Chromatin Accessibility Mapping Research Articles

High-Resolution Genome-Wide Mapping of Chromatin Accessibility and Torsional Stress.

Torsional stress in chromatin plays a fundamental role in cellular functions, influencing key processes such as transcription, replication, and chromatin organization. Transcription and other processes may generate and be regulated by torsional stