Transcription Factor Occupancy Research Articles

Altered chromatin occupancy of master regulators underlies evolutionary divergence in the transcriptional landscape of erythroid differentiation.

Erythropoiesis is one of the best understood examples of cellular differentiation. Morphologically, erythroid differentiation proceeds in a nearly identical fashion between humans and mice, but recent evidence has shown that networks of gene expression governing this process are divergent between species. We undertook a systematic comparative analysis of six histone modifications and four transcriptional master regulators in primary proerythroblasts and erythroid cell lines to better understand the underlying basis of these transcriptional differences. Our analyses suggest that while chromatin structure across orthologous promoters is strongly conserved, subtle differences are associated with transcriptional divergence between species. Many transcription factor (TF) occupancy sites were poorly conserved across species (∼25% for GATA1, TAL1, and NFE2) but were more conserved between proerythroblasts and cell lines derived from the same species. We found that certain cis-regulatory modules co-occupied by GATA1, TAL1, and KLF1 are under strict evolutionary constraint and localize to genes necessary for erythroid cell identity. More generally, we show that conserved TF occupancy sites are indicative of active regulatory regions and strong gene expression that is sustained during maturation. Our results suggest that evolutionary turnover of TF binding sites associates with changes in the underlying chromatin structure, driving transcriptional divergence. We provide examples of how this framework can be applied to understand epigenomic variation in specific regulatory regions, such as the β-globin gene locus. Our findings have important implications for understanding epigenomic changes that mediate variation in cellular differentiation across species, while also providing a valuable resource for studies of hematopoiesis.

Transcriptional regulation of the human TNFSF11 gene in T cells via a cell type-selective set of distal enhancers.

In addition to osteoblast lineage cells, the TNF-like factor receptor activator of NF-κB ligand (RANKL) is expressed in both B and T cells and may play a role in bone resorption. Rankl gene (Tnfsf11) expression in mouse T cells is mediated through multiple distal elements marked by increased transcription factor occupancy, histone tail acetylation, and RNA polymerase II recruitment. Little is known, however, of the regulation of human TNFSF11 in T cells. Accordingly, we examined the consequence of T cell activation on the expression of this factor both in Jurkat cells and in primary human T cells. We then explored the mechanism of this regulation by scanning over 400 kb of DNA surrounding the TNFSF11 locus for regulatory enhancers using ChIP-chip analysis. Histone H3/H4 acetylation enrichment identified putative regulatory regions located between -170 and -220 kb upstream of the human TNFSF11 TSS that we designated the human T cell control region (hTCCR). This region showed high sequence conservation with the mouse TCCR. Inhibition of MEK1/2 by U0126 resulted in decreased RANKL expression suggesting that stimulation through MEK1/2 was a prerequisite. ChIP-chip analysis also revealed that c-FOS was recruited to the hTCCR as well. Importantly, both the human TNFSF11 D5a/b (RLD5a/b) enhancer and segments of the hTCCR mediated robust inducible reporter activity following TCR activation. Finally, SNPs implicated in diseases characterized by dysregulated BMD co-localized to the hTCCR region. We conclude that the hTCCR region contains a cell-selective set of enhancers that plays an integral role in the transcriptional regulation of the TNFSF11 gene in human T cells.

Conservation of trans-acting circuitry during mammalian regulatory evolution.

The basic body plan and major physiological axes have been highly conserved during mammalian evolution, yet only a small fraction of the human genome sequence appears to be subject to evolutionary constraint. To quantify cis- versus trans-acting contributions to mammalian regulatory evolution, we performed genomic DNase I footprinting of the mouse genome across 25 cell and tissue types, collectively defining ∼8.6 million transcription factor (TF) occupancy sites at nucleotide resolution. Here we show that mouse TF footprints conjointly encode a regulatory lexicon that is ∼95% similar with that derived from human TF footprints. However, only ∼20% of mouse TF footprints have human orthologues. Despite substantial turnover of the cis-regulatory landscape, nearly half of all pairwise regulatory interactions connecting mouse TF genes have been maintained in orthologous human cell types through evolutionary innovation of TF recognition sequences. Furthermore, the higher-level organization of mouse TF-to-TF connections into cellular network architectures is nearly identical with human. Our results indicate that evolutionary selection on mammalian gene regulation is targeted chiefly at the level of trans-regulatory circuitry, enabling and potentiating cis-regulatory plasticity.Supplementary informationThe online version of this article (doi:10.1038/nature13972) contains supplementary material, which is available to authorized users.

