bruneau, B. G., Krogan, N. J., Pollard, K. S., Srivastava, D. 2022

  Abstract

  Congenital heart disease (CHD) is present in 1% of live births, yet identification of causal mutations remains challenging. We hypothesized that genetic determinants for CHDs may lie in the protein interactomes of transcription factors whose mutations cause CHDs. Defining the interactomes of two transcription factors haplo-insufficient in CHD, GATA4 and TBX5, within human cardiac progenitors, and integrating the results with nearly 9,000 exomes from proband-parent trios revealed an enrichment of de novo missense variants associated with CHD within the interactomes. Scoring variants of interactome members based on residue, gene, and proband features identified likely CHD-causing genes, including the epigenetic reader GLYR1. GLYR1 and GATA4 widely co-occupied and co-activated cardiac developmental genes, and the identified GLYR1 missense variant disrupted interaction with GATA4, impairing in vitro and in vivo function in mice. This integrative proteomic and genetic approach provides a framework for prioritizing and interrogating genetic variants in heart disease.

  View details for DOI 10.1016/j.cell.2022.01.021

  View details for PubMedID 35182466

• SARS-CoV-2 Susceptibility and ACE2 Gene Variations Within Diverse Ethnic Backgrounds. Frontiers in genetics Vadgama, N., Kreymerman, A., Campbell, J., Shamardina, O., Brugger, C., Research Consortium, G. E., Deaconescu, A. M., Lee, R. T., Penkett, C. J., Gifford, C. A., Mercola, M., Nasir, J., Karakikes, I. 2022; 13: 888025

  Abstract

  There is considerable variability in the susceptibility and progression for COVID-19 and it appears to be strongly correlated with age, gender, ethnicity and pre-existing health conditions. However, to our knowledge, cohort studies of COVID-19 in clinically vulnerable groups are lacking. Host genetics has also emerged as a major risk factor for COVID-19, and variation in the ACE2 receptor, which facilitates entry of the SARS-CoV-2 virus into the cell, has become a major focus of attention. Thus, we interrogated an ethnically diverse cohort of National Health Service (NHS) patients in the United Kingdom (United Kingdom) to assess the association between variants in the ACE2 locus and COVID-19 risk. We analysed whole-genome sequencing (WGS) data of 1,837 cases who were tested positive for SARS-CoV-2, and 37,207 controls who were not tested, from the UK's 100,000 Genomes Project (100KGP) for the presence of ACE2 coding variants and extract expression quantitative trait loci (eQTLs). We identified a splice site variant (rs2285666) associated with increased ACE2 expression with an overrepresentation in SARS-CoV-2 positive patients relative to 100KGP controls (p = 0.015), and in hospitalised European patients relative to outpatients in intra-ethnic comparisons (p = 0.029). We also compared the prevalence of 288 eQTLs, of which 23 were enriched in SARS-CoV-2 positive patients. The eQTL rs12006793 had the largest effect size (d = 0.91), which decreases ACE2 expression and is more prevalent in controls, thus potentially reducing the risk of COVID-19. We identified three novel nonsynonymous variants predicted to alter ACE2 function, and showed that three variants (p.K26R, p. H378R, p. Y515N) alter receptor affinity for the viral Spike (S) protein. Variant p. N720D, more prevalent in the European population (p < 0.001), potentially increases viral entry by affecting the ACE2-TMPRSS2 complex. The spectrum of genetic variants in ACE2 may inform risk stratification of COVID-19 patients and could partially explain the differences in disease susceptibility and severity among different ethnic groups.

  View details for DOI 10.3389/fgene.2022.888025

  View details for PubMedID 35571054

• Early-career researchers in the time of COVID-19: Starting a new lab during a pandemic CELL STEM CELL Tikhonova, A. N., Xiang, Y., Gifford, C. 2021; 28 (5): 808-810

  Abstract

  New investigators launching their labs in the midst of the pandemic face unique challenges. Here, Cell Stem Cell talks with three participants from our year-long "Introductions to the community: Early-career researchers in the time of COVID-19" Voices series about hurdles they faced while starting their independent careers during shutdowns.

