Bio


William Greenleaf is an Associate Professor in the Genetics Department at Stanford University School of Medicine, with a courtesy appointment in the Applied Physics Department. He is a member of Bio-X, the Biophysics Program, the Biomedical Informatics Program, and the Cancer Center. He received an A.B. in physics from Harvard University (summa cum laude) in 2002, and received a Gates Fellowship to study computer science for one year in Trinity College, Cambridge, UK (with distinction). After this experience abroad, he returned to Stanford to carry out his Ph.D. in Applied Physics in the laboratory of Steven Block, where he investigated, at the single molecule level, the chemo-mechanics of RNA polymerase and the folding of RNA transcripts. He conducted postdoctoral work in the laboratory of X. Sunney Xie in the Chemistry and Chemical Biology Department at Harvard University, where he was awarded a Damon Runyon Cancer Research Foundation Fellowship, and developed new fluorescence-based high-throughput sequencing methodologies. He moved to Stanford as an Assistant Professor in November 2011. Since beginning his lab, he has been named a Rita Allen Foundation Young Scholar, an Ellison Foundation Young Scholar in Aging (declined), a Baxter Foundation Scholar, and a Chan-Zuckerberg Investigator. His highly interdisciplinary research links molecular biology, computer science, bioengineering, and genomics a to understand how the physical state of the human genome controls gene regulation and biological state. Efforts in his lab are split between building new tools to leverage the power of high-throughput sequencing and cutting-edge microscopies, and bringing these new technologies to bear against basic biological questions of genomic and epigenomic variation. His long-term goal is to unlock an understanding of the physical “regulome” — i.e. the factors that control how the genetic information is read into biological instructions — profoundly impacting our understanding of how cells maintain, or fail to maintain, their state in health and disease.

Academic Appointments


Honors & Awards


  • Chan-Zuckerberg Fellow, Chan-Zuckerberg Foundation (2017-)
  • Baxter Foundation Faculty Fellow, Baxter Foundation (2014)
  • Rita Allen Scholar, Rita Allen Foundation (2011-)
  • Damon Runyon Cancer Research Fellowship, Damon Runyon Foundation (2009-2011)
  • ARCS Fellowship, ARCS (2006)
  • Graduate Fellowship, National Science Foundation (2003-2005)
  • Gates Cambridge Trust Scholar, Gates Foundation (2002-2003)

Professional Education


  • Postdoctoral Fellow, Harvard University, Chemistry and Chemical Biology
  • PhD, Stanford University, Applied Physics (2008)
  • Dip Comp Sci, Trinity College, Cambridge University, UK, Computer Science (2003)
  • AB, Harvard University, Physics (2002)

Current Research and Scholarly Interests


Our lab focuses on developing methods to probe both the structure and function of molecules encoded by the genome, as well as the physical compaction and folding of the genome itself. Our efforts are split between building new tools to leverage the power of high-throughput sequencing technologies and cutting-edge optical microscopies, and bringing these technologies to bear against basic biological questions by linking DNA sequence, structure, and function.

2018-19 Courses


Stanford Advisees


Graduate and Fellowship Programs


All Publications


  • Epigenetic signature of PD-1+ TCF1+ CD8 T cells that act as resource cells during chronic viral infection and respond to PD-1 blockade. Proceedings of the National Academy of Sciences of the United States of America Jadhav, R. R., Im, S. J., Hu, B., Hashimoto, M., Li, P., Lin, J., Leonard, W. J., Greenleaf, W. J., Ahmed, R., Goronzy, J. J. 2019

    Abstract

    We have recently defined a novel population of PD-1 (programmed cell death 1)+ TCF1 (T cell factor 1)+ virus-specific CD8 T cells that function as resource cells during chronic LCMV infection and provide the proliferative burst seen after PD-1 blockade. Such CD8 T cells have been found in other chronic infections and also in cancer in mice and humans. These CD8 T cells exhibit stem-like properties undergoing self-renewal and also differentiating into the terminally exhausted CD8 T cells. Here we compared the epigenetic signature of stem-like CD8 T cells with exhausted CD8 T cells. ATAC-seq analysis showed that stem-like CD8 T cells had a unique signature implicating activity of HMG (TCF) and RHD (NF-kappaB) transcription factor family members in contrast to higher accessibility to ETS and RUNX motifs in exhausted CD8 T cells. In addition, regulatory regions of the transcription factors Tcf7 and Id3 were more accessible in stem-like cells whereas Prdm1 and Id2 were more accessible in exhausted CD8 T cells. We also compared the epigenetic signatures of the 2 CD8 T cell subsets from chronically infected mice with effector and memory CD8 T cells generated after an acute LCMV infection. Both CD8 T cell subsets generated during chronic infection were strikingly different from CD8 T cell subsets from acute infection. Interestingly, the stem-like CD8 T cell subset from chronic infection, despite sharing key functional properties with memory CD8 T cells, had a very distinct epigenetic program. These results show that the chronic stem-like CD8 T cell program represents a specific adaptation of the T cell response to persistent antigenic stimulation.

    View details for DOI 10.1073/pnas.1903520116

    View details for PubMedID 31227606

  • Demonstration of protein cooperativity mediated by RNA structure using the human protein PUM2 RNA Becker, W. R., Jarmoskaite, I., Vaidyanathan, P. P., Greenleaf, W. J., Herschlag, D. 2019; 25 (6): 702–12
  • Cell cycle dynamics of human pluripotent stem cells primed for differentiation. Stem cells (Dayton, Ohio) Shcherbina, A., Li, J., Narayanan, C., Greenleaf, W., Kundaje, A., Chetty, S. 2019

    Abstract

    Understanding the molecular properties of the cell cycle of human pluripotent stem cells (hPSCs) is critical for effectively promoting differentiation. Here, we use the Fluorescence Ubiquitin Cell Cycle Indicator (FUCCI) system adapted into hPSCs and perform RNA-sequencing on cell cycle sorted hPSCs primed and unprimed for differentiation. Gene expression patterns of signaling factors and developmental regulators change in a cell cycle-specific manner in cells primed for differentiation without altering genes associated with pluripotency. Furthermore, we identify an important role for PI3K signaling in regulating the early transitory states of hPSCs toward differentiation. SIGNIFICANCE STATEMENT: Generating differentiated cell types from human pluripotent stem cells (hPSCs) holds great therapeutic promise, but has proven to be challenging in practice. The cell cycle may play an important role in enhancing the differentiation potential of hPSCs. Here, the authors track and isolate hPSCs from different phases of the cell cycle and perform RNA-sequencing. The data show that gene expression patterns of signaling factors and developmental regulators change in a cell cycle-specific manner as hPSCs transition toward differentiation and highlight an important role for PI3K signaling in regulating these early transitory states. © AlphaMed Press 2019.

    View details for DOI 10.1002/stem.3041

    View details for PubMedID 31135093

  • HiChIRP reveals RNA-associated chromosome conformation. Nature methods Mumbach, M. R., Granja, J. M., Flynn, R. A., Roake, C. M., Satpathy, A. T., Rubin, A. J., Qi, Y., Jiang, Z., Shams, S., Louie, B. H., Guo, J. K., Gennert, D. G., Corces, M. R., Khavari, P. A., Atianand, M. K., Artandi, S. E., Fitzgerald, K. A., Greenleaf, W. J., Chang, H. Y. 2019

    Abstract

    Modular domains of long non-coding RNAs can serve as scaffolds to bring distant regions of the linear genome into spatial proximity. Here, we present HiChIRP, a method leveraging bio-orthogonal chemistry and optimized chromosome conformation capture conditions, which enables interrogation of chromatin architecture focused around a specific RNA of interest down to approximately ten copies per cell. HiChIRP of three nuclear RNAs reveals insights into promoter interactions (7SK), telomere biology (telomerase RNA component) and inflammatory gene regulation (lincRNA-EPS).

    View details for DOI 10.1038/s41592-019-0407-x

    View details for PubMedID 31133759

  • Blind tests of RNA-protein binding affinity prediction. Proceedings of the National Academy of Sciences of the United States of America Kappel, K., Jarmoskaite, I., Vaidyanathan, P. P., Greenleaf, W. J., Herschlag, D., Das, R. 2019

    Abstract

    Interactions between RNA and proteins are pervasive in biology, driving fundamental processes such as protein translation and participating in the regulation of gene expression. Modeling the energies of RNA-protein interactions is therefore critical for understanding and repurposing living systems but has been hindered by complexities unique to RNA-protein binding. Here, we bring together several advances to complete a calculation framework for RNA-protein binding affinities, including a unified free energy function for bound complexes, automated Rosetta modeling of mutations, and use of secondary structure-based energetic calculations to model unbound RNA states. The resulting Rosetta-Vienna RNP-DeltaDeltaG method achieves root-mean-squared errors (RMSEs) of 1.3 kcal/mol on high-throughput MS2 coat protein-RNA measurements and 1.5 kcal/mol on an independent test set involving the signal recognition particle, human U1A, PUM1, and FOX-1. As a stringent test, the method achieves RMSE accuracy of 1.4 kcal/mol in blind predictions of hundreds of human PUM2-RNA relative binding affinities. Overall, these RMSE accuracies are significantly better than those attained by prior structure-based approaches applied to the same systems. Importantly, Rosetta-Vienna RNP-DeltaDeltaG establishes a framework for further improvements in modeling RNA-protein binding that can be tested by prospective high-throughput measurements on new systems.

    View details for PubMedID 30962376

  • Demonstration of Protein Cooperativity Mediated by RNA Structure Using the Human Protein PUM2. RNA (New York, N.Y.) Becker, W. R., Jarmoskaite, I., Vaidyanathan, P. P., Greenleaf, W. J., Herschlag, D. 2019

    Abstract

    Post-translational gene regulation requires a complex network of RNA/protein interactions. Cooperativity, which tunes response sensitivities, originates from protein-protein interactions in many systems. For RNA binding proteins, cooperativity can also be mediated through RNA structure. RNA structural cooperativity (RSC) arises when binding of one protein induces a redistribution of RNA conformational states that enhance access (positive cooperativity) or block access (negative cooperativity) to additional binding sites. As RSC does not require direct protein-protein interactions, it allows cooperativity to be tuned for individual RNAs, via alterations in sequence that alter structural stability. Given the potential importance of this mechanism of control and our desire to quantitatively dissect features that underlie physiological regulation, we developed a statistical mechanical framework for RSC, and tested this model by performing equilibrium binding measurements of the human PUF family protein PUM2. Using 68 RNAs that contain 2-5 PUM2 binding sites and RNA structures of varying stabilities, we observed a range of structure-dependent cooperative behaviors. To test our ability to account for this cooperativity with known physical constants, we used PUM2 affinity and nearest-neighbor RNA secondary structure predictions. Our model gave qualitative agreement for our disparate set of 68 RNAs across two temperatures, but quantitative deviations arise from overestimation of RNA structural stability. Our results demonstrate cooperativity mediated by RNA structure and underscore the power of quantitative stepwise experimental evaluation of mechanisms and computational tools.

    View details for PubMedID 30914482

  • Large-Scale, Quantitative Protein Assays on a High-Throughput DNA Sequencing Chip. Molecular cell Layton, C. J., McMahon, P. L., Greenleaf, W. J. 2019; 73 (5): 1075

    Abstract

    High-throughput DNA sequencing techniques haveenabled diverse approaches for linking DNA sequence to biochemical function. In contrast, assays of protein function have substantial limitations in terms of throughput, automation, and widespread availability. We have adapted an Illumina high-throughput sequencing chip to display an immense diversity of ribosomally translated proteins and peptides and then carried out fluorescence-based functional assays directly on this flow cell, demonstrating that a single, widely available high-throughput platform can perform both sequencing-by-synthesis and protein assays. We quantified the binding of the M2 anti-FLAG antibody to a library of 1.3* 104 variant FLAG peptides, exploring non-additive effects of combinations of mutations and discovering a "superFLAG" epitope variant. We also measured the enzymatic activity of 1.56* 105 molecular variants of full-length human O6-alkylguanine-DNA alkyltransferase (SNAP-tag). This comprehensive corpus of catalytic rates revealed amino acid interactionnetworks and cooperativity, linked positive cooperativity to structural proximity, and revealed ubiquitous positively cooperative interactions with histidine residues.

    View details for PubMedID 30849388

  • Chromatin accessibility and the regulatory epigenome. Nature reviews. Genetics Klemm, S. L., Shipony, Z., Greenleaf, W. J. 2019

    Abstract

    Physical access to DNA is a highly dynamic property of chromatin that plays an essential role in establishing and maintaining cellular identity. The organization of accessible chromatin across the genome reflects a network of permissible physical interactions through which enhancers, promoters, insulators and chromatin-binding factors cooperatively regulate gene expression. This landscape of accessibility changes dynamically in response to both external stimuli and developmental cues, and emerging evidence suggests that homeostatic maintenance of accessibility is itself dynamically regulated through a competitive interplay between chromatin-binding factors and nucleosomes. In this Review, we examine how the accessible genome is measured and explore the role of transcription factors in initiating accessibility remodelling; our goal is to illustrate how chromatin accessibility defines regulatory elements within the genome and how these epigenetic features are dynamically established to control gene expression.