Genomics: mice in the ENCODE spotlight.

Following on from affiliated projects in humans and model invertebrates, the Mouse ENCODE Project presents comprehensive data sets on genome regulation in this key mammalian model. See Articles p.355, p.365, p.371 & Letter p.402 The mouse is the premier model organism in biomedical research. To gain greater insights into the shared and species-specific transcriptional and cellular regulatory programs, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types. These finding are compared with the corresponding human data to confirm substantial conservation in the newly annotated potential functional sequences, and to reveal pronounced divergence of other sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. The data and their analyses provide a valuable resource for research into mammalian biology and mechanisms of human diseases. Having generated genomic DNase I footprinting data of the mouse genome across 25 cell and tissue types, these authors use these data to quantify cis-versus-trans regulatory contributions to mammalian regulatory evolution. They describe more than 600 motifs that collectively are over 95% similar to that recognized in vivo by human transcription factors (TFs). Despite substantial turnover of the cis-regulatory landscape around each TF gene, nearly half of all pairwise regulatory interactions connecting mouse TF genes have been maintained in orthologous human cell types through evolutionary innovation of TF recognition sequences. Conservation between mouse and human TF regulatory networks is particularly similar at the highest organization level. The work was performed as part of the mouse ENCODE project. As part of the mouse ENCODE project Mike Snyder and colleagues studied the genome-wide transcription factor (TF) occupancy repertoires, associated epigenetic signals, and TF co-association patterns in mice and humans to broaden our understanding of the evolution of gene regulation mechanisms in mammals. The results indicate that although many aspects of TF occupied sequences are conserved in both species, the extent to which orthologous DNA segments are bound by orthologous TFs in human and mouse varies both among TFs and with genomic location. Importantly, TF occupied sequences with conserved occupancy tend to be pleiotropic; they are also enriched for single nucleotide variants (SNVs) that are known to have regulatory potential or are associated with known phenotypes. As part of the mouse ENCODE project, David Gilbert and colleagues study the relationship between replication timing and higher order chromatin domains in mouse and human. They find that boundaries of replication domains — domains within the genome which replicate at the same time — share a near one-to-one correlation with topology associated domains. These and other results reconcile cell-type specific sub-nuclear compartmentalization with developmentally stable chromosome domains and offer a unified model for large-scale chromosome structure and function.

Principles of regulatory information conservation between mouse and human.

To broaden our understanding of the evolution of gene regulation mechanisms, we generated occupancy profiles for 34 orthologous transcription factors (TFs) in human–mouse erythroid progenitor, lymphoblast and embryonic stem-cell lines. By combining the genome-wide transcription factor occupancy repertoires, associated epigenetic signals, and co-association patterns, here we deduce several evolutionary principles of gene regulatory features operating since the mouse and human lineages diverged. The genomic distribution profiles, primary binding motifs, chromatin states, and DNA methylation preferences are well conserved for TF-occupied sequences. However, the extent to which orthologous DNA segments are bound by orthologous TFs varies both among TFs and with genomic location: binding at promoters is more highly conserved than binding at distal elements. Notably, occupancy-conserved TF-occupied sequences tend to be pleiotropic; they function in several tissues and also co-associate with many TFs. Single nucleotide variants at sites with potential regulatory functions are enriched in occupancy-conserved TF-occupied sequences.Supplementary informationThe online version of this article (doi:10.1038/nature13985) contains supplementary material, which is available to authorized users.