  View details for DOI 10.1016/j.stem.2021.04.021

  View details for Web of Science ID 000648283600011

  View details for PubMedID 33961766

  View details for PubMedCentralID PMC8101062

• Introductions to the Community: Early-Career Researchers in the Time of COVID-19 CELL STEM CELL Ganesh, K., Patel, J., Orlova, V. V., Gifford, C., Elias, S., Vaughan, A. 2021; 28 (1): 17–19

  Abstract

  COVID-19 has unfortunately halted lab work, conferences, and in-person networking, which is especially detrimental to researchers just starting their labs. Through social media and our reviewer networks, we met some early-career stem cell investigators impacted by the closures. Here, they introduce themselves and their research to our readers.

  View details for Web of Science ID 000606536600007

  View details for PubMedID 33417867

• A transcriptional switch governs fibroblast activation in heart disease. Nature Alexanian, M., Przytycki, P. F., Micheletti, R., Padmanabhan, A., Ye, L., Travers, J. G., Gonzalez-Teran, B., Silva, A. C., Duan, Q., Ranade, S. S., Felix, F., Linares-Saldana, R., Li, L., Lee, C. Y., Sadagopan, N., Pelonero, A., Huang, Y., Andreoletti, G., Jain, R., McKinsey, T. A., Rosenfeld, M. G., Gifford, C. A., Pollard, K. S., Haldar, S. M., Srivastava, D. 2021

  Abstract

  In diseased organs, stress-activated signalling cascades alter chromatin, thereby triggering maladaptive cell state transitions. Fibroblast activation is a common stress response in tissues that worsens lung, liver, kidney and heart disease, yet its mechanistic basis remains unclear1,2. Pharmacological inhibition of bromodomain and extra-terminal domain (BET) proteins alleviates cardiac dysfunction3-7, providing a tool to interrogate and modulate cardiac cell states as a potential therapeutic approach. Here we use single-cell epigenomic analyses of hearts dynamically exposed to BET inhibitors to reveal a reversible transcriptional switch that underlies the activation of fibroblasts. Resident cardiac fibroblasts demonstrated robust toggling between the quiescent and activated state in a manner directly correlating with BET inhibitor exposure and cardiac function. Single-cell chromatin accessibility revealed previously undescribed DNA elements, the accessibility of which dynamically correlated with cardiac performance. Among the most dynamic elements was an enhancer that regulated the transcription factor MEOX1, which was specifically expressed in activated fibroblasts, occupied putative regulatory elements of a broad fibrotic gene program and was required for TGFβ-induced fibroblast activation. Selective CRISPR inhibition of the single most dynamic cis-element within the enhancer blocked TGFβ-induced Meox1 activation. We identify MEOX1 as a central regulator of fibroblast activation associated with cardiac dysfunction and demonstrate its upregulation after activation of human lung, liver and kidney fibroblasts. The plasticity and specificity of BET-dependent regulation of MEOX1 in tissue fibroblasts provide previously unknown trans- and cis-targets for treating fibrotic disease.

  View details for DOI 10.1038/s41586-021-03674-1

  View details for PubMedID 34163071

• Network-based screen in iPSC-derived cells reveals therapeutic candidate for heart valve disease. Science (New York, N.Y.) Theodoris, C. V., Zhou, P., Liu, L., Zhang, Y., Nishino, T., Huang, Y., Kostina, A., Ranade, S. S., Gifford, C. A., Uspenskiy, V., Malaschicheva, A., Ding, S., Srivastava, D. 2020

  Abstract

  Mapping the gene regulatory networks dysregulated in human disease would allow the design of network-correcting therapies that treat the core disease mechanism. However, small molecules are traditionally screened for their effects on one to several outputs at most, biasing discovery and limiting the likelihood of true disease-modifying drug candidates. Here, we developed a machine learning approach to identify small molecules that broadly correct gene networks dysregulated in a human induced pluripotent stem cell (iPSC) disease model of a common form of heart disease involving the aortic valve. Gene network correction by the most efficacious therapeutic candidate, XCT790, generalized to patient-derived primary aortic valve cells and was sufficient to prevent and treat aortic valve disease in vivo in a mouse model. This strategy, made feasible by human iPSC technology, network analysis, and machine learning, may represent an effective path for drug discovery.