    View details for PubMedID 30675018

  • Satb1 integrates DNA binding site geometry and torsional stress to differentially target nucleosome-dense regions. Nature communications Ghosh, R. P., Shi, Q., Yang, L., Reddick, M. P., Nikitina, T., Zhurkin, V. B., Fordyce, P., Stasevich, T. J., Chang, H. Y., Greenleaf, W. J., Liphardt, J. T. 2019; 10 (1): 3221

    Abstract

    The Satb1 genome organizer regulates multiple cellular and developmental processes. It is not yet clear how Satb1 selects different sets of targets throughout the genome. Here we have used live-cell single molecule imaging and deep sequencing to assess determinants of Satb1 binding-site selectivity. We have found that Satb1 preferentially targets nucleosome-dense regions and can directly bind consensus motifs within nucleosomes. Some genomic regions harbor multiple, regularly spaced Satb1 binding motifs (typical separation ~1 turn of the DNA helix) characterized by highly cooperative binding. The Satb1 homeodomain is dispensable for high affinity binding but is essential for specificity. Finally, we find that Satb1-DNA interactions are mechanosensitive. Increasing negative torsional stress in DNA enhances Satb1 binding and Satb1 stabilizes base unpairing regions against melting by molecular machines. The ability of Satb1 to control diverse biological programs may reflect its ability to combinatorially use multiple site selection criteria.

    View details for DOI 10.1038/s41467-019-11118-8

    View details for PubMedID 31324780

  • OPEN CHROMATIN PROFILING IDENTIFIES FUNCTIONAL NONCODING RISK VARIANTS IN HUMAN IPSC MODEL OF PSYCHIATRIC DISORDERS Duan, J., Forrest, M., Zhang, H., Moy, W., McGowan, H., Leites, C., Shi, J., Sanders, A., Greenleaf, W., Cowan, C., Pang, Z., Gejman, P., Penzes, P. ELSEVIER SCIENCE BV. 2019: S765
  • High-Throughput Analysis Reveals Rules for Target RNA Binding and Cleavage by AGO2. Molecular cell Becker, W. R., Ober-Reynolds, B., Jouravleva, K., Jolly, S. M., Zamore, P. D., Greenleaf, W. J. 2019

    Abstract

    Argonaute proteins loaded with microRNAs (miRNAs) or small interfering RNAs (siRNAs) form the RNA-induced silencing complex (RISC), which represses target RNA expression. Predicting the biological targets, specificity, and efficiency of both miRNAs and siRNAs has been hamstrung by an incomplete understanding of the sequence determinants of RISC binding and cleavage. We applied high-throughput methods to measure the association kinetics, equilibrium binding energies, and single-turnover cleavage rates of RISC. We find that RISC readily tolerates insertions of up to 7 nt in its target opposite the central region of the guide. Our data uncover specific guide:target mismatches that enhance the rate of target cleavage, suggesting novel siRNA design strategies. Using these data, we derive quantitative models for RISC binding and target cleavage and show that our in vitro measurements and models predict knockdown in an engineered cellular system.

    View details for DOI 10.1016/j.molcel.2019.06.012

    View details for PubMedID 31324449

  • A Quantitative and Predictive Model for RNA Binding by Human Pumilio Proteins. Molecular cell Jarmoskaite, I., Denny, S. K., Vaidyanathan, P. P., Becker, W. R., Andreasson, J. O., Layton, C. J., Kappel, K., Shivashankar, V., Sreenivasan, R., Das, R., Greenleaf, W. J., Herschlag, D. 2019

    Abstract

    High-throughput methodologies have enabled routine generation of RNA target sets and sequence motifs for RNA-binding proteins (RBPs). Nevertheless, quantitative approaches are needed to capture the landscape of RNA-RBP interactions responsible for cellular regulation. We have used the RNA-MaP platform to directly measure equilibrium binding for thousands of designed RNAs and to construct a predictive model for RNA recognition by the human Pumilio proteins PUM1 and PUM2. Despite prior findings of linear sequence motifs, our measurements revealed widespread residue flipping and instances of positional coupling. Application of our thermodynamic model to published in vivo crosslinking data reveals quantitative agreement between predicted affinities and in vivo occupancies. Our analyses suggest a thermodynamically driven, continuous Pumilio-binding landscape that is negligibly affected by RNA structure or kinetic factors, such as displacement by ribosomes. This work provides a quantitative foundation for dissecting the cellular behavior of RBPs and cellular features that impact their occupancies.

    View details for PubMedID 31078383

  • High-resolution mapping of cancer cell networks using co-functional interactions. Molecular systems biology Boyle, E. A., Pritchard, J. K., Greenleaf, W. J. 2018; 14 (12): e8594

    Abstract

    Powerful new technologies for perturbing genetic elements have recently expanded the study of genetic interactions in model systems ranging from yeast to human cell lines. However, technical artifacts can confound signal across genetic screens and limit the immense potential of parallel screening approaches. To address this problem, we devised a novel PCA-based method for correcting genome-wide screening data, bolstering the sensitivity and specificity of detection for genetic interactions. Applying this strategy to a set of 436 whole genome CRISPR screens, we report more than 1.5 million pairs of correlated "co-functional" genes that provide finer-scale information about cell compartments, biological pathways, and protein complexes than traditional gene sets. Lastly, we employed a gene community detection approach to implicate core genes for cancer growth and compress signal from functionally related genes in the same community into a single score. This work establishes new algorithms for probing cancer cell networks and motivates the acquisition of further CRISPR screen data across diverse genotypes and cell types to further resolve complex cellular processes.

    View details for PubMedID 30573688

  • Coupled Single-Cell CRISPR Screening and Epigenomic Profiling Reveals Causal Gene Regulatory Networks. Cell Rubin, A. J., Parker, K. R., Satpathy, A. T., Qi, Y., Wu, B., Ong, A. J., Mumbach, M. R., Ji, A. L., Kim, D. S., Cho, S. W., Zarnegar, B. J., Greenleaf, W. J., Chang, H. Y., Khavari, P. A. 2018

    Abstract

    Here, we present Perturb-ATAC, a method that combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in 4,300 single cells, encompassing more than 63 genotype-phenotype relationships. Perturb-ATAC in human Blymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning and identified a hierarchy of TFs that govern B cell state, variation, and disease-associated cis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules of cis-elements that specify keratinocyte fate. Combinatorial deletion of all pairs of these TFsuncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful strategy to dissect gene regulatory networks in development and disease.

    View details for PubMedID 30580963

  • Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules. Cell stem cell Zviran, A., Mor, N., Rais, Y., Gingold, H., Peles, S., Chomsky, E., Viukov, S., Buenrostro, J. D., Scognamiglio, R., Weinberger, L., Manor, Y. S., Krupalnik, V., Zerbib, M., Hezroni, H., Jaitin, D. A., Larastiaso, D., Gilad, S., Benjamin, S., Gafni, O., Mousa, A., Ayyash, M., Sheban, D., Bayerl, J., Aguilera-Castrejon, A., Massarwa, R., Maza, I., Hanna, S., Stelzer, Y., Ulitsky, I., Greenleaf, W. J., Tanay, A., Trumpp, A., Amit, I., Pilpel, Y., Novershtern, N., Hanna, J. H. 2018

    Abstract

    The epigenetic dynamics of induced pluripotent stem cell (iPSC) reprogramming in correctly reprogrammed cells at high resolution and throughout the entire process remain largely undefined. Here, we characterize conversion of mouse fibroblasts into iPSCs using Gatad2a-Mbd3/NuRD-depleted and highly efficient reprogramming systems. Unbiased high-resolution profiling of dynamic changes in levels of gene expression, chromatin engagement, DNA accessibility, and DNA methylation were obtained. We identified two distinct and synergistic transcriptional modules that dominate successful reprogramming, which are associated with cell identity and biosynthetic genes. The pluripotency module is governed by dynamic alterations in epigenetic modifications to promoters and binding by Oct4, Sox2, and Klf4, but not Myc. Early DNA demethylation at certain enhancers prospectively marks cells fated to reprogram. Myc activity drives expression of the essential biosynthetic module and is associated with optimized changes in tRNA codon usage. Our functional validations highlight interweaved epigenetic- and Myc-governed essential reconfigurations that rapidly commission and propel deterministic reprogramming toward naive pluripotency.

    View details for PubMedID 30554962

  • Identification of phagocytosis regulators using magnetic genome-wide CRISPR screens. Nature genetics Haney, M. S., Bohlen, C. J., Morgens, D. W., Ousey, J. A., Barkal, A. A., Tsui, C. K., Ego, B. K., Levin, R., Kamber, R. A., Collins, H., Tucker, A., Li, A., Vorselen, D., Labitigan, L., Crane, E., Boyle, E., Jiang, L., Chan, J., Rincon, E., Greenleaf, W. J., Li, B., Snyder, M. P., Weissman, I. L., Theriot, J. A., Collins, S. R., Barres, B. A., Bassik, M. C. 2018

    Abstract

    Phagocytosis is required for a broad range of physiological functions, from pathogen defense to tissue homeostasis, but the mechanisms required for phagocytosis of diverse substrates remain incompletely understood. Here, we developed a rapid magnet-based phenotypic screening strategy, and performed eight genome-wide CRISPR screens in human cells to identify genes regulating phagocytosis of distinct substrates. After validating select hits in focused miniscreens, orthogonal assays and primary human macrophages, we show that (1) the previously uncharacterized gene NHLRC2 is a central player in phagocytosis, regulating RhoA-Rac1 signaling cascades that control actin polymerization and filopodia formation, (2) very-long-chain fatty acids are essential for efficient phagocytosis of certain substrates and (3) the previously uncharacterized Alzheimer's disease-associated gene TM2D3 can preferentially influence uptake of amyloid-beta aggregates. These findings illuminate new regulators and core principles of phagocytosis, and more generally establish an efficient method for unbiased identification of cellular uptake mechanisms across diverse physiological and pathological contexts.

    View details for PubMedID 30397336

  • Joint single-cell DNA accessibility and protein epitope profiling reveals environmental regulation of epigenomic heterogeneity. Nature communications Chen, X., Litzenburger, U. M., Wei, Y., Schep, A. N., LaGory, E. L., Choudhry, H., Giaccia, A. J., Greenleaf, W. J., Chang, H. Y. 2018; 9 (1): 4590

    Abstract

    Here we introduce Protein-indexed Assay of Transposase Accessible Chromatin with sequencing (Pi-ATAC) that combines single-cell chromatin and proteomic profiling. In conjunction with DNA transposition, the levels of multiple cell surface or intracellular protein epitopes are recorded by index flow cytometry and positions in arrayed microwells, and then subject to molecular barcoding for subsequent pooled analysis. Pi-ATAC simultaneously identifies the epigenomic and proteomic heterogeneity in individual cells. Pi-ATAC reveals a casual link between transcription factor abundance and DNA motif access, and deconvolute cell types and states in the tumor microenvironment in vivo. We identify a dominant role for hypoxia, marked by HIF1alpha protein, in the tumor microvenvironment for shaping the regulome in a subset of epithelial tumor cells.

    View details for PubMedID 30389926

  • Linking RNA Sequence, Structure, and Function on Massively Parallel High-Throughput Sequencers. Cold Spring Harbor perspectives in biology Denny, S. K., Greenleaf, W. J. 2018

    Abstract

    SUMMARYHigh-throughput sequencing methods have revolutionized our ability to catalog the diversity of RNAs and RNA-protein interactions that can exist in our cells. However, the relationship between RNA sequence, structure, and function is enormously complex, demonstrating the need for methods that can provide quantitative thermodynamic and kinetic measurements of macromolecular interaction with RNA, at a scale commensurate with the sequence diversity of RNA. Here, we discuss a class of methods that extend the core functionality of DNA sequencers to enable high-throughput measurements of RNA folding and RNA-protein interactions. Topics discussed include a description of the method and multiple applications to RNA-binding proteins, riboswitch design and engineering, and RNA tertiary structure energetics.

    View details for PubMedID 30322887

  • Intertumoral Heterogeneity in SCLC Is Influenced by the Cell Type of Origin. Cancer discovery Yang, D., Denny, S. K., Greenside, P. G., Chaikovsky, A. C., Brady, J. J., Ouadah, Y., Granja, J. M., Jahchan, N. S., Lim, J. S., Kwok, S., Kong, C. S., Berghoff, A. S., Schmitt, A., Reinhardt, H. C., Park, K., Preusser, M., Kundaje, A., Greenleaf, W. J., Sage, J., Winslow, M. M. 2018

    Abstract

    The extent to which early events shape tumor evolution is largely uncharacterized, even though a better understanding of these early events may help identify key vulnerabilities in advanced tumors. Here, using genetically defined mouse models of small cell lung cancer (SCLC), we uncovered distinct metastatic programs attributable to the cell type of origin. In one model, tumors gain metastatic ability through amplification of the transcription factor NFIB and a widespread increase in chromatin accessibility, whereas in the other model, tumors become metastatic in the absence of NFIB-driven chromatin alterations. Gene-expression and chromatin accessibility analyses identify distinct mechanisms as well as markers predictive of metastatic progression in both groups. Underlying the difference between the two programs was the cell type of origin of the tumors, with NFIB-independent metastases arising from mature neuroendocrine cells. Our findings underscore the importance of the identity of cell type of origin in influencing tumor evolution and metastatic mechanisms.SIGNIFICANCE: We show that SCLC can arise from different cell types of origin, which profoundly influences the eventual genetic and epigenetic changes that enable metastatic progression. Understanding intertumoral heterogeneity in SCLC, and across cancer types, may illuminate mechanisms of tumor progression and uncover how the cell type of origin affects tumor evolution. Cancer Discov; 8(10); 1-16. ©2018 AACR.See related commentary by Pozo et al., p. 1216.

    View details for PubMedID 30228179

  • High-throughput chromatin accessibility profiling at single-cell resolution. Nature communications Mezger, A., Klemm, S., Mann, I., Brower, K., Mir, A., Bostick, M., Farmer, A., Fordyce, P., Linnarsson, S., Greenleaf, W. 2018; 9 (1): 3647

    Abstract

    Here we develop a high-throughput single-cell ATAC-seq (assay for transposition of accessible chromatin) method to measure physical access to DNA in whole cells. Our approach integrates fluorescence imaging and addressable reagent deposition across a massively parallel (5184) nano-well array, yielding a nearly 20-fold improvement in throughput (up to ~1800 cells/chip, 4-5h on-chip processing time) and library preparationcost (~81 per cell) compared to prior microfluidic implementations. We apply this method to measure regulatory variation in peripheral blood mononuclear cells (PBMCs) and show robust, de novo clustering of single cells by hematopoietic cell type.

    View details for PubMedID 30194434

  • Neutralizing Gatad2a-Chd4-Mbd3/NuRD Complex Facilitates Deterministic Induction of Naive Pluripotency CELL STEM CELL Mor, N., Rais, Y., Sheban, D., Peles, S., Aguilera-Castrejon, A., Zviran, A., Elinger, D., Viukov, S., Geula, S., Krupalnik, V., Zerbib, M., Chomsky, E., Lasman, L., Shani, T., Bayerl, J., Gafni, O., Hanna, S., Buenrostro, J. D., Hagai, T., Masika, H., Vainorius, G., Bergman, Y., Greenleaf, W. J., Esteban, M. A., Elling, U., Levin, Y., Massarwa, R., Merbl, Y., Novershtern, N., Hanna, J. H. 2018; 23 (3): 412-+

    Abstract

    Mbd3, a member of nucleosome remodeling and deacetylase (NuRD) co-repressor complex, was previously identified as an inhibitor for deterministic induced pluripotent stem cell (iPSC) reprogramming, where up to 100% of donor cells successfully complete the process. NuRD can assume multiple mutually exclusive conformations, and it remains unclear whether this deterministic phenotype can be attributed to a specific Mbd3/NuRD subcomplex. Moreover, since complete ablation of Mbd3 blocks somatic cell proliferation, we aimed to explore functionally relevant alternative ways to neutralize Mbd3-dependent NuRD activity. We identify Gatad2a, a NuRD-specific subunit, whose complete deletion specifically disrupts Mbd3/NuRD repressive activity on the pluripotency circuitry during iPSC differentiation and reprogramming without ablating somatic cell proliferation. Inhibition of Gatad2a facilitates deterministic murine iPSC reprogramming within 8 days. We validate a distinct molecular axis, Gatad2a-Chd4-Mbd3, within Mbd3/NuRD as being critical for blocking reestablishment of naive pluripotency and further highlight signaling-dependent and post-translational modifications of Mbd3/NuRD that influence its interactions and assembly.

    View details for PubMedID 30122475

  • A Chromatin Basis for Cell Lineage and Disease Risk in the Human Pancreas. Cell systems Arda, H. E., Tsai, J., Rosli, Y. R., Giresi, P., Bottino, R., Greenleaf, W. J., Chang, H. Y., Kim, S. K. 2018

    Abstract

    Understanding the genomic logic that underlies cellular diversity and developmental potential in the human pancreas will accelerate the growth of cell replacement therapies and reveal genetic risk mechanisms in diabetes. Here, we identified and characterized thousands of chromatin regions governing cell-specific gene regulation in human pancreatic endocrine and exocrine lineages, including islet betacells, alpha cells, duct, and acinar cells. Our findings have captured cellular ontogenies at the chromatin level, identified lineage-specific regulators potentially acting on these sites, and uncovered hallmarks of regulatory plasticity between cell types that suggest mechanisms to regenerate beta cells from pancreatic endocrine or exocrine cells. Our work shows that disease risk variants related to pancreas are significantly enriched in these regulatory regions and reveals previously unrecognized links between endocrine and exocrine pancreas in diabetes risk.

    View details for PubMedID 30145115

  • Discovery of stimulation-responsive immune enhancers with CRISPR activation (vol 549, pg 111, 2017) NATURE Simeonov, D. R., Gowen, B. G., Boontanrart, M., Roth, T. L., Gagnon, J. D., Mumbach, M. R., Satpathy, A. T., Lee, Y., Bray, N. L., Chan, A. Y., Lituiev, D. S., Nguyen, M. L., Gate, R. E., Subramaniam, M., Li, Z., Woo, J. M., Mitros, T., Ray, G. J., Curie, G. L., Naddaf, N., Chu, J. S., Ma, H., Boyer, E., Van Gool, F., Huang, H., Liu, R., Tobin, V. R., Schumann, K., Daly, M. J., Farh, K. K., Ansel, K., Ye, C. J., Greenleaf, W. J., Anderson, M. S., Bluestone, J. A., Chang, H. Y., Corn, J. E., Marson, A. 2018; 559 (7715): E13

    Abstract

    In this Letter, analysis of steady-state regulatory T (Treg) cell percentages from Il2ra enhancer deletion (EDEL) and wild-type (WT) mice revealed no differences between them (Extended Data Fig. 9d). This analysis included two mice whose genotypes were incorrectly assigned. Even after correction of the genotypes, no significant differences in Treg cell percentages were seen when data across experimental cohorts were averaged (as was done in Extended Data Fig. 9d). However, if we normalize the corrected data to account for variation among experimental cohorts, a subtle decrease in EDEL Treg cell percentages is revealed and, using the corrected and normalized data, we have redrawn Extended Data Fig. 9d in Supplementary Fig. 1. The Supplementary Information to this Amendment contains the corrected and reanalysed Extended Data Fig. 9d. The sentence "This enhancer deletion (EDEL) strain also had no obvious T cell phenotypes at steady state (Extended Data Fig. 9)." should read: "This enhancer deletion (EDEL) strain had a small decrease in the percentage of Treg cells (Extended Data Fig. 9).". This error does not affect any of the main figures in the Letter or the data from mice with the human autoimmune-associated single nucleotide polymorphism (SNP) knocked in or with a 12-base-pair deletion at the site (12DEL). In addition, we stated in the Methods that we observed consistent immunophenotypes of EDEL mice across three founders, but in fact, we observed consistent phenotypes in mice from two founders. This does not change any of our conclusions and the original Letter has not been corrected.

    View details for PubMedID 29899441

  • High-Throughput Investigation of Diverse Junction Elements in RNA Tertiary Folding. Cell Denny, S. K., Bisaria, N., Yesselman, J. D., Das, R., Herschlag, D., Greenleaf, W. J. 2018

    Abstract

    RNAs fold into defined tertiary structures to function in critical biological processes. While quantitative models can predict RNA secondary structure stability, we are still unable to predict the thermodynamic stability of RNA tertiary structure. Here, we probe conformational preferences of diverse RNA two-way junctions to develop a predictive model for the formation of RNA tertiary structure. We quantitatively measured tertiary assembly energetics of >1,000 of RNA junctions inserted in multiple structural scaffolds to generate a "thermodynamic fingerprint" for each junction. Thermodynamic fingerprints enabled comparison of junction conformational preferences, revealing principles for how sequence influences 3-dimensional conformations. Utilizing fingerprints of junctions with known crystal structures, we generated ensembles for related junctions that predicted their thermodynamic effects on assembly formation. This work reveals sequence-structure-energeticrelationships in RNA, demonstrates the capacity fordiverse compensation strategies within tertiary structures, and provides a path to quantitative modeling of RNA folding energetics based on "ensemble modularity."

    View details for PubMedID 29961580

  • Unsupervised clustering and epigenetic classification of single cells NATURE COMMUNICATIONS Zamanighomi, M., Lin, Z., Daley, T., Chen, X., Duren, Z., Schep, A., Greenleaf, W. J., Wong, W. 2018; 9: 2410

    Abstract

    Characterizing epigenetic heterogeneity at the cellular level is a critical problem in the modern genomics era. Assays such as single cell ATAC-seq (scATAC-seq) offer an opportunity to interrogate cellular level epigenetic heterogeneity through patterns of variability in open chromatin. However, these assays exhibit technical variability that complicates clear classification and cell type identification in heterogeneous populations. We present scABC, an R package for the unsupervised clustering of single-cell epigenetic data, to classify scATAC-seq data and discover regions of open chromatin specific to cell identity.

    View details for PubMedID 29925875

  • Integrated Single-Cell Analysis Maps the Continuous Regulatory Landscape of Human Hematopoietic Differentiation CELL Buenrostro, J. D., Corces, M., Lareau, C. A., Wu, B., Schep, A. N., Aryee, M. J., Majeti, R., Chang, H. Y., Greenleaf, W. J. 2018; 173 (6): 1535-+

    Abstract

    Human hematopoiesis involves cellular differentiation of multipotent cells into progressively more lineage-restricted states. While the chromatin accessibility landscape of this process has been explored in defined populations, single-cell regulatory variation has been hidden by ensemble averaging. We collected single-cell chromatin accessibility profiles across 10 populations of immunophenotypically defined human hematopoietic cell types and constructed a chromatin accessibility landscape of human hematopoiesis to characterize differentiation trajectories. We find variation consistent with lineage bias toward different developmental branches in multipotent cell types. We observe heterogeneity within common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) and develop a strategy to partition GMPs along their differentiation trajectory. Furthermore, we integrated single-cell RNA sequencing (scRNA-seq) data to associate transcription factors to chromatin accessibility changes and regulatory elements to target genes through correlations of expression and regulatory element accessibility. Overall, this work provides a framework for integrative exploration of complex regulatory dynamics in a primary human tissue at single-cell resolution.

    View details for PubMedID 29706549

  • Diff-seq: A high throughput sequencing-based mismatch detection assay for DNA variant enrichment and discovery NUCLEIC ACIDS RESEARCH Aggeli, D., Karas, V. O., Sinnott-Armstrong, N. A., Varghese, V., Shafer, R. W., Greenleaf, W. J., Sherlock, G. 2018; 46 (7)

    Abstract

    Much of the within species genetic variation is in the form of single nucleotide polymorphisms (SNPs), typically detected by whole genome sequencing (WGS) or microarray-based technologies. However, WGS produces mostly uninformative reads that perfectly match the reference, while microarrays require genome-specific reagents. We have developed Diff-seq, a sequencing-based mismatch detection assay for SNP discovery without the requirement for specialized nucleic-acid reagents. Diff-seq leverages the Surveyor endonuclease to cleave mismatched DNA molecules that are generated after cross-annealing of a complex pool of DNA fragments. Sequencing libraries enriched for Surveyor-cleaved molecules result in increased coverage at the variant sites. Diff-seq detected all mismatches present in an initial test substrate, with specific enrichment dependent on the identity and context of the variation. Application to viral sequences resulted in increased observation of variant alleles in a biologically relevant context. Diff-Seq has the potential to increase the sensitivity and efficiency of high-throughput sequencing in the detection of variation.

    View details for PubMedID 29361139

    View details for PubMedCentralID PMC5909455

  • Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts ELIFE Bell, J. C., Jukam, D., Teran, N. A., Risca, V. I., Smith, O. K., Johnson, W. L., Skotheim, J. M., Greenleaf, W., Straight, A. F. 2018; 7

    Abstract

    RNA is a critical component of chromatin in eukaryotes, both as a product of transcription, and as an essential constituent of ribonucleoprotein complexes that regulate both local and global chromatin states. Here, we present a proximity ligation and sequencing method called Chromatin-Associated RNA sequencing (ChAR-seq) that maps all RNA-to-DNA contacts across the genome. Using Drosophila cells, we show that ChAR-seq provides unbiased, de novo identification of targets of chromatin-bound RNAs including nascent transcripts, chromosome-specific dosage compensation ncRNAs, and genome-wide trans-associated RNAs involved in co-transcriptional RNA processing.