Regression analysis of combined gene expression regulation in acute myeloid leukemia.

Gene expression is a combinatorial function of genetic/epigenetic factors such as copy number variation (CNV), DNA methylation (DM), transcription factors (TF) occupancy, and microRNA (miRNA) post-transcriptional regulation. At the maturity of microarray/sequencing technologies, large amounts of data measuring the genome-wide signals of those factors became available from Encyclopedia of DNA Elements (ENCODE) and The Cancer Genome Atlas (TCGA). However, there is a lack of an integrative model to take full advantage of these rich yet heterogeneous data. To this end, we developed RACER (Regression Analysis of Combined Expression Regulation), which fits the mRNA expression as response using as explanatory variables, the TF data from ENCODE, and CNV, DM, miRNA expression signals from TCGA. Briefly, RACER first infers the sample-specific regulatory activities by TFs and miRNAs, which are then used as inputs to infer specific TF/miRNA-gene interactions. Such a two-stage regression framework circumvents a common difficulty in integrating ENCODE data measured in generic cell-line with the sample-specific TCGA measurements. As a case study, we integrated Acute Myeloid Leukemia (AML) data from TCGA and the related TF binding data measured in K562 from ENCODE. As a proof-of-concept, we first verified our model formalism by 10-fold cross-validation on predicting gene expression. We next evaluated RACER on recovering known regulatory interactions, and demonstrated its superior statistical power over existing methods in detecting known miRNA/TF targets. Additionally, we developed a feature selection procedure, which identified 18 regulators, whose activities clustered consistently with cytogenetic risk groups. One of the selected regulators is miR-548p, whose inferred targets were significantly enriched for leukemia-related pathway, implicating its novel role in AML pathogenesis. Moreover, survival analysis using the inferred activities identified C-Fos as a potential AML prognostic marker. Together, we provided a novel framework that successfully integrated the TCGA and ENCODE data in revealing AML-specific regulatory program at global level.

Dynamic GATA4 enhancers shape the chromatin landscape central to heart development and disease

How stage-specific enhancer dynamics modulate gene expression patterns essential for organ development, homesostasis, and disease is not well understood. Here, we addressed this question by mapping chromatin occupancy of GATA4—a master cardiac transcription factor—in heart development and disease. We find that GATA4 binds and participaes in establishing active chromatin regions by stimulating H3K27ac deposition, which facilitates GATA4-driven gene expression. GATA4 chromatin occupancy changes markedly between fetal and adult heart, with a limitted binding sites overlap. Cardiac stress restored GATA4 occupancy to a subset of fetal sites, but many stress-associated GATA4 binding sites localized to loci not occupied by GATA4 during normal heart development. Collectively, our data show that dynamic, context-specific transcription factors occupancy underlies stage-specific events in development, homeostasis, and disease.

DNase Footprint Signatures Are Dictated by Factor Dynamics and DNA Sequence

Genomic footprinting has emerged as an unbiased discovery method for transcription factor (TF) occupancy at cognate DNA in vivo. A basic premise of footprinting is that sequence-specific TF-DNA interactions are associated with localized resistance to nucleases, leaving observable signatures of cleavage within accessible chromatin. This phenomenon is interpreted to imply protection of the critical nucleotides by the stably bound protein factor. However, this model conflicts with previous reports of many TFs exchanging with specific binding sites in living cells on a timescale of seconds. We show that TFs with short DNA residence times have no footprints at bound motif elements. Moreover, the nuclease cleavage profile within a footprint originates from the DNA sequence in the factor-binding site, rather than from the protein occupying specific nucleotides. These findings suggest a revised understanding of TF footprinting and reveal limitations in comprehensive reconstruction of the TF regulatory network using this approach.

SignalSpider: probabilistic pattern discovery on multiple normalized ChIP-Seq signal profiles.

Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-Seq) measures the genome-wide occupancy of transcription factors in vivo. Different combinations of DNA-binding protein occupancies may result in a gene being expressed in different tissues or at different developmental stages. To fully understand the functions of genes, it is essential to develop probabilistic models on multiple ChIP-Seq profiles to decipher the combinatorial regulatory mechanisms by multiple transcription factors. In this work, we describe a probabilistic model (SignalSpider) to decipher the combinatorial binding events of multiple transcription factors. Comparing with similar existing methods, we found SignalSpider performs better in clustering promoter and enhancer regions. Notably, SignalSpider can learn higher-order combinatorial patterns from multiple ChIP-Seq profiles. We have applied SignalSpider on the normalized ChIP-Seq profiles from the ENCODE consortium and learned model instances. We observed different higher-order enrichment and depletion patterns across sets of proteins. Those clustering patterns are supported by Gene Ontology (GO) enrichment, evolutionary conservation and chromatin interaction enrichment, offering biological insights for further focused studies. We also proposed a specific enrichment map visualization method to reveal the genome-wide transcription factor combinatorial patterns from the models built, which extend our existing fine-scale knowledge on gene regulation to a genome-wide level. The matrix-algebra-optimized executables and source codes are available at the authors' websites: http://www.cs.toronto.edu/∼wkc/SignalSpider.

Mapping and Dynamics of Regulatory DNA and Transcription Factor Networks in A. thaliana

Our understanding of gene regulation in plants is constrained by our limited knowledge of plant cis-regulatory DNA and its dynamics. We mapped DNase I hypersensitive sites (DHSs) in A. thaliana seedlings and used genomic footprinting to delineate ∼ 700,000 sites of in vivo transcription factor (TF) occupancy at nucleotide resolution. We show that variation associated with 72 diverse quantitative phenotypes localizes within DHSs. TF footprints encode an extensive cis-regulatory lexicon subject to recent evolutionary pressures, and widespread TF binding within exons may have shaped codon usage patterns. The architecture of A. thaliana TF regulatory networks is strikingly similar to that of animals in spite of diverged regulatory repertoires. We analyzed regulatory landscape dynamics during heat shock and photomorphogenesis, disclosing thousands of environmentally sensitive elements and enabling mapping of key TF regulatory circuits underlying these fundamental responses. Our results provide an extensive resource for the study of A. thaliana gene regulation and functional biology.

Architectural proteins CTCF and cohesin have distinct roles in modulating the higher order structure and expression of the CFTR locus.

Higher order chromatin structures across the genome are maintained in part by the architectural proteins CCCTC binding factor (CTCF) and the cohesin complex, which co-localize at many sites across the genome. Here, we examine the role of these proteins in mediating chromatin structure at the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encompasses nearly 200 kb flanked by CTCF-binding enhancer-blocking insulator elements and is regulated by cell-type-specific intronic enhancers, which loop to the promoter in the active locus. SiRNA-mediated depletion of CTCF or the cohesin component, RAD21, showed that these two factors have distinct roles in regulating the higher order organization of CFTR. CTCF mediates the interactions between CTCF/cohesin binding sites, some of which have enhancer-blocking insulator activity. Cohesin shares this tethering role, but in addition stabilizes interactions between the promoter and cis-acting intronic elements including enhancers, which are also dependent on the forkhead box A1/A2 (FOXA1/A2) transcription factors (TFs). Disruption of the three-dimensional structure of the CFTR gene by depletion of CTCF or RAD21 increases gene expression, which is accompanied by alterations in histone modifications and TF occupancy across the locus, and causes internalization of the gene from the nuclear periphery.

Promoter-proximal transcription factor binding is transcriptionally active when coupled with nucleosome repositioning in immediate vicinity.