  View details for DOI 10.1126/science.abd0724

  View details for PubMedID 33303684

• Single-cell analysis of cardiogenesis reveals basis for organ-level developmental defects. Nature de Soysa, T. Y., Ranade, S. S., Okawa, S., Ravichandran, S., Huang, Y., Salunga, H. T., Schricker, A., Del Sol, A., Gifford, C. A., Srivastava, D. 2019; 572 (7767): 120-124

  Abstract

  Organogenesis involves integration of diverse cell types; dysregulation of cell-type-specific gene networks results in birth defects, which affect 5% of live births. Congenital heart defects are the most common malformations, and result from disruption of discrete subsets of cardiac progenitor cells1, but the transcriptional changes in individual progenitors that lead to organ-level defects remain unknown. Here we used single-cell RNA sequencing to interrogate early cardiac progenitor cells as they become specified during normal and abnormal cardiogenesis, revealing how dysregulation of specific cellular subpopulations has catastrophic consequences. A network-based computational method for single-cell RNA-sequencing analysis that predicts lineage-specifying transcription factors2,3 identified Hand2 as a specifier of outflow tract cells but not right ventricular cells, despite the failure of right ventricular formation in Hand2-null mice4. Temporal single-cell-transcriptome analysis of Hand2-null embryos revealed failure of outflow tract myocardium specification, whereas right ventricular myocardium was specified but failed to properly differentiate and migrate. Loss of Hand2 also led to dysregulation of retinoic acid signalling and disruption of anterior-posterior patterning of cardiac progenitors. This work reveals transcriptional determinants that specify fate and differentiation in individual cardiac progenitor cells, and exposes mechanisms of disrupted cardiac development at single-cell resolution, providing a framework for investigating congenital heart defects.

  View details for DOI 10.1038/s41586-019-1414-x

  View details for PubMedID 31341279

  View details for PubMedCentralID PMC6719697

• Context-Specific Transcription Factor Functions Regulate Epigenomic and Transcriptional Dynamics during Cardiac Reprogramming. Cell stem cell Stone, N. R., Gifford, C. A., Thomas, R., Pratt, K. J., Samse-Knapp, K., Mohamed, T. M., Radzinsky, E. M., Schricker, A., Ye, L., Yu, P., van Bemmel, J. G., Ivey, K. N., Pollard, K. S., Srivastava, D. 2019; 25 (1): 87-102.e9

  Abstract

  Ectopic expression of combinations of transcription factors (TFs) can drive direct lineage conversion, thereby reprogramming a somatic cell's identity. To determine the molecular mechanisms by which Gata4, Mef2c, and Tbx5 (GMT) induce conversion from a cardiac fibroblast toward an induced cardiomyocyte, we performed comprehensive transcriptomic, DNA-occupancy, and epigenomic interrogation throughout the reprogramming process. Integration of these datasets identified new TFs involved in cardiac reprogramming and revealed context-specific roles for GMT, including the ability of Mef2c and Tbx5 to independently promote chromatin remodeling at previously inaccessible sites. We also find evidence for cooperative facilitation and refinement of each TF's binding profile in a combinatorial setting. A reporter assay employing newly defined regulatory elements confirmed that binding of a single TF can be sufficient for gene activation, suggesting that co-binding events do not necessarily reflect synergy. These results shed light on fundamental mechanisms by which combinations of TFs direct lineage conversion.

  View details for DOI 10.1016/j.stem.2019.06.012

  View details for PubMedID 31271750

  View details for PubMedCentralID PMC6632093

• Oligogenic inheritance of a human heart disease involving a genetic modifier. Science (New York, N.Y.) Gifford, C. A., Ranade, S. S., Samarakoon, R., Salunga, H. T., de Soysa, T. Y., Huang, Y., Zhou, P., Elfenbein, A., Wyman, S. K., Bui, Y. K., Cordes Metzler, K. R., Ursell, P., Ivey, K. N., Srivastava, D. 2019; 364 (6443): 865-870

  Abstract

  Complex genetic mechanisms are thought to underlie many human diseases, yet experimental proof of this model has been elusive. Here, we show that a human cardiac anomaly can be caused by a combination of rare, inherited heterozygous mutations. Whole-exome sequencing of a nuclear family revealed that three offspring with childhood-onset cardiomyopathy had inherited three missense single-nucleotide variants in the MKL2, MYH7, and NKX2-5 genes. The MYH7 and MKL2 variants were inherited from the affected, asymptomatic father and the rare NKX2-5 variant (minor allele frequency, 0.0012) from the unaffected mother. We used CRISPR-Cas9 to generate mice encoding the orthologous variants and found that compound heterozygosity for all three variants recapitulated the human disease phenotype. Analysis of murine hearts and human induced pluripotent stem cell-derived cardiomyocytes provided histologic and molecular evidence for the NKX2-5 variant's contribution as a genetic modifier.

  View details for DOI 10.1126/science.aat5056

  View details for PubMedID 31147515

  View details for PubMedCentralID PMC6557373

• Genetic determinants and epigenetic effects of pioneer-factor occupancy NATURE GENETICS Donaghey, J., Thakurela, S., Charlton, J., Chen, J. S., Smith, Z. D., Gu, H., Pop, R., Clement, K., Stamenova, E. K., Karnik, R., Kelley, D. R., Gifford, C. A., Cacchiarelli, D., Rinn, J. L., Gnirke, A., Ziller, M. J., Meissner, A. 2018; 50 (2): 250-+

  Abstract

  Transcription factors (TFs) direct developmental transitions by binding to target DNA sequences, influencing gene expression and establishing complex gene-regultory networks. To systematically determine the molecular components that enable or constrain TF activity, we investigated the genomic occupancy of FOXA2, GATA4 and OCT4 in several cell types. Despite their classification as pioneer factors, all three TFs exhibit cell-type-specific binding, even when supraphysiologically and ectopically expressed. However, FOXA2 and GATA4 can be distinguished by low enrichment at loci that are highly occupied by these factors in alternative cell types. We find that expression of additional cofactors increases enrichment at a subset of these sites. Finally, FOXA2 occupancy and changes to DNA accessibility can occur in G1-arrested cells, but subsequent loss of DNA methylation requires DNA replication.

  View details for DOI 10.1038/s41588-017-0034-3

  View details for Web of Science ID 000424519200016

  View details for PubMedID 29358654

  View details for PubMedCentralID PMC6517675

• Differentiation of V2a interneurons from human pluripotent stem cells. Proceedings of the National Academy of Sciences of the United States of America Butts, J. C., McCreedy, D. A., Martinez-Vargas, J. A., Mendoza-Camacho, F. N., Hookway, T. A., Gifford, C. A., Taneja, P., Noble-Haeusslein, L., McDevitt, T. C. 2017; 114 (19): 4969-4974

  Abstract

  The spinal cord consists of multiple neuronal cell types that are critical to motor control and arise from distinct progenitor domains in the developing neural tube. Excitatory V2a interneurons in particular are an integral component of central pattern generators that control respiration and locomotion; however, the lack of a robust source of human V2a interneurons limits the ability to molecularly profile these cells and examine their therapeutic potential to treat spinal cord injury (SCI). Here, we report the directed differentiation of CHX10+ V2a interneurons from human pluripotent stem cells (hPSCs). Signaling pathways (retinoic acid, sonic hedgehog, and Notch) that pattern the neural tube were sequentially perturbed to identify an optimized combination of small molecules that yielded ∼25% CHX10+ cells in four hPSC lines. Differentiated cultures expressed much higher levels of V2a phenotypic markers (CHX10 and SOX14) than other neural lineage markers. Over time, CHX10+ cells expressed neuronal markers [neurofilament, NeuN, and vesicular glutamate transporter 2 (VGlut2)], and cultures exhibited increased action potential frequency. Single-cell RNAseq analysis confirmed CHX10+ cells within the differentiated population, which consisted primarily of neurons with some glial and neural progenitor cells. At 2 wk after transplantation into the spinal cord of mice, hPSC-derived V2a cultures survived at the site of injection, coexpressed NeuN and VGlut2, extended neurites >5 mm, and formed putative synapses with host neurons. These results provide a description of V2a interneurons differentiated from hPSCs that may be used to model central nervous system development and serve as a potential cell therapy for SCI.

  View details for DOI 10.1073/pnas.1608254114

  View details for PubMedID 28438991

  View details for PubMedCentralID PMC5441696

• Transcriptional and Chromatin Dynamics of Muscle Regeneration after Severe Trauma STEM CELL REPORTS Aguilar, C. A., Pop, R., Shcherbina, A., Watts, A., Matheny, R. W., Cacchiarelli, D., Han, W. M., Shin, E., Nakhai, S. A., Jang, Y. C., Carrigan, C. T., Gifford, C. A., Kottke, M. A., Cesana, M., Lee, J., Urso, M. L., Meissner, A. 2016; 7 (5): 983–97

  Abstract

  Following injury, adult skeletal muscle undergoes a well-coordinated sequence of molecular and physiological events to promote repair and regeneration. However, a thorough understanding of the in vivo epigenomic and transcriptional mechanisms that control these reparative events is lacking. To address this, we monitored the in vivo dynamics of three histone modifications and coding and noncoding RNA expression throughout the regenerative process in a mouse model of traumatic muscle injury. We first illustrate how both coding and noncoding RNAs in tissues and sorted satellite cells are modified and regulated during various stages after trauma. Next, we use chromatin immunoprecipitation followed by sequencing to evaluate the chromatin state of cis-regulatory elements (promoters and enhancers) and view how these elements evolve and influence various muscle repair and regeneration transcriptional programs. These results provide a comprehensive view of the central factors that regulate muscle regeneration and underscore the multiple levels through which both transcriptional and epigenetic patterns are regulated to enact appropriate repair and regeneration.

  View details for DOI 10.1016/j.stemcr.2016.09.009

  View details for Web of Science ID 000389509000013

  View details for PubMedID 27773702

  View details for PubMedCentralID PMC5106515

• Heart disease modelling adds a Notch to its belt. Nature cell biology Gifford, C. A., Srivastava, D. 2016; 18 (1): 3-5

  Abstract

  The heart is a complex organ, consisting of multiple cell types that coordinately regulate blood flow. Reciprocal Notch pathway signalling in endocardial and myocardial cells is now shown to promote maturation of the ventricular chambers. These insights reveal mechanisms that, when disrupted, can lead to cardiomyopathies.

  View details for DOI 10.1038/ncb3294

  View details for PubMedID 26693916

• A qPCRCR ScoreCard quantifies the differentiation potential of human pluripotent stem cells NATURE BIOTECHNOLOGY Tsankov, A. M., Akopian, V., Pop, R., Chetty, S., Gifford, C. A., Daheron, L., Tsankova, N. M., Meissner, A. 2015; 33 (11): 1182-U117

  Abstract

  Research on human pluripotent stem cells has been hampered by the lack of a standardized, quantitative, scalable assay of pluripotency. We previously described an assay called ScoreCard that used gene expression signatures to quantify differentiation efficiency. Here we report an improved version of the assay based on qPCR that enables faster, more quantitative assessment of functional pluripotency. We provide an in-depth characterization of the revised signature panel (commercially available as the TaqMan hPSC Scorecard Assay) through embryoid body and directed differentiation experiments as well as a detailed comparison to the teratoma assay. We further show that the improved ScoreCard enables a wider range of applications, such as screening of small molecules, genetic perturbations and assessment of culture conditions. Our approach can be extended beyond stem cell applications to characterize and assess the utility of other cell types and lineages.

  View details for DOI 10.1038/nbt.3387

  View details for Web of Science ID 000364916000026

  View details for PubMedID 26501952

  View details for PubMedCentralID PMC4636964

• In vivo Monitoring of Transcriptional Dynamics After Lower-Limb Muscle Injury Enables Quantitative Classification of Healing SCIENTIFIC REPORTS Aguilar, C. A., Shcherbina, A., Ricke, D. O., Pop, R., Carrigan, C. T., Gifford, C. A., Urso, M. L., Kottke, M. A., Meissner, A. 2015; 5: 13885

  Abstract

  Traumatic lower-limb musculoskeletal injuries are pervasive amongst athletes and the military and typically an individual returns to activity prior to fully healing, increasing a predisposition for additional injuries and chronic pain. Monitoring healing progression after a musculoskeletal injury typically involves different types of imaging but these approaches suffer from several disadvantages. Isolating and profiling transcripts from the injured site would abrogate these shortcomings and provide enumerative insights into the regenerative potential of an individual's muscle after injury. In this study, a traumatic injury was administered to a mouse model and healing progression was examined from 3 hours to 1 month using high-throughput RNA-Sequencing (RNA-Seq). Comprehensive dissection of the genome-wide datasets revealed the injured site to be a dynamic, heterogeneous environment composed of multiple cell types and thousands of genes undergoing significant expression changes in highly regulated networks. Four independent approaches were used to determine the set of genes, isoforms, and genetic pathways most characteristic of different time points post-injury and two novel approaches were developed to classify injured tissues at different time points. These results highlight the possibility to quantitatively track healing progression in situ via transcript profiling using high- throughput sequencing.

  View details for DOI 10.1038/srep13885

  View details for Web of Science ID 000361376900001

  View details for PubMedID 26381351

  View details for PubMedCentralID PMC4585378

• Integrative Analyses of Human Reprogramming Reveal Dynamic Nature of Induced Pluripotency CELL Cacchiarelli, D., Trapnell, C., Ziller, M. J., Soumillon, M., Cesana, M., Karnik, R., Donaghey, J., Smith, Z. D., Ratanasirintrawoot, S., Zhang, X., Sui, S., Wu, Z., Akopian, V., Gifford, C. A., Doench, J., Rinn, J. L., Daley, G. Q., Meissner, A., Lander, E. S., Mikkelsen, T. S. 2015; 162 (2): 412–24

  Abstract

  Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here, we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. PAPERCLIP.

  View details for DOI 10.1016/j.cell.2015.06.016

  View details for Web of Science ID 000358087700020

  View details for PubMedID 26186193

  View details for PubMedCentralID PMC4511597

• Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells NATURE GENETICS Liao, J., Karnik, R., Gu, H., Ziller, M. J., Clement, K., Tsankov, A. M., Akopian, V., Gifford, C. A., Donaghey, J., Galonska, C., Pop, R., Reyon, D., Tsai, S. Q., Mallard, W., Joung, J., Rinn, J. L., Gnirke, A., Meissner, A. 2015; 47 (5): 469–U64

  Abstract

  DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Here we inactivated all three catalytically active DNA methyltransferases (DNMTs) in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing to further investigate the roles and genomic targets of these enzymes. Disruption of DNMT3A or DNMT3B individually as well as of both enzymes in tandem results in viable, pluripotent cell lines with distinct effects on the DNA methylation landscape, as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to findings in mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome this immediate lethality, we generated a doxycycline-responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1-mutant lines. However, doxycycline-mediated repression of exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death. Our data provide a comprehensive characterization of DNMT-mutant ESCs, including single-base genome-wide maps of the targets of these enzymes.