    View details for PubMedID 29648534

  • Rapid chromatin repression by Aire provides precise control of immune tolerance NATURE IMMUNOLOGY Koh, A. S., Miller, E. L., Buenrostro, J. D., Moskowitz, D. M., Wang, J., Greenleaf, W. J., Chang, H. Y., Crabtree, G. R. 2018; 19 (2): 162-+

    Abstract

    Aire mediates the expression of tissue-specific antigens in thymic epithelial cells to promote tolerance against self-reactive T lymphocytes. However, the mechanism that allows expression of tissue-specific genes at levels that prevent harm is unknown. Here we show that Brg1 generates accessibility at tissue-specific loci to impose central tolerance. We found that Aire has an intrinsic repressive function that restricts chromatin accessibility and opposes Brg1 across the genome. Aire exerted this repressive influence within minutes after recruitment to chromatin and restrained the amplitude of active transcription. Disease-causing mutations that impair Aire-induced activation also impair the protein's repressive function, which indicates dual roles for Aire. Together, Brg1 and Aire fine-tune the expression of tissue-specific genes at levels that prevent toxicity yet promote immune tolerance.

    View details for PubMedID 29335648

  • INO80 Chromatin Remodeling Coordinates Metabolic Homeostasis with Cell Division CELL REPORTS Gowans, G. J., Schep, A. N., Wong, K., King, D. A., Greenleaf, W. J., Morrison, A. J. 2018; 22 (3): 611–23

    Abstract

    Adaptive survival requires the coordination of nutrient availability with expenditure of cellular resources. For example, in nutrient-limited environments, 50% of all S. cerevisiae genes synchronize and exhibit periodic bursts of expression in coordination with respiration and cell division in the yeast metabolic cycle (YMC). Despite the importance of metabolic and proliferative synchrony, the majority of YMC regulators are currently unknown. Here, we demonstrate that the INO80 chromatin-remodeling complex is required to coordinate respiration and cell division with periodic gene expression. Specifically, INO80 mutants have severe defects in oxygen consumption and promiscuous cell division that is no longer coupled with metabolic status. In mutant cells, chromatin accessibility of periodic genes, including TORC1-responsive genes, is relatively static, concomitant with severely attenuated gene expression. Collectively, these results reveal that the INO80 complex mediates metabolic signaling to chromatin to restrict proliferation to metabolically optimal states.

    View details for PubMedID 29346761

  • Transcript-indexed ATAC-seq for precision immune profiling. Nature medicine Satpathy, A. T., Saligrama, N., Buenrostro, J. D., Wei, Y., Wu, B., Rubin, A. J., Granja, J. M., Lareau, C. A., Li, R., Qi, Y., Parker, K. R., Mumbach, M. R., Serratelli, W. S., Gennert, D. G., Schep, A. N., Corces, M. R., Khodadoust, M. S., Kim, Y. H., Khavari, P. A., Greenleaf, W. J., Davis, M. M., Chang, H. Y. 2018

    Abstract

    T cells create vast amounts of diversity in the genes that encode their T cell receptors (TCRs), which enables individual clones to recognize specific peptide-major histocompatibility complex (MHC) ligands. Here we combined sequencing of the TCR-encoding genes with assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis at the single-cell level to provide information on the TCR specificity and epigenomic state of individual T cells. By using this approach, termed transcript-indexed ATAC-seq (T-ATAC-seq), we identified epigenomic signatures in immortalized leukemic T cells, primary human T cells from healthy volunteers and primary leukemic T cells from patient samples. In peripheral blood CD4+ T cells from healthy individuals, we identified cis and trans regulators of naive and memory T cell states and found substantial heterogeneity in surface-marker-defined T cell populations. In patients with a leukemic form of cutaneous T cell lymphoma, T-ATAC-seq enabled identification of leukemic and nonleukemic regulatory pathways in T cells from the same individual by allowing separation of the signals that arose from the malignant clone from the background T cell noise. Thus, T-ATAC-seq is a new tool that enables analysis of epigenomic landscapes in clonal T cells and should be valuable for studies of T cell malignancy, immunity and immunotherapy.

    View details for PubMedID 29686426

  • The chromatin accessibility landscape of primary human cancers. Science (New York, N.Y.) Corces, M. R., Granja, J. M., Shams, S., Louie, B. H., Seoane, J. A., Zhou, W., Silva, T. C., Groeneveld, C., Wong, C. K., Cho, S. W., Satpathy, A. T., Mumbach, M. R., Hoadley, K. A., Robertson, A. G., Sheffield, N. C., Felau, I., Castro, M. A., Berman, B. P., Staudt, L. M., Zenklusen, J. C., Laird, P. W., Curtis, C., Greenleaf, W. J., Chang, H. Y. 2018; 362 (6413)

    Abstract

    We present the genome-wide chromatin accessibility profiles of 410 tumor samples spanning 23 cancer types from The Cancer Genome Atlas (TCGA). We identify 562,709 transposase-accessible DNA elements that substantially extend the compendium of known cis-regulatory elements. Integration of ATAC-seq (the assay for transposase-accessible chromatin using sequencing) with TCGA multi-omic data identifies a large number of putative distal enhancers that distinguish molecular subtypes of cancers, uncovers specific driving transcription factors via protein-DNA footprints, and nominates long-range gene-regulatory interactions in cancer. These data reveal genetic risk loci of cancer predisposition as active DNA regulatory elements in cancer, identify gene-regulatory interactions underlying cancer immune evasion, and pinpoint noncoding mutations that drive enhancer activation and may affect patient survival. These results suggest a systematic approach to understanding the noncoding genome in cancer to advance diagnosis and therapy.

    View details for DOI 10.1126/science.aav1898

    View details for PubMedID 30361341

  • Origin and differentiation of human memory CD8 T cells after vaccination NATURE Akondy, R. S., Fitch, M., Edupuganti, S., Yang, S., Kissick, H. T., Li, K. W., Youngblood, B. A., Abdelsamed, H. A., McGuire, D. J., Cohen, K. W., Alexe, G., Nagar, S., McCausland, M. M., Gupta, S., Tata, P., Haining, W., McElrath, M., Zhang, D., Hu, B., Greenleaf, W. J., Goronzy, J. J., Mulligan, M. J., Hellerstein, M., Ahmed, R. 2017; 552 (7685): 362-+

    Abstract

    The differentiation of human memory CD8 T cells is not well understood. Here we address this issue using the live yellow fever virus (YFV) vaccine, which induces long-term immunity in humans. We used in vivo deuterium labelling to mark CD8 T cells that proliferated in response to the virus and then assessed cellular turnover and longevity by quantifying deuterium dilution kinetics in YFV-specific CD8 T cells using mass spectrometry. This longitudinal analysis showed that the memory pool originates from CD8 T cells that divided extensively during the first two weeks after infection and is maintained by quiescent cells that divide less than once every year (doubling time of over 450 days). Although these long-lived YFV-specific memory CD8 T cells did not express effector molecules, their epigenetic landscape resembled that of effector CD8 T cells. This open chromatin profile at effector genes was maintained in memory CD8 T cells isolated even a decade after vaccination, indicating that these cells retain an epigenetic fingerprint of their effector history and remain poised to respond rapidly upon re-exposure to the pathogen.

    View details for PubMedID 29236685

  • Challenges and recommendations for epigenomics in precision health NATURE BIOTECHNOLOGY Carter, A. C., Chang, H. Y., Church, G., Dombkowski, A., Ecker, J. R., Gil, E., Giresi, P. G., Greely, H., Greenleaf, W. J., Hacohen, N., He, C., Hill, D., Ko, J., Kohane, I., Kundaje, A., Palmer, M., Snyder, M. P., Tung, J., Urban, A., Vidal, M., Wong, W. 2017; 35 (12): 1128–32

    View details for PubMedID 29220033

  • Chromatin accessibility dynamics reveal novel functional enhancers in C. elegans GENOME RESEARCH Daugherty, A. C., Yeo, R. W., Buenrostro, J. D., Greenleaf, W. J., Kundaje, A., Brunet, A. 2017; 27 (12): 2096–2107

    Abstract

    Chromatin accessibility, a crucial component of genome regulation, has primarily been studied in homogeneous and simple systems, such as isolated cell populations or early-development models. Whether chromatin accessibility can be assessed in complex, dynamic systems in vivo with high sensitivity remains largely unexplored. In this study, we use ATAC-seq to identify chromatin accessibility changes in a whole animal, the model organism Caenorhabditis elegans, from embryogenesis to adulthood. Chromatin accessibility changes between developmental stages are highly reproducible, recapitulate histone modification changes, and reveal key regulatory aspects of the epigenomic landscape throughout organismal development. We find that over 5000 distal noncoding regions exhibit dynamic changes in chromatin accessibility between developmental stages and could thereby represent putative enhancers. When tested in vivo, several of these putative enhancers indeed drive novel cell-type- and temporal-specific patterns of expression. Finally, by integrating transcription factor binding motifs in a machine learning framework, we identify EOR-1 as a unique transcription factor that may regulate chromatin dynamics during development. Our study provides a unique resource for C. elegans, a system in which the prevalence and importance of enhancers remains poorly characterized, and demonstrates the power of using whole organism chromatin accessibility to identify novel regulatory regions in complex systems.

    View details for PubMedID 29141961

  • Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens NATURE COMMUNICATIONS Morgens, D. W., Wainberg, M., Boyle, E. A., Ursu, O., Araya, C. L., Tsui, C. K., Haney, M. S., Hess, G. T., Han, K., Jeng, E. E., Li, A., Snyder, M. P., Greenleaf, W. J., Kundaje, A., Bassik, M. C. 2017; 8

    Abstract

    CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens.

    View details for DOI 10.1038/ncomms15178

    View details for PubMedID 28474669

  • Comprehensive and quantitative mapping of RNA-protein interactions across a transcribed eukaryotic genome PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA She, R., Chakravarty, A. K., Layton, C. J., Chircus, L. M., Andreasson, J. O., Damaraju, N., McMahon, P. L., Buenrostro, J. D., Jarosz, D. F., Greenleaf, W. J. 2017; 114 (14): 3619-3624

    Abstract

    RNA-binding proteins (RBPs) control the fate of nearly every transcript in a cell. However, no existing approach for studying these posttranscriptional gene regulators combines transcriptome-wide throughput and biophysical precision. Here, we describe an assay that accomplishes this. Using commonly available hardware, we built a customizable, open-source platform that leverages the inherent throughput of Illumina technology for direct biophysical measurements. We used the platform to quantitatively measure the binding affinity of the prototypical RBP Vts1 for every transcript in the Saccharomyces cerevisiae genome. The scale and precision of these measurements revealed many previously unknown features of this well-studied RBP. Our transcribed genome array (TGA) assayed both rare and abundant transcripts with equivalent proficiency, revealing hundreds of low-abundance targets missed by previous approaches. These targets regulated diverse biological processes including nutrient sensing and the DNA damage response, and implicated Vts1 in de novo gene "birth." TGA provided single-nucleotide resolution for each binding site and delineated a highly specific sequence and structure motif for Vts1 binding. Changes in transcript levels in vts1Δ cells established the regulatory function of these binding sites. The impact of Vts1 on transcript abundance was largely independent of where it bound within an mRNA, challenging prevailing assumptions about how this RBP drives RNA degradation. TGA thus enables a quantitative description of the relationship between variant RNA structures, affinity, and in vivo phenotype on a transcriptome-wide scale. We anticipate that TGA will provide similarly comprehensive and quantitative insights into the function of virtually any RBP.

    View details for DOI 10.1073/pnas.1618370114

    View details for Web of Science ID 000398159000041

    View details for PubMedID 28325876

  • Landscape of monoallelic DNA accessibility in mouse embryonic stem cells and neural progenitor cells. Nature genetics Xu, J., Carter, A. C., Gendrel, A., Attia, M., Loftus, J., Greenleaf, W. J., Tibshirani, R., Heard, E., Chang, H. Y. 2017; 49 (3): 377-386

    Abstract

    We developed an allele-specific assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) to genotype and profile active regulatory DNA across the genome. Using a mouse hybrid F1 system, we found that monoallelic DNA accessibility across autosomes was pervasive, developmentally programmed and composed of several patterns. Genetically determined accessibility was enriched at distal enhancers, but random monoallelically accessible (RAMA) elements were enriched at promoters and may act as gatekeepers of monoallelic mRNA expression. Allelic choice at RAMA elements was stable across cell generations and bookmarked through mitosis. RAMA elements in neural progenitor cells were biallelically accessible in embryonic stem cells but premarked with bivalent histone modifications; one allele was silenced during differentiation. Quantitative analysis indicated that allelic choice at the majority of RAMA elements is consistent with a stochastic process; however, up to 30% of RAMA elements may deviate from the expected pattern, suggesting a regulated or counting mechanism.