Previous studies have analyzed patterns of transcription, transcription factor (TF) binding or mapped nucleosome occupancy across the genome. These suggest that the three aspects are genetically connected but the cause and effect relationships are still unknown. For example, physiologic TF binding studies involve many TFs, consequently, it is difficult to assign nucleosome reorganization to the binding site occupancy of any particular TF. Therefore, several aspects remain unclear: does TF binding influence nucleosome (re)organizations locally or impact the chromatin landscape at a more global level; are all or only a fraction of TF binding a result of reorganization in nucleosome occupancy and do all TF binding and associated changes in nucleosome occupancy result in altered gene expression? With these in mind, following characterization of two states (before and after induction of a single TF of choice) we determined: (i) genomic binding sites of the TF, (ii) promoter nucleosome occupancy and (iii) transcriptome profiles. Results demonstrated that promoter-proximal TF binding influenced expression of the target gene when it was coupled to nucleosome repositioning at or close to its binding site in most cases. In contrast, only in few cases change in target gene expression was found when TF binding occurred without local nucleosome reorganization.

Fully automated high-throughput chromatin immunoprecipitation for ChIP-seq: identifying ChIP-quality p300 monoclonal antibodies.

Chromatin immunoprecipitation coupled with DNA sequencing (ChIP-seq) is the major contemporary method for mapping in vivo protein-DNA interactions in the genome. It identifies sites of transcription factor, cofactor and RNA polymerase occupancy, as well as the distribution of histone marks. Consortia such as the ENCyclopedia Of DNA Elements (ENCODE) have produced large datasets using manual protocols. However, future measurements of hundreds of additional factors in many cell types and physiological states call for higher throughput and consistency afforded by automation. Such automation advances, when provided by multiuser facilities, could also improve the quality and efficiency of individual small-scale projects. The immunoprecipitation process has become rate-limiting, and is a source of substantial variability when performed manually. Here we report a fully automated robotic ChIP (R-ChIP) pipeline that allows up to 96 reactions. A second bottleneck is the dearth of renewable ChIP-validated immune reagents, which do not yet exist for most mammalian transcription factors. We used R-ChIP to screen new mouse monoclonal antibodies raised against p300, a histone acetylase, well-known as a marker of active enhancers, for which ChIP-competent monoclonal reagents have been lacking. We identified, validated for ChIP-seq, and made publicly available a monoclonal reagent called ENCITp300-1.

Transcription, epigenetics and ameliorative strategies in Huntington's Disease: a genome-wide perspective.

Transcriptional dysregulation in Huntington’s disease (HD) is an early event that shapes the brain transcriptome by both the depletion and ectopic activation of gene products that eventually affect survival and neuronal functions. Disruption in the activity of gene expression regulators, such as transcription factors, chromatin-remodeling proteins, and noncoding RNAs, accounts for the expression changes observed in multiple animal and cellular models of HD and in samples from patients. Here, I review the recent advances in the study of HD transcriptional dysregulation and its causes to finally discuss the possible implications in ameliorative strategies from a genome-wide perspective. To date, the use of genome-wide approaches, predominantly based on microarray platforms, has been successful in providing an extensive catalog of differentially regulated genes, including biomarkers aimed at monitoring the progress of the pathology. Although still incipient, the introduction of combined next-generation sequencing techniques is enhancing our comprehension of the mechanisms underlying altered transcriptional dysregulation in HD by providing the first genomic landscapes associated with epigenetics and the occupancy of transcription factors. In addition, the use of genome-wide approaches is becoming more and more necessary to evaluate the efficacy and safety of ameliorative strategies and to identify novel mechanisms of amelioration that may help in the improvement of current preclinical therapeutics. Finally, the major conclusions obtained from HD transcriptomics studies have the potential to be extrapolated to other neurodegenerative disorders.