  View details for DOI 10.1038/ng.3258

  View details for Web of Science ID 000353635800009

  View details for PubMedID 25822089

  View details for PubMedCentralID PMC4414868

• Dissecting neural differentiation regulatory networks through epigenetic footprinting NATURE Ziller, M. J., Edri, R., Yaffe, Y., Donaghey, J., Pop, R., Mallard, W., Issner, R., Gifford, C. A., Goren, A., Xing, J., Gu, H., Cacchiarelli, D., Tsankov, A. M., Epstein, C., Rinn, J. L., Mikkelsen, T. S., Kohlbacher, O., Gnirke, A., Bernstein, B. E., Elkabetz, Y., Meissner, A. 2015; 518 (7539): 355–59

  Abstract

  Models derived from human pluripotent stem cells that accurately recapitulate neural development in vitro and allow for the generation of specific neuronal subtypes are of major interest to the stem cell and biomedical community. Notch signalling, particularly through the Notch effector HES5, is a major pathway critical for the onset and maintenance of neural progenitor cells in the embryonic and adult nervous system. Here we report the transcriptional and epigenomic analysis of six consecutive neural progenitor cell stages derived from a HES5::eGFP reporter human embryonic stem cell line. Using this system, we aimed to model cell-fate decisions including specification, expansion and patterning during the ontogeny of cortical neural stem and progenitor cells. In order to dissect regulatory mechanisms that orchestrate the stage-specific differentiation process, we developed a computational framework to infer key regulators of each cell-state transition based on the progressive remodelling of the epigenetic landscape and then validated these through a pooled short hairpin RNA screen. We were also able to refine our previous observations on epigenetic priming at transcription factor binding sites and suggest here that they are mediated by combinations of core and stage-specific factors. Taken together, we demonstrate the utility of our system and outline a general framework, not limited to the context of the neural lineage, to dissect regulatory circuits of differentiation.

  View details for DOI 10.1038/nature13990

  View details for Web of Science ID 000349547400033

  View details for PubMedID 25533951

  View details for PubMedCentralID PMC4336237

• Transcriptional and Epigenetic Dynamics during Specification of Human Embryonic Stem Cells CELL Gifford, C. A., Ziller, M. J., Gu, H., Trapnell, C., Donaghey, J., Tsankov, A., Shalek, A. K., Kelley, D. R., Shishkin, A. A., Issner, R., Zhang, X., Coyne, M., Fostel, J. L., Holmes, L., Meldrim, J., Guttman, M., Epstein, C., Park, H., Kohlbacher, O., Rinn, J., Gnirke, A., Lander, E. S., Bernstein, B. E., Meissner, A. 2013; 153 (5): 1149–63

  Abstract

  Differentiation of human embryonic stem cells (hESCs) provides a unique opportunity to study the regulatory mechanisms that facilitate cellular transitions in a human context. To that end, we performed comprehensive transcriptional and epigenetic profiling of populations derived through directed differentiation of hESCs representing each of the three embryonic germ layers. Integration of whole-genome bisulfite sequencing, chromatin immunoprecipitation sequencing, and RNA sequencing reveals unique events associated with specification toward each lineage. Lineage-specific dynamic alterations in DNA methylation and H3K4me1 are evident at putative distal regulatory elements that are frequently bound by pluripotency factors in the undifferentiated hESCs. In addition, we identified germ-layer-specific H3K27me3 enrichment at sites exhibiting high DNA methylation in the undifferentiated state. A better understanding of these initial specification events will facilitate identification of deficiencies in current approaches, leading to more faithful differentiation strategies as well as providing insights into the rewiring of human regulatory programs during cellular transitions.

  View details for DOI 10.1016/j.cell.2013.04.037

  View details for Web of Science ID 000319456800020

  View details for PubMedID 23664763

  View details for PubMedCentralID PMC3709577

• Epigenetic obstacles encountered by transcription factors: reprogramming against all odds CURRENT OPINION IN GENETICS & DEVELOPMENT Gifford, C. A., Meissner, A. 2012; 22 (5): 409–15

  Abstract

  Reprogramming of a somatic nucleus to an induced pluripotent state can be achieved in vitro through ectopic expression of Oct4 (Pou5f1), Sox2, Klf4 and c-Myc. While the ability of these factors to regulate transcription in a pluripotent context has been studied extensively, their ability to interact with and remodel a somatic genome remains underexplored. Several recent studies have begun to provide mechanistic insights that will eventually lead to a more rational design and improved understanding of nuclear reprogramming.