    View details for DOI 10.1038/ng.3769

    View details for PubMedID 28112738

  • TOP2 synergizes with BAF chromatin remodeling for both resolution and formation of facultative heterochromatin. Nature structural & molecular biology Miller, E. L., Hargreaves, D. C., Kadoch, C., Chang, C., Calarco, J. P., Hodges, C., Buenrostro, J. D., Cui, K., Greenleaf, W. J., Zhao, K., Crabtree, G. R. 2017

    Abstract

    The resolution and formation of facultative heterochromatin are essential for development, reprogramming, and oncogenesis. The mechanisms underlying these changes are poorly understood owing to the difficulty of studying heterochromatin dynamics and structure in vivo. We devised an in vivo approach to investigate these mechanisms and found that topoisomerase II (TOP2), but not TOP1, synergizes with BAF (mSWI/SNF) ATP-dependent chromatin remodeling complexes genome-wide to resolve facultative heterochromatin to accessible chromatin independent of transcription. This indicates that changes in DNA topology that take place through (de-)catenation rather than the release of torsional stress through swiveling are necessary for heterochromatin resolution. TOP2 and BAF cooperate to recruit pluripotency factors, which explains some of the instructive roles of BAF complexes. Unexpectedly, we found that TOP2 also plays a role in the re-formation of facultative heterochromatin; this finding suggests that facultative heterochromatin and accessible chromatin exist at different states of catenation or other topologies, which might be critical to their structures.

    View details for DOI 10.1038/nsmb.3384

    View details for PubMedID 28250416

  • Epigenomics of human CD8 T cell differentiation and aging. Science immunology Moskowitz, D. M., Zhang, D. W., Hu, B., Le Saux, S., Yanes, R. E., Ye, Z., Buenrostro, J. D., Weyand, C. M., Greenleaf, W. J., Goronzy, J. J. 2017; 2 (8)

    Abstract

    The efficacy of the adaptive immune response declines dramatically with age, but the cell-intrinsic mechanisms driving immune aging in humans remain poorly understood. Immune aging is characterized by a loss of self-renewing naïve cells and the accumulation of differentiated but dysfunctional cells within the CD8 T cell compartment. Using ATAC-seq, we inferred the transcription factor binding activities correlated with naive and central and effector memory CD8 T cell states in young adults. Integrating our results with RNA-seq, we identified transcription networks associated with CD8 T cell differentiation, with prominent roles implicated for BATF, ETS1, Eomes, and Sp1. Extending our analysis to aged humans, we found that the differences between the memory and naive subsets were largely preserved across age, but that naive and central memory cells from older individuals exhibited a shift toward more differentiated patterns of chromatin openness. Additionally, aged naive cells displayed a loss in chromatin accessibility at gene promoters, largely associated with a decrease in NRF1 binding. This shift was implicated in a marked drop-off in the ability of the aged naive cells to transcribe respiratory chain genes, which may explain the reduced capacity of oxidative phosphorylation in older naïve cells. Our findings identify BATF- and NRF1-driven gene regulation as potential targets for delaying CD8 T cell aging and restoring function.

    View details for DOI 10.1126/sciimmunol.aag0192

    View details for PubMedID 28439570

  • Single-cell epigenomic variability reveals functional cancer heterogeneity. Genome biology Litzenburger, U. M., Buenrostro, J. D., Wu, B., Shen, Y., Sheffield, N. C., Kathiria, A., Greenleaf, W. J., Chang, H. Y. 2017; 18 (1): 15-?

    Abstract

    Cell-to-cell heterogeneity is a major driver of cancer evolution, progression, and emergence of drug resistance. Epigenomic variation at the single-cell level can rapidly create cancer heterogeneity but is difficult to detect and assess functionally.We develop a strategy to bridge the gap between measurement and function in single-cell epigenomics. Using single-cell chromatin accessibility and RNA-seq data in K562 leukemic cells, we identify the cell surface marker CD24 as co-varying with chromatin accessibility changes linked to GATA transcription factors in single cells. Fluorescence-activated cell sorting of CD24 high versus low cells prospectively isolated GATA1 and GATA2 high versus low cells. GATA high versus low cells express differential gene regulatory networks, differential sensitivity to the drug imatinib mesylate, and differential self-renewal capacity. Lineage tracing experiments show that GATA/CD24hi cells have the capability to rapidly reconstitute the heterogeneity within the entire starting population, suggesting that GATA expression levels drive a phenotypically relevant source of epigenomic plasticity.Single-cell chromatin accessibility can guide prospective characterization of cancer heterogeneity. Epigenomic subpopulations in cancer impact drug sensitivity and the clonal dynamics of cancer evolution.

    View details for DOI 10.1186/s13059-016-1133-7

    View details for PubMedID 28118844

  • Multiparameter Particle Display (MPPD): A Quantitative Screening Method for the Discovery of Highly Specific Aptamers. Angewandte Chemie (International ed. in English) Wang, J., Yu, J., Yang, Q., McDermott, J., Scott, A., Vukovich, M., Lagrois, R., Gong, Q., Greenleaf, W., Eisenstein, M., Ferguson, B. S., Soh, H. T. 2017; 56 (3): 744-747

    Abstract

    Aptamers are a promising class of affinity reagents because they are chemically synthesized, thus making them highly reproducible and distributable as sequence information rather than a physical entity. Although many high-quality aptamers have been previously reported, it is difficult to routinely generate aptamers that possess both high affinity and specificity. One of the reasons is that conventional aptamer selection can only be performed either for affinity (positive selection) or for specificity (negative selection), but not both simultaneously. In this work, we harness the capacity of fluorescence activated cell sorting (FACS) for multicolor sorting to simultaneously screen for affinity and specificity at a throughput of 10(7) aptamers per hour. As a proof of principle, we generated DNA aptamers that exhibit picomolar to low nanomolar affinity in human serum for three diverse proteins, and show that these aptamers are capable of outperforming high-quality monoclonal antibodies in a standard ELISA detection assay.

    View details for DOI 10.1002/anie.201608880

    View details for PubMedID 27933702

    View details for PubMedCentralID PMC5225111

  • Variable chromatin structure revealed by in situ spatially correlated DNA cleavage mapping. Nature Risca, V. I., Denny, S. K., Straight, A. F., Greenleaf, W. J. 2017; 541 (7636): 237-241

    Abstract

    Chromatin structure at the length scale encompassing local nucleosome-nucleosome interactions is thought to play a crucial role in regulating transcription and access to DNA. However, this secondary structure of chromatin remains poorly understood compared with the primary structure of single nucleosomes or the tertiary structure of long-range looping interactions. Here we report the first genome-wide map of chromatin conformation in human cells at the 1-3 nucleosome (50-500 bp) scale, obtained using ionizing radiation-induced spatially correlated cleavage of DNA with sequencing (RICC-seq) to identify DNA-DNA contacts that are spatially proximal. Unbiased analysis of RICC-seq signal reveals regional enrichment of DNA fragments characteristic of alternating rather than adjacent nucleosome interactions in tri-nucleosome units, particularly in H3K9me3-marked heterochromatin. We infer differences in the likelihood of nucleosome-nucleosome contacts among open chromatin, H3K27me3-marked, and H3K9me3-marked repressed chromatin regions. After calibrating RICC-seq signal to three-dimensional distances, we show that compact two-start helical fibre structures with stacked alternating nucleosomes are consistent with RICC-seq fragmentation patterns from H3K9me3-marked chromatin, while non-compact structures and solenoid structures are consistent with open chromatin. Our data support a model of chromatin architecture in intact interphase nuclei consistent with variable longitudinal compaction of two-start helical fibres.

    View details for DOI 10.1038/nature20781

    View details for PubMedID 28024297

  • Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements. Nature genetics Mumbach, M. R., Satpathy, A. T., Boyle, E. A., Dai, C., Gowen, B. G., Cho, S. W., Nguyen, M. L., Rubin, A. J., Granja, J. M., Kazane, K. R., Wei, Y., Nguyen, T., Greenside, P. G., Corces, M. R., Tycko, J., Simeonov, D. R., Suliman, N., Li, R., Xu, J., Flynn, R. A., Kundaje, A., Khavari, P. A., Marson, A., Corn, J. E., Quertermous, T., Greenleaf, W. J., Chang, H. Y. 2017

    Abstract

    The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer-promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases.

    View details for PubMedID 28945252

  • An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nature methods Corces, M. R., Trevino, A. E., Hamilton, E. G., Greenside, P. G., Sinnott-Armstrong, N. A., Vesuna, S., Satpathy, A. T., Rubin, A. J., Montine, K. S., Wu, B., Kathiria, A., Cho, S. W., Mumbach, M. R., Carter, A. C., Kasowski, M., Orloff, L. A., Risca, V. I., Kundaje, A., Khavari, P. A., Montine, T. J., Greenleaf, W. J., Chang, H. Y. 2017

    Abstract

    We present Omni-ATAC, an improved ATAC-seq protocol for chromatin accessibility profiling that works across multiple applications with substantial improvement of signal-to-background ratio and information content. The Omni-ATAC protocol generates chromatin accessibility profiles from archival frozen tissue samples and 50-μm sections, revealing the activities of disease-associated DNA elements in distinct human brain structures. The Omni-ATAC protocol enables the interrogation of personal regulomes in tissue context and translational studies.

    View details for PubMedID 28846090

  • chromVAR: inferring transcription-factor-associated accessibility from single-cell epigenomic data. Nature methods Schep, A. N., Wu, B., Buenrostro, J. D., Greenleaf, W. J. 2017; 14 (10): 975–78

    Abstract

    Single-cell ATAC-seq (scATAC) yields sparse data that make conventional analysis challenging. We developed chromVAR (http://www.github.com/GreenleafLab/chromVAR), an R package for analyzing sparse chromatin-accessibility data by estimating gain or loss of accessibility within peaks sharing the same motif or annotation while controlling for technical biases. chromVAR enables accurate clustering of scATAC-seq profiles and characterization of known and de novo sequence motifs associated with variation in chromatin accessibility.

    View details for PubMedID 28825706

  • Open Chromatin Profiling in hiPSC-Derived Neurons Prioritizes Functional Noncoding Psychiatric Risk Variants and Highlights Neurodevelopmental Loci. Cell stem cell Forrest, M. P., Zhang, H., Moy, W., McGowan, H., Leites, C., Dionisio, L. E., Xu, Z., Shi, J., Sanders, A. R., Greenleaf, W. J., Cowan, C. A., Pang, Z. P., Gejman, P. V., Penzes, P., Duan, J. 2017; 21 (3): 305–18.e8

    Abstract

    Most disease variants lie within noncoding genomic regions, making their functional interpretation challenging. Because chromatin openness strongly influences transcriptional activity, we hypothesized that cell-type-specific open chromatin regions (OCRs) might highlight disease-relevant noncoding sequences. To investigate, we mapped global OCRs in neurons differentiating from hiPSCs, a cellular model for studying neurodevelopmental disorders such as schizophrenia (SZ). We found that the OCRs are highly dynamic and can stratify GWAS-implicated SZ risk variants. Of the more than 3,500 SZ-associated variants analyzed, we prioritized ∼100 putatively functional ones located in neuronal OCRs, including rs1198588, at a leading risk locus flanking MIR137. Excitatory neurons derived from hiPSCs with CRISPR/Cas9-edited rs1198588 or a rare proximally located SZ risk variant showed altered MIR137 expression, dendrite arborization, and synapse maturation. Our study shows that noncoding disease variants in OCRs can affect neurodevelopment, and that analysis of open chromatin regions can help prioritize functionally relevant noncoding variants identified by GWAS.

    View details for PubMedID 28803920

  • Chromatin Accessibility Landscape of Cutaneous T Cell Lymphoma and Dynamic Response to HDAC Inhibitors. Cancer cell Qu, K., Zaba, L. C., Satpathy, A. T., Giresi, P. G., Li, R., Jin, Y., Armstrong, R., Jin, C., Schmitt, N., Rahbar, Z., Ueno, H., Greenleaf, W. J., Kim, Y. H., Chang, H. Y. 2017

    Abstract

    Here, we define the landscape and dynamics of active regulatory DNA in cutaneous T cell lymphoma (CTCL) by ATAC-seq. Analysis of 111 human CTCL and control samples revealed extensive chromatin signatures that distinguished leukemic, host, and normal CD4(+) T cells. We identify three dominant patterns of transcription factor (TF) activation that drive leukemia regulomes, as well as TF deactivations that alter host T cells in CTCL patients. Clinical response to histone deacetylase inhibitors (HDACi) is strongly associated with a concurrent gain in chromatin accessibility. HDACi causes distinct chromatin responses in leukemic and host CD4(+) T cells, reprogramming host T cells toward normalcy. These results provide a foundational framework to study personal regulomes in human cancer and epigenetic therapy.