Dynamic shifts in transcription factor occupancy drive reprogramming of gene expression during hematopoietic differentiation (LB181)

Global views of biochemical mechanisms regulating gene expression during cell lineage commitment and maturation can be obtained by analyzing chromatin immunoprecipitation and gene expression on a genome-wide scale, enabled by massively parallel sequencing. We have conducted ChIP-seq and RNA-seq assays on cells from many informative stages of mouse hematopoiesis, ranging from multilineage progenitor cells to mature megakaryocytes and erythroblasts. We find that histone modification patterns characteristic of repressed or active chromatin are established during lineage commitment. The active chromatin forms an accessible stage on which many transcription factors change their binding patterns dynamically. Sites of binding by transcription factors, such as GATA2/1 and TAL1, change both during commitment to specific lineages and during terminal maturation. These shifts are strongly associated with changes in gene expression, allowing active proliferation in early stages and dramatic changes in morphology and function during maturation. Motif searches coupled with ChIP-seq against many transcription factors reveal co-associating proteins that help drive the dynamic binding of the key factors. Grant Funding Source: Supported by NIH grants R01DK065806 and RC2HG005573

The Transcriptional Complex Between the BCL2 i-Motif and hnRNP LL Is a Molecular Switch for Control of Gene Expression That Can Be Modulated by Small Molecules

In a companion paper (DOI: 10.021/ja410934b) we demonstrate that the C-rich strand of the cis-regulatory element in the BCL2 promoter element is highly dynamic in nature and can form either an i-motif or a flexible hairpin. Under physiological conditions these two secondary DNA structures are found in an equilibrium mixture, which can be shifted by the addition of small molecules that trap out either the i-motif (IMC-48) or the flexible hairpin (IMC-76). In cellular experiments we demonstrate that the addition of these molecules has opposite effects on BCL2 gene expression and furthermore that these effects are antagonistic. In this contribution we have identified a transcriptional factor that recognizes and binds to the BCL2 i-motif to activate transcription. The molecular basis for the recognition of the i-motif by hnRNP LL is determined, and we demonstrate that the protein unfolds the i-motif structure to form a stable single-stranded complex. In subsequent experiments we show that IMC-48 and IMC-76 have opposite, antagonistic effects on the formation of the hnRNP LL–i-motif complex as well as on the transcription factor occupancy at the BCL2 promoter. For the first time we propose that the i-motif acts as a molecular switch that controls gene expression and that small molecules that target the dynamic equilibrium of the i-motif and the flexible hairpin can differentially modulate gene expression.

Global discovery of erythroid long noncoding RNAs reveals novel regulators of red cell maturation

AbstractErythropoiesis is regulated at multiple levels to ensure the proper generation of mature red cells under multiple physiological conditions. To probe the contribution of long noncoding RNAs (lncRNAs) to this process, we examined >1 billion RNA-seq reads of polyadenylated and nonpolyadenylated RNA from differentiating mouse fetal liver red blood cells and identified 655 lncRNA genes including not only intergenic, antisense, and intronic but also pseudogene and enhancer loci. More than 100 of these genes are previously unrecognized and highly erythroid specific. By integrating genome-wide surveys of chromatin states, transcription factor occupancy, and tissue expression patterns, we identify multiple lncRNAs that are dynamically expressed during erythropoiesis, show epigenetic regulation, and are targeted by key erythroid transcription factors GATA1, TAL1, or KLF1. We focus on 12 such candidates and find that they are nuclear-localized and exhibit complex developmental expression patterns. Depleting them severely impaired erythrocyte maturation, inhibiting cell size reduction and subsequent enucleation. One of them, alncRNA-EC7, is transcribed from an enhancer and is specifically needed for activation of the neighboring gene encoding BAND 3. Our study provides an annotated catalog of erythroid lncRNAs, readily available through an online resource, and shows that diverse types of lncRNAs participate in the regulatory circuitry underlying erythropoiesis.Key Points

CgChIP: a cell type- and gene-specific method for chromatin analysis.

Hox and other homeobox-containing genes encode critical transcriptional regulators of animal development. Although these genes are well known for their roles in the body axis and appendage development, little is known regarding the mechanisms by which these factors influence chromatin landscapes. Chromatin structure can have a profound influence on gene expression during animal body formation. However, when applied to developing embryos, conventional chromatin analysis of genes and cis-regulatory modules (CRMs) typically lacks the required cell type-specific resolution due to the heterogeneous nature of animal bodies. Here we present a strategy to analyze both the composition and conformation of in vivo-tagged CRM sequences in a cell type-specific manner, using as a system Drosophila embryos. We term this method cgChIP (cell- and gene-specific Chromatin Immunoprecipitation) by which we access and analyze regulatory chromatin in specific cell types. cgChIP is an in vivo method designed to analyze genetic elements derived from limited cell populations. cgChIP can be used for both the analysis of chromatin structure (e.g., long-distance interactions between DNA elements) and the composition of histones and histone modifications and the occupancy of transcription factors and chromatin modifiers. This method was applied to the Hox target gene Distalless (Dll), which encodes for a homeodomain-containing transcription factor critical for the formation of appendages in Drosophila. However, cgChIP can be applied in diverse animal models to better dissect CRM-dependent gene regulation and body formation in developing animals.

Transposition of Native Chromatin for Fast and Sensitive Mulitmodal Analysis of Chromatin Architecture

Eukaryotic genomes are hierarchically packaged into chromatin, and the nature of this packaging plays a central role in gene regulation. Major insights into the nucleoprotein structure of chromatin have come from high-throughput, genome-wide methods for separately assaying the chromatin accessibility (open chromatin), nucleosome positioning, and transcription factor (TF) occupancy. However, published protocols for these existing methods require millions of cells as starting material, complex and time-consuming sample preparations, and cannot simultaneously probe the interplay of nucleosome positioning, chromatin accessibility, and TF binding. These limitations 1) average over and “drown out” heterogeneity in cellular populations, 2) often require cells to be grown ex vivo to obtain sufficient biomaterials, modulating the epigenetic state in unknown ways, and 3) often prevent application of these assays to well-defined clinical samples, precluding generation of “personal epigenomes” in diagnostic timescales. Here we describe an Assay for Transposase Accessible Chromatin using sequencing (ATAC-seq) based on direct in vitro transposition of sequencing adapters into native chromatin - as a rapid and sensitive method for integrative epigenomic analysis. ATAC-seq captures open chromatin sites using a simple 2-step protocol from 500 to 50,000 cells, and reveals the interplay between genomic locations of open chromatin, DNA binding proteins, individual nucleosomes, and higher-order compaction at regulatory regions. Using this method, we discover classes of DNA binding factor that strictly avoid, can tolerate, or tend to overlap with nucleosomes. The method enabled serial daily epigenomes of resting human T cells to be observed using standard blood draws. We show that ATAC-seq is compatible with FACS, enabling studies on carefully sorted and rare subpopulations from primary tissues. The combination of speed, simplicity, and low input requirements of ATAC-seq will enable new gene regulatory insights into biology and medicine.

Transcription Factor Occupancy Can Mediate Active Turnover of DNA Methylation at Regulatory Regions

Distal regulatory elements, including enhancers, play a critical role in regulating gene activity. Transcription factor binding to these elements correlates with Low Methylated Regions (LMRs) in a process that is poorly understood. Here we ask whether and how actual occupancy of DNA-binding factors is linked to DNA methylation at the level of individual molecules. Using CTCF as an example, we observe that frequency of binding correlates with the likelihood of a demethylated state and sites of low occupancy display heterogeneous DNA methylation within the CTCF motif. In line with a dynamic model of binding and DNA methylation turnover, we find that 5-hydroxymethylcytosine (5hmC), formed as an intermediate state of active demethylation, is enriched at LMRs in stem and somatic cells. Moreover, a significant fraction of changes in 5hmC during differentiation occurs at these regions, suggesting that transcription factor activity could be a key driver for active demethylation. Since deletion of CTCF is lethal for embryonic stem cells, we used genetic deletion of REST as another DNA-binding factor implicated in LMR formation to test this hypothesis. The absence of REST leads to a decrease of hydroxymethylation and a concomitant increase of DNA methylation at its binding sites. These data support a model where DNA-binding factors can mediate turnover of DNA methylation as an integral part of maintenance and reprogramming of regulatory regions.