  View details for DOI 10.1016/j.gde.2012.08.002

  View details for Web of Science ID 000311470900003

  View details for PubMedID 22922161

  View details for PubMedCentralID PMC3490009

• Epigenomics and chromatin dynamics. Akopian, V., Chan, M. M., Clement, K., Galonska, C., Gifford, C. A., Lehtola, E., Liao, J., Samavarchi-Tehrani, P., Sindhu, C., Smith, Z. D., Tsankov, A. M., Webster, J., Zhang, Y., Ziller, M. J., Meissner, A. 2012: 313

  Abstract

  A report of the 'Joint Keystone Symposium on Epigenomics and Chromatin Dynamics', Keystone, Colorado, 17-22 January 2012.

  View details for DOI 10.1186/gb-2012-13-2-313

  View details for PubMedID 22364154

• Threonine phosphorylation post-translationally regulates protein secretion in Pseudomonas aeruginosa NATURE CELL BIOLOGY Mougous, J. D., Gifford, C. A., Ramsdell, T. L., Mekalanos, J. J. 2007; 9 (7): 797–U121

  Abstract

  Secreted proteins are crucial to the arsenal of bacterial pathogens. Although optimal activity of these proteins is likely to require precise regulation of release, the signalling events that trigger secretion are poorly understood. Here, we identify a threonine phosphorylation event that post-translationally regulates the Hcp secretion island-I-encoded type VI secretion system of Pseudomonas aeruginosa (H-T6SS). We show that a serine-threonine kinase, PpkA, is required for assembly of the H-T6SS and for secretion of Hcp1. PpkA activity is antagonized by PppA, a Ser-Thr phosphatase. These proteins exhibit reciprocal effects on the H-T6SS by acting on an FHA domain-containing protein, termed Fha1. Colocalization experiments with the T6S AAA+ family protein, ClpV1, indicate that Fha1 is a core scaffolding protein of the H-T6SS. Mutations affecting this H-T6S regulatory pathway provide a molecular explanation for the variation in Hcp1 secretion among clinical P. aeruginosa isolates. This mechanism of triggering secretion may be general, as many T6SSs contain orthologues of these proteins. Post-translational regulation of protein secretion by Thr phosphorylation is unprecedented in bacteria, and is likely to reflect the requirement for T6S to respond rapidly and reversibly to its environment.

  View details for DOI 10.1038/ncb1605

  View details for Web of Science ID 000247760700015

  View details for PubMedID 17558395

• A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus SCIENCE Mougous, J. D., Cuff, M. E., Raunser, S., Shen, A., Zhou, M., Gifford, C. A., Goodman, A. L., Joachimiak, G., Ordonez, C. L., Lory, S., Walz, T., Joachimiak, A., Mekalanos, J. J. 2006; 312 (5779): 1526–30

  Abstract

  Bacterial pathogens frequently use protein secretion to mediate interactions with their hosts. Here we found that a virulence locus (HSI-I) of Pseudomonas aeruginosa encodes a protein secretion apparatus. The apparatus assembled in discrete subcellular locations and exported Hcp1, a hexameric protein that forms rings with a 40 angstrom internal diameter. Regulatory patterns of HSI-I suggested that the apparatus functions during chronic infections. We detected Hcp1 in pulmonary secretions of cystic fibrosis (CF) patients and Hcp1-specific antibodies in their sera. Thus, HSI-I likely contributes to the pathogenesis of P. aeruginosa in CF patients. HSI-I-related loci are widely distributed among bacterial pathogens and may play a general role in mediating host interactions.

  View details for DOI 10.1126/science.1128393

  View details for Web of Science ID 000238124100049

  View details for PubMedID 16763151

  View details for PubMedCentralID PMC2800167