    View details for PubMedID 28625481

  • Discovery of stimulation-responsive immune enhancers with CRISPR activation. Nature Simeonov, D. R., Gowen, B. G., Boontanrart, M., Roth, T. L., Gagnon, J. D., Mumbach, M. R., Satpathy, A. T., Lee, Y., Bray, N. L., Chan, A. Y., Lituiev, D. S., Nguyen, M. L., Gate, R. E., Subramaniam, M., Li, Z., Woo, J. M., Mitros, T., Ray, G. J., Curie, G. L., Naddaf, N., Chu, J. S., Ma, H., Boyer, E., Van Gool, F., Huang, H., Liu, R., Tobin, V. R., Schumann, K., Daly, M. J., Farh, K. K., Ansel, K. M., Ye, C. J., Greenleaf, W. J., Anderson, M. S., Bluestone, J. A., Chang, H. Y., Corn, J. E., Marson, A. 2017

    Abstract

    The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (TH17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.

    View details for PubMedID 28854172

  • Lineage-specific dynamic and pre-established enhancer-promoter contacts cooperate in terminal differentiation. Nature genetics Rubin, A. J., Barajas, B. C., Furlan-Magaril, M., Lopez-Pajares, V., Mumbach, M. R., Howard, I., Kim, D. S., Boxer, L. D., Cairns, J., Spivakov, M., Wingett, S. W., Shi, M., Zhao, Z., Greenleaf, W. J., Kundaje, A., Snyder, M., Chang, H. Y., Fraser, P., Khavari, P. A. 2017; 49 (10): 1522–28

    Abstract

    Chromosome conformation is an important feature of metazoan gene regulation; however, enhancer-promoter contact remodeling during cellular differentiation remains poorly understood. To address this, genome-wide promoter capture Hi-C (CHi-C) was performed during epidermal differentiation. Two classes of enhancer-promoter contacts associated with differentiation-induced genes were identified. The first class ('gained') increased in contact strength during differentiation in concert with enhancer acquisition of the H3K27ac activation mark. The second class ('stable') were pre-established in undifferentiated cells, with enhancers constitutively marked by H3K27ac. The stable class was associated with the canonical conformation regulator cohesin, whereas the gained class was not, implying distinct mechanisms of contact formation and regulation. Analysis of stable enhancers identified a new, essential role for a constitutively expressed, lineage-restricted ETS-family transcription factor, EHF, in epidermal differentiation. Furthermore, neither class of contacts was observed in pluripotent cells, suggesting that lineage-specific chromatin structure is established in tissue progenitor cells and is further remodeled in terminal differentiation.

    View details for PubMedID 28805829

  • HiChIP: efficient and sensitive analysis of protein-directed genome architecture. Nature methods Mumbach, M. R., Rubin, A. J., Flynn, R. A., Dai, C., Khavari, P. A., Greenleaf, W. J., Chang, H. Y. 2016; 13 (11): 919-922

    Abstract

    Genome conformation is central to gene control but challenging to interrogate. Here we present HiChIP, a protein-centric chromatin conformation method. HiChIP improves the yield of conformation-informative reads by over 10-fold and lowers the input requirement over 100-fold relative to that of ChIA-PET. HiChIP of cohesin reveals multiscale genome architecture with greater signal-to-background ratios than those of in situ Hi-C.

    View details for DOI 10.1038/nmeth.3999

    View details for PubMedID 27643841

  • ATAC-see reveals the accessible genome by transposase-mediated imaging and sequencing. Nature methods Chen, X., Shen, Y., Draper, W., Buenrostro, J. D., Litzenburger, U., Cho, S. W., Satpathy, A. T., Carter, A. C., Ghosh, R. P., East-Seletsky, A., Doudna, J. A., Greenleaf, W. J., Liphardt, J. T., Chang, H. Y. 2016

    Abstract

    Spatial organization of the genome plays a central role in gene expression, DNA replication, and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see), a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ, cell sorting, and deep sequencing to reveal the identity of the imaged elements. ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion, termed NETosis. Integration of ATAC-see with flow cytometry enables automated quantitation and prospective cell isolation as a function of chromatin accessibility, and it reveals a cell-cycle dependence of chromatin accessibility that is especially dynamic in G1 phase. The integration of imaging and epigenomics provides a general and scalable approach for deciphering the spatiotemporal architecture of gene control.

    View details for DOI 10.1038/nmeth.4031

    View details for PubMedID 27749837

  • Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution. Nature genetics Corces, M. R., Buenrostro, J. D., Wu, B., Greenside, P. G., Chan, S. M., Koenig, J. L., Snyder, M. P., Pritchard, J. K., Kundaje, A., Greenleaf, W. J., Majeti, R., Chang, H. Y. 2016; 48 (10): 1193-1203

    Abstract

    We define the chromatin accessibility and transcriptional landscapes in 13 human primary blood cell types that span the hematopoietic hierarchy. Exploiting the finding that the enhancer landscape better reflects cell identity than mRNA levels, we enable 'enhancer cytometry' for enumeration of pure cell types from complex populations. We identify regulators governing hematopoietic differentiation and further show the lineage ontogeny of genetic elements linked to diverse human diseases. In acute myeloid leukemia (AML), chromatin accessibility uncovers unique regulatory evolution in cancer cells with a progressively increasing mutation burden. Single AML cells exhibit distinctive mixed regulome profiles corresponding to disparate developmental stages. A method to account for this regulatory heterogeneity identified cancer-specific deviations and implicated HOX factors as key regulators of preleukemic hematopoietic stem cell characteristics. Thus, regulome dynamics can provide diverse insights into hematopoietic development and disease.

    View details for DOI 10.1038/ng.3646

    View details for PubMedID 27526324

  • Nfib Promotes Metastasis through a Widespread Increase in Chromatin Accessibility CELL Denny, S. K., Yang, D., Chuang, C., Brady, J. J., Lim, J. S., Gruner, B. M., Chiou, S., Schep, A. N., Baral, J., Hamard, C., Antoine, M., Wislez, M., Kong, C. S., Connolly, A. J., Park, K., Sage, J., Greenleaf, W. J., Winslow, M. M. 2016; 166 (2): 328-342

    Abstract

    Metastases are the main cause of cancer deaths, but the mechanisms underlying metastatic progression remain poorly understood. We isolated pure populations of cancer cells from primary tumors and metastases from a genetically engineered mouse model of human small cell lung cancer (SCLC) to investigate the mechanisms that drive the metastatic spread of this lethal cancer. Genome-wide characterization of chromatin accessibility revealed the opening of large numbers of distal regulatory elements across the genome during metastatic progression. These changes correlate with copy number amplification of the Nfib locus, and differentially accessible sites were highly enriched for Nfib transcription factor binding sites. Nfib is necessary and sufficient to increase chromatin accessibility at a large subset of the intergenic regions. Nfib promotes pro-metastatic neuronal gene expression programs and drives the metastatic ability of SCLC cells. The identification of widespread chromatin changes during SCLC progression reveals an unexpected global reprogramming during metastatic progression.

    View details for DOI 10.1016/j.cell.2016.05.052

    View details for PubMedID 27374332

  • Identification of significantly mutated regions across cancer types highlights a rich landscape of functional molecular alterations. Nature genetics Araya, C. L., Cenik, C., Reuter, J. A., Kiss, G., Pande, V. S., Snyder, M. P., Greenleaf, W. J. 2016; 48 (2): 117-125

    Abstract

    Cancer sequencing studies have primarily identified cancer driver genes by the accumulation of protein-altering mutations. An improved method would be annotation independent, sensitive to unknown distributions of functions within proteins and inclusive of noncoding drivers. We employed density-based clustering methods in 21 tumor types to detect variably sized significantly mutated regions (SMRs). SMRs reveal recurrent alterations across a spectrum of coding and noncoding elements, including transcription factor binding sites and untranslated regions mutated in up to ∼15% of specific tumor types. SMRs demonstrate spatial clustering of alterations in molecular domains and at interfaces, often with associated changes in signaling. Mutation frequencies in SMRs demonstrate that distinct protein regions are differentially mutated across tumor types, as exemplified by a linker region of PIK3CA in which biophysical simulations suggest that mutations affect regulatory interactions. The functional diversity of SMRs underscores both the varied mechanisms of oncogenic misregulation and the advantage of functionally agnostic driver identification.

    View details for DOI 10.1038/ng.3471

    View details for PubMedID 26691984

  • Structured nucleosome fingerprints enable high-resolution mapping of chromatin architecture within regulatory regions GENOME RESEARCH Schep, A. N., Buenrostro, J. D., Denny, S. K., Schwartz, K., Sherlock, G., Greenleaf, W. J. 2015; 25 (11): 1757-1770

    View details for DOI 10.1101/gr.192294.115

    View details for PubMedID 26314830

  • Individuality and Variation of Personal Regulomes in Primary Human T Cells CELL SYSTEMS Qu, K., Zaba, L. C., Giresi, P. G., Li, R., Longmire, M., Kim, Y. H., Greenleaf, W. J., Chang, H. Y. 2015; 1 (1): 51-61

    Abstract

    Here we survey variation and dynamics of active regulatory elements genome-wide using longitudinal samples from human individuals. We applied Assay of Transposase Accessible Chromatin with sequencing (ATAC-seq) to map chromatin accessibility in primary CD4+ T cells isolated from standard blood draws of 12 healthy volunteers over time, from cancer patients, and during T cell activation. Over 4,000 predicted regulatory elements (7.2%) showed reproducible variation in accessibility between individuals. Gender was the most significant attributable source of variation. ATAC-seq revealed previously undescribed elements that escape X chromosome inactivation and predicted gender-specific gene regulatory networks across autosomes, which coordinately affect genes with immune function. Noisy regulatory elements with personal variation in accessibility are significantly enriched for autoimmune disease loci. Over one third of regulome variation lacked genetic variation in cis, suggesting contributions from environmental or epigenetic factors. These results refine concepts of human individuality and provide a foundational reference for comparing disease-associated regulomes.

    View details for DOI 10.1016/j.cels.2015.06.003

    View details for Web of Science ID 000209925400012

    View details for PubMedCentralID PMC4522940

  • Individuality and variation of personal regulomes in primary human T cells. Cell systems Qu, K., Zaba, L. C., Giresi, P. G., Li, R., Longmire, M., Kim, Y. H., Greenleaf, W. J., Chang, H. Y. 2015; 1 (1): 51-61

    Abstract

    Here we survey variation and dynamics of active regulatory elements genome-wide using longitudinal samples from human individuals. We applied Assay of Transposase Accessible Chromatin with sequencing (ATAC-seq) to map chromatin accessibility in primary CD4+ T cells isolated from standard blood draws of 12 healthy volunteers over time, from cancer patients, and during T cell activation. Over 4,000 predicted regulatory elements (7.2%) showed reproducible variation in accessibility between individuals. Gender was the most significant attributable source of variation. ATAC-seq revealed previously undescribed elements that escape X chromosome inactivation and predicted gender-specific gene regulatory networks across autosomes, which coordinately affect genes with immune function. Noisy regulatory elements with personal variation in accessibility are significantly enriched for autoimmune disease loci. Over one third of regulome variation lacked genetic variation in cis, suggesting contributions from environmental or epigenetic factors. These results refine concepts of human individuality and provide a foundational reference for comparing disease-associated regulomes.

    View details for PubMedID 26251845

  • Single-cell chromatin accessibility reveals principles of regulatory variation NATURE Buenostro, J. D., Wu, B., Litzenburger, U. M., Ruff, D., Gonzales, M. L., Snyder, M. P., Chang, H. Y., Greenleaf, W. J. 2015; 523 (7561): 486-U264

    Abstract

    Cell-to-cell variation is a universal feature of life that affects a wide range of biological phenomena, from developmental plasticity to tumour heterogeneity. Although recent advances have improved our ability to document cellular phenotypic variation, the fundamental mechanisms that generate variability from identical DNA sequences remain elusive. Here we reveal the landscape and principles of mammalian DNA regulatory variation by developing a robust method for mapping the accessible genome of individual cells by assay for transposase-accessible chromatin using sequencing (ATAC-seq) integrated into a programmable microfluidics platform. Single-cell ATAC-seq (scATAC-seq) maps from hundreds of single cells in aggregate closely resemble accessibility profiles from tens of millions of cells and provide insights into cell-to-cell variation. Accessibility variance is systematically associated with specific trans-factors and cis-elements, and we discover combinations of trans-factors associated with either induction or suppression of cell-to-cell variability. We further identify sets of trans-factors associated with cell-type-specific accessibility variance across eight cell types. Targeted perturbations of cell cycle or transcription factor signalling evoke stimulus-specific changes in this observed variability. The pattern of accessibility variation in cis across the genome recapitulates chromosome compartments de novo, linking single-cell accessibility variation to three-dimensional genome organization. Single-cell analysis of DNA accessibility provides new insight into cellular variation of the 'regulome'.

    View details for DOI 10.1038/nature14590

    View details for Web of Science ID 000358378900042

  • Unraveling the 3D genome: genomics tools for multiscale exploration. Trends in genetics Risca, V. I., Greenleaf, W. J. 2015; 31 (7): 357-372

    Abstract

    A decade of rapid method development has begun to yield exciting insights into the 3D architecture of the metazoan genome and the roles it may play in regulating transcription. Here we review core methods and new tools in the modern genomicist's toolbox at three length scales, ranging from single base pairs to megabase-scale chromosomal domains, and discuss the emerging picture of the 3D genome that these tools have revealed. Blind spots remain, especially at intermediate length scales spanning a few nucleosomes, but thanks in part to new technologies that permit targeted alteration of chromatin states and time-resolved studies, the next decade holds great promise for hypothesis-driven research into the mechanisms that drive genome architecture and transcriptional regulation.

    View details for DOI 10.1016/j.tig.2015.03.010

    View details for PubMedID 25887733

    View details for PubMedCentralID PMC4490074

  • Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors NATURE BIOTECHNOLOGY Maza, I., Caspi, I., Zviran, A., Chomsky, E., Rais, Y., Viukov, S., Geula, S., Buenrostro, J. D., Weinberger, L., Krupalnik, V., Hanna, S., Zerbib, M., Dutton, J. R., Greenleaf, W. J., Massarwa, R., Novershtern, N., Hanna, J. H. 2015; 33 (7): 769-774

    Abstract

    Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced 'transdifferentiation' pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by various methods.

    View details for DOI 10.1038/nbt.3270

    View details for Web of Science ID 000358396100034

    View details for PubMedCentralID PMC4500825

  • Assaying the epigenome in limited numbers of cells. Methods Greenleaf, W. J. 2015; 72: 51-56

    Abstract

    Spectacular advances in the throughput of DNA sequencing have allowed genome-wide analysis of epigenetic features such as methylation, nucleosome position and post-translational modification, chromatin accessibility and connectivity, and transcription factor binding. However, for rare or precious biological samples, input requirements of many of these methods limit their application. In this review we discuss recent advances for low-input genome-wide analysis of chromatin immunoprecipitation, methylation, DNA accessibility, and chromatin conformation.

    View details for DOI 10.1016/j.ymeth.2014.10.010

    View details for PubMedID 25461774

  • Beyond the Linear Genome: Paired-End Sequencing as a Biophysical Tool. Trends in cell biology Risca, V. I., Greenleaf, W. J. 2015; 25 (12): 716–19

    Abstract

    Paired-end sequencing has enabled a variety of new methods for high-throughput interrogation of both genome structure and chromatin architecture. Here, we discuss how the paired-end paradigm can be used to interpret sequencing data as biophysical measurements of in vivo chromatin structure that report on single molecules in single cells.

    View details for PubMedID 26437592

  • ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide. Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] Buenrostro, J. D., Wu, B., Chang, H. Y., Greenleaf, W. J. 2015; 109: 21 29 1-9

    Abstract

    This unit describes Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq), a method for mapping chromatin accessibility genome-wide. This method probes DNA accessibility with hyperactive Tn5 transposase, which inserts sequencing adapters into accessible regions of chromatin. Sequencing reads can then be used to infer regions of increased accessibility, as well as to map regions of transcription-factor binding and nucleosome position. The method is a fast and sensitive alternative to DNase-seq for assaying chromatin accessibility genome-wide, or to MNase-seq for assaying nucleosome positions in accessible regions of the genome. © 2015 by John Wiley & Sons, Inc.

    View details for DOI 10.1002/0471142727.mb2129s109

    View details for PubMedID 25559105

  • Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors. Nature biotechnology Maza, I., Caspi, I., Zviran, A., Chomsky, E., Rais, Y., Viukov, S., Geula, S., Buenrostro, J. D., Weinberger, L., Krupalnik, V., Hanna, S., Zerbib, M., Dutton, J. R., Greenleaf, W. J., Massarwa, R., Novershtern, N., Hanna, J. H. 2015; 33 (7): 769–74

    Abstract

    Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced 'transdifferentiation' pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by various methods.

    View details for PubMedID 26098448

  • A Conditional System to Specifically Link Disruption of Protein-Coding Function with Reporter Expression in Mice CELL REPORTS Chiou, S., Kim-Kiselak, C., Risca, V. I., Heimann, M. K., Chuang, C., Burds, A. A., Greenleaf, W. J., Jacks, T. E., Feldser, D. M., Winslow, M. M. 2014; 7 (6): 2078-2086
  • A conditional system to specifically link disruption of protein-coding function with reporter expression in mice. Cell reports Chiou, S., Kim-Kiselak, C., Risca, V. I., Heimann, M. K., Chuang, C., Burds, A. A., Greenleaf, W. J., Jacks, T. E., Feldser, D. M., Winslow, M. M. 2014; 7 (6): 2078-2086

    Abstract

    Conditional gene deletion in mice has contributed immensely to our understanding of many biological and biomedical processes. Despite an increasing awareness of nonprotein-coding functional elements within protein-coding transcripts, current gene-targeting approaches typically involve simultaneous ablation of noncoding elements within targeted protein-coding genes. The potential for protein-coding genes to have additional noncoding functions necessitates the development of novel genetic tools capable of precisely interrogating individual functional elements. We present a strategy that couples Cre/loxP-mediated conditional gene disruption with faithful GFP reporter expression in mice in which Cre-mediated stable inversion of a splice acceptor-GFP-splice donor cassette concurrently disrupts protein production and creates a GFP fusion product. Importantly, cassette inversion maintains physiologic transcript structure, thereby ensuring proper microRNA-mediated regulation of the GFP reporter, as well as maintaining expression of nonprotein-coding elements. To test this potentially generalizable strategy, we generated and analyzed mice with this conditional knockin reporter targeted to the Hmga2 locus.

    View details for DOI 10.1016/j.celrep.2014.05.031

    View details for PubMedID 24931605

    View details for PubMedCentralID PMC4113058

  • Quantitative analysis of RNA-protein interactions on a massively parallel array reveals biophysical and evolutionary landscapes. Nature biotechnology Buenrostro, J. D., Araya, C. L., Chircus, L. M., Layton, C. J., Chang, H. Y., Snyder, M. P., Greenleaf, W. J. 2014; 32 (6): 562-568

    Abstract

    RNA-protein interactions drive fundamental biological processes and are targets for molecular engineering, yet quantitative and comprehensive understanding of the sequence determinants of affinity remains limited. Here we repurpose a high-throughput sequencing instrument to quantitatively measure binding and dissociation of a fluorescently labeled protein to >10(7) RNA targets generated on a flow cell surface by in situ transcription and intermolecular tethering of RNA to DNA. Studying the MS2 coat protein, we decompose the binding energy contributions from primary and secondary RNA structure, and observe that differences in affinity are often driven by sequence-specific changes in both association and dissociation rates. By analyzing the biophysical constraints and modeling mutational paths describing the molecular evolution of MS2 from low- to high-affinity hairpins, we quantify widespread molecular epistasis and a long-hypothesized, structure-dependent preference for G:U base pairs over C:A intermediates in evolutionary trajectories. Our results suggest that quantitative analysis of RNA on a massively parallel array (RNA-MaP) provides generalizable insight into the biophysical basis and evolutionary consequences of sequence-function relationships.

    View details for DOI 10.1038/nbt.2880

    View details for PubMedID 24727714

  • A pause sequence enriched at translation start sites drives transcription dynamics in vivo. Science Larson, M. H., Mooney, R. A., Peters, J. M., Windgassen, T., Nayak, D., Gross, C. A., Block, S. M., Greenleaf, W. J., Landick, R., Weissman, J. S. 2014; 344 (6187): 1042-1047

    Abstract

    Transcription by RNA polymerase (RNAP) is interrupted by pauses that play diverse regulatory roles. Although individual pauses have been studied in vitro, the determinants of pauses in vivo and their distribution throughout the bacterial genome remain unknown. Using nascent transcript sequencing, we identified a 16-nucleotide consensus pause sequence in Escherichia coli that accounts for known regulatory pause sites as well as ~20,000 new in vivo pause sites. In vitro single-molecule and ensemble analyses demonstrate that these pauses result from RNAP-nucleic acid interactions that inhibit next-nucleotide addition. The consensus sequence also leads to pausing by RNAPs from diverse lineages and is enriched at translation start sites in both E. coli and Bacillus subtilis. Our results thus reveal a conserved mechanism unifying known and newly identified pause events.

    View details for DOI 10.1126/science.1251871

    View details for PubMedID 24789973

  • Exome sequencing identifies a DNAJB6 mutation in a family with dominantly-inherited limb-girdle muscular dystrophy. Neuromuscular disorders Couthouis, J., Raphael, A. R., Siskind, C., Findlay, A. R., Buenrostro, J. D., Greenleaf, W. J., Vogel, H., Day, J. W., Flanigan, K. M., Gitler, A. D. 2014; 24 (5): 431-435

    Abstract

    Limb-girdle muscular dystrophy primarily affects the muscles of the hips and shoulders (the "limb-girdle" muscles), although it is a heterogeneous disorder that can present with varying symptoms. There is currently no cure. We sought to identify the genetic basis of limb-girdle muscular dystrophy type 1 in an American family of Northern European descent using exome sequencing. Exome sequencing was performed on DNA samples from two affected siblings and one unaffected sibling and resulted in the identification of eleven candidate mutations that co-segregated with the disease. Notably, this list included a previously reported mutation in DNAJB6, p.Phe89Ile, which was recently identified as a cause of limb-girdle muscular dystrophy type 1D. Additional family members were Sanger sequenced and the mutation in DNAJB6 was only found in affected individuals. Subsequent haplotype analysis indicated that this DNAJB6 p.Phe89Ile mutation likely arose independently of the previously reported mutation. Since other published mutations are located close by in the G/F domain of DNAJB6, this suggests that the area may represent a mutational hotspot. Exome sequencing provided an unbiased and effective method for identifying the genetic etiology of limb-girdle muscular dystrophy type 1 in a previously genetically uncharacterized family. This work further confirms the causative role of DNAJB6 mutations in limb-girdle muscular dystrophy type 1D.

    View details for DOI 10.1016/j.nmd.2014.01.014

    View details for PubMedID 24594375

  • Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position NATURE METHODS Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y., Greenleaf, W. J. 2013; 10 (12): 1213-?

    Abstract

    We describe an assay for transposase-accessible chromatin using sequencing (ATAC-seq), based on direct in vitro transposition of sequencing adaptors into native chromatin, as a rapid and sensitive method for integrative epigenomic analysis. ATAC-seq captures open chromatin sites using a simple two-step protocol with 500-50,000 cells and reveals the interplay between genomic locations of open chromatin, DNA-binding proteins, individual nucleosomes and chromatin compaction at nucleotide resolution. We discovered classes of DNA-binding factors that strictly avoided, could tolerate or tended to overlap with nucleosomes. Using ATAC-seq maps of human CD4(+) T cells from a proband obtained on consecutive days, we demonstrated the feasibility of analyzing an individual's epigenome on a timescale compatible with clinical decision-making.

    View details for DOI 10.1038/NMETH.2688

    View details for Web of Science ID 000327698100025

    View details for PubMedID 24097267

  • Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA Sequencing Libraries AMERICAN JOURNAL OF HUMAN GENETICS Carpenter, M. L., Buenrostro, J. D., Valdiosera, C., Schroeder, H., Allentoft, M. E., Sikora, M., Rasmussen, M., Gravel, S., Guillen, S., Nekhrizov, G., Leshtakov, K., Dimitrova, D., Theodossiev, N., Pettener, D., Luiselli, D., Sandoval, K., Moreno-Estrada, A., Li, Y., Wang, J., Gilbert, M. T., Willerslev, E., Greenleaf, W. J., Bustamante, C. D. 2013; 93 (5): 852-864

    Abstract

    Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062-147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217-73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples.

    View details for DOI 10.1016/j.ajhg.2013.10.002

    View details for Web of Science ID 000326996600006

  • Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA Sequencing Libraries. American journal of human genetics Carpenter, M. L., Buenrostro, J. D., Valdiosera, C., Schroeder, H., Allentoft, M. E., Sikora, M., Rasmussen, M., Gravel, S., Guillén, S., Nekhrizov, G., Leshtakov, K., Dimitrova, D., Theodossiev, N., Pettener, D., Luiselli, D., Sandoval, K., Moreno-Estrada, A., Li, Y., Wang, J., Gilbert, M. T., Willerslev, E., Greenleaf, W. J., Bustamante, C. D. 2013; 93 (5): 852–64

    Abstract

    Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062-147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217-73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples.

    View details for PubMedID 24568772

  • Digital Polymerase Chain Reaction in an Array of Femtoliter Polydimethylsiloxane Microreactors ANALYTICAL CHEMISTRY Men, Y., Fu, Y., Chen, Z., Sims, P. A., Greenleaf, W. J., Huang, Y. 2012; 84 (10): 4262-4266

    Abstract

    We developed a simple, compact microfluidic device to perform high dynamic-range digital polymerase chain reaction (dPCR) in an array of isolated 36-femtoliter microreactors. The density of the microreactors exceeded 20000/mm(2). This device, made from polydimethylsiloxane (PDMS), allows the samples to be loaded into all microreactors simultaneously. The microreactors are completely sealed through the deformation of a PDMS membrane. The small volume of the microreactors ensures a compact device with high reaction efficiency and low reagent and sample consumption. Future potential applications of this platform include multicolor dPCR and massively parallel dPCR for next generation sequencing library preparation.

    View details for DOI 10.1021/ac300761n

    View details for Web of Science ID 000303965500005

    View details for PubMedID 22482776

  • Fluorogenic DNA sequencing in PDMS microreactors NATURE METHODS Sims, P. A., Greenleaf, W. J., Duan, H., Xie, S. 2011; 8 (7): 575-U84

    Abstract

    We developed a multiplex sequencing-by-synthesis method combining terminal phosphate-labeled fluorogenic nucleotides (TPLFNs) and resealable polydimethylsiloxane (PDMS) microreactors. In the presence of phosphatase, primer extension by DNA polymerase using nonfluorescent TPLFNs generates fluorophores, which are confined in the microreactors and detected. We immobilized primed DNA templates in the microreactors, then sequentially introduced one of the four identically labeled TPLFNs, sealed the microreactors and recorded a fluorescence image after template-directed primer extension. With cycle times of <10 min, we demonstrate 30 base reads with ∼99% raw accuracy. Our 'fluorogenic pyrosequencing' offers benefits of pyrosequencing, such as rapid turnaround, one-color detection and generation of native DNA, along with high detection sensitivity and simplicity of parallelization because simultaneous real-time monitoring of all microreactors is not required.

    View details for DOI 10.1038/NMETH.1629

    View details for Web of Science ID 000292194500021

    View details for PubMedID 21666670

  • AN OPTICAL APPARATUS FOR ROTATION AND TRAPPING METHODS IN ENZYMOLOGY, VOL 475: SINGLE MOLECULE TOOLS, PT B Gutierrez-Medina, B., Andreasson, J. O., Greenleaf, W. J., Laporta, A., Block, S. M. 2010; 475: 377-404

    Abstract

    We present details of the design, construction, and testing of a single-beam optical tweezers apparatus capable of measuring and exerting torque, as well as force, on microfabricated, optically anisotropic particles (an "optical torque wrench"). The control of angular orientation is achieved by rotating the linear polarization of a trapping laser with an electro-optic modulator (EOM), which affords improved performance over previous designs. The torque imparted to the trapped particle is assessed by measuring the difference between left- and right-circular components of the transmitted light, and constant torque is maintained by feeding this difference signal back into a custom-designed electronic servo loop. The limited angular range of the EOM (+/-180 degrees ) is extended by rapidly reversing the polarization once a threshold angle is reached, enabling the torque clamp to function over unlimited, continuous rotations at high bandwidth. In addition, we developed particles suitable for rotation in this apparatus using microfabrication techniques. Altogether, the system allows for the simultaneous application of forces (approximately 0.1-100 pN) and torques (approximately 1-10,000 pN nm) in the study of biomolecules. As a proof of principle, we demonstrate how our instrument can be used to study the supercoiling of single DNA molecules.

    View details for DOI 10.1016/S0076-6879(10)75015-1

    View details for Web of Science ID 000280733800015

    View details for PubMedID 20627165

    View details for PubMedCentralID PMC2965466

  • Applied force reveals mechanistic and energetic details of transcription termination CELL Larson, M. H., Greenleaf, W. J., Landick, R., Block, S. M. 2008; 132 (6): 971-982

    Abstract

    Transcription termination by bacterial RNA polymerase (RNAP) occurs at sequences coding for a GC-rich RNA hairpin followed by a U-rich tract. We used single-molecule techniques to investigate the mechanism by which three representative terminators (his, t500, and tR2) destabilize the elongation complex (EC). For his and tR2 terminators, loads exerted to bias translocation did not affect termination efficiency (TE). However, the force-dependent kinetics of release and the force-dependent TE of a mutant imply a forward translocation mechanism for the t500 terminator. Tension on isolated U-tracts induced transcript release in a manner consistent with RNA:DNA hybrid shearing. We deduce that different mechanisms, involving hypertranslocation or shearing, operate at terminators with different U-tracts. Tension applied to RNA at terminators suggests that closure of the final 2-3 hairpin bases destabilizes the hybrid and that competing RNA structures modulate TE. We propose a quantitative, energetic model that predicts the behavior for these terminators and mutant variants.

    View details for DOI 10.1016/j.cell.2008.01.027

    View details for Web of Science ID 000254273600016

    View details for PubMedID 18358810

    View details for PubMedCentralID PMC2295211

  • Direct observation of hierarchical folding in single riboswitch aptamers SCIENCE Greenleaf, W. J., Frieda, K. L., Foster, D. A., Woodside, M. T., Block, S. M. 2008; 319 (5863): 630-633

    Abstract

    Riboswitches regulate genes through structural changes in ligand-binding RNA aptamers. With the use of an optical-trapping assay based on in situ transcription by a molecule of RNA polymerase, single nascent RNAs containing pbuE adenine riboswitch aptamers were unfolded and refolded. Multiple folding states were characterized by means of both force-extension curves and folding trajectories under constant force by measuring the molecular contour length, kinetics, and energetics with and without adenine. Distinct folding steps correlated with the formation of key secondary or tertiary structures and with ligand binding. Adenine-induced stabilization of the weakest helix in the aptamer, the mechanical switch underlying regulatory action, was observed directly. These results provide an integrated view of hierarchical folding in an aptamer, demonstrating how complex folding can be resolved into constituent parts, and supply further insights into tertiary structure formation.

    View details for DOI 10.1126/science.1151298

    View details for Web of Science ID 000252772000044

    View details for PubMedID 18174398

    View details for PubMedCentralID PMC2640945

  • Single-molecule studies of RNA polymerase: Motoring along ANNUAL REVIEW OF BIOCHEMISTRY Herbert, K. M., Greenleaf, W. J., Block, S. M. 2008; 77: 149-176

    Abstract

    Single-molecule techniques have advanced our understanding of transcription by RNA polymerase (RNAP). A new arsenal of approaches, including single-molecule fluorescence, atomic-force microscopy, magnetic tweezers, and optical traps (OTs) have been employed to probe the many facets of the transcription cycle. These approaches supply fresh insights into the means by which RNAP identifies a promoter, initiates transcription, translocates and pauses along the DNA template, proofreads errors, and ultimately terminates transcription. Results from single-molecule experiments complement the knowledge gained from biochemical and genetic assays by facilitating the observation of states that are otherwise obscured by ensemble averaging, such as those resulting from heterogeneity in molecular structure, elongation rate, or pause propensity. Most studies to date have been performed with bacterial RNAP, but work is also being carried out with eukaryotic polymerase (Pol II) and single-subunit polymerases from bacteriophages. We discuss recent progress achieved by single-molecule studies, highlighting some of the unresolved questions and ongoing debates.

    View details for DOI 10.1146/annurev.biochem.77.073106.100741

    View details for Web of Science ID 000257596800008

    View details for PubMedID 18410247

    View details for PubMedCentralID PMC2854675

  • Molecule by molecule, the physics and chemistry of life: SMB 2007. Nature chemical biology Block, S. M., Larson, M. H., Greenleaf, W. J., Herbert, K. M., Guydosh, N. R., Anthony, P. C. 2007; 3 (4): 193-197

    Abstract

    Interdisciplinary work in the life sciences at the boundaries of biology, chemistry and physics is making enormous strides. This progress was showcased at the recent Single Molecule Biophysics conference.

    View details for PubMedID 17372599

  • High-resolution, single-molecule measurements of biomolecular motion ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE Greenleaf, W. J., Woodside, M. T., Block, S. M. 2007; 36: 171-190

    Abstract

    Many biologically important macromolecules undergo motions that are essential to their function. Biophysical techniques can now resolve the motions of single molecules down to the nanometer scale or even below, providing new insights into the mechanisms that drive molecular movements. This review outlines the principal approaches that have been used for high-resolution measurements of single-molecule motion, including centroid tracking, fluorescence resonance energy transfer, magnetic tweezers, atomic force microscopy, and optical traps. For each technique, the principles of operation are outlined, the capabilities and typical applications are examined, and various practical issues for implementation are considered. Extensions to these methods are also discussed, with an eye toward future application to outstanding biological problems.

    View details for DOI 10.1146/annurev.biophys.36.101106.101451

    View details for Web of Science ID 000247773000009

    View details for PubMedID 17328679

    View details for PubMedCentralID PMC1945240

  • High-resolution, single-molecule optical trapping measurements of transcription with basepair accuracy: Instrumentation and methods Conference on Optical Trapping and Optical Micromanipulation IV Greenleaf, W. J., Frieda, K. L., Abbondanzieri, E. A., Woodside, M. T., Block, S. M. SPIE-INT SOC OPTICAL ENGINEERING. 2007

    View details for DOI 10.1117/12.739631

    View details for Web of Science ID 000251162100004

  • Single-molecule, motion-based DNA sequencing using RNA polymerase SCIENCE Greenleaf, W. J., Block, S. M. 2006; 313 (5788): 801-801

    Abstract

    We present a method for sequencing DNA that relies on the motion of single RNA polymerase molecules. When a given nucleotide species limits the rate of transcription, polymerase molecules pause at positions corresponding to the rare base. An ultrastable optical trapping apparatus capable of base pair resolution was used to monitor transcription under limiting amounts of each of the four nucleotide species. From the aligned patterns of pauses recorded from as few as four molecules, we determined the DNA sequence. This proof of principle demonstrates that the motion of a processive nucleic acid enzyme may be used to extract sequence information directly from DNA.

    View details for DOI 10.1126/science.1130105

    View details for Web of Science ID 000239671300049

    View details for PubMedID 16902131

    View details for PubMedCentralID PMC1865524

  • Direct observation of base-pair stepping by RNA polymerase NATURE Abbondanzieri, E. A., Greenleaf, W. J., Shaevitz, J. W., Landick, R., Block, S. M. 2005; 438 (7067): 460-465

    Abstract

    During transcription, RNA polymerase (RNAP) moves processively along a DNA template, creating a complementary RNA. Here we present the development of an ultra-stable optical trapping system with ångström-level resolution, which we used to monitor transcriptional elongation by single molecules of Escherichia coli RNAP. Records showed discrete steps averaging 3.7 +/- 0.6 A, a distance equivalent to the mean rise per base found in B-DNA. By combining our results with quantitative gel analysis, we conclude that RNAP advances along DNA by a single base pair per nucleotide addition to the nascent RNA. We also determined the force-velocity relationship for transcription at both saturating and sub-saturating nucleotide concentrations; fits to these data returned a characteristic distance parameter equivalent to one base pair. Global fits were inconsistent with a model for movement incorporating a power stroke tightly coupled to pyrophosphate release, but consistent with a brownian ratchet model incorporating a secondary NTP binding site.

    View details for DOI 10.1038/nature04268

    View details for Web of Science ID 000233458200041

    View details for PubMedID 16284617

    View details for PubMedCentralID PMC1356566

  • Passive all-optical force clamp for high-resolution laser trapping PHYSICAL REVIEW LETTERS Greenleaf, W. J., Woodside, M. T., Abbondanzieri, E. A., Block, S. M. 2005; 95 (20)

    Abstract

    Optical traps are useful for studying the effects of forces on single molecules. Feedback-based force clamps are often used to maintain a constant load, but the response time of the feedback limits bandwidth and can introduce instability. We developed a novel force clamp that operates without feedback, taking advantage of the anharmonic region of the trapping potential where the differential stiffness vanishes. We demonstrate the utility of such a force clamp by measuring the unfolding of DNA hairpins and the effect of trap stiffness on opening distance and transition rates.

    View details for DOI 10.1103/PhysRevLett.95.208102

    View details for Web of Science ID 000233243500069

    View details for PubMedID 16384102

    View details for PubMedCentralID PMC1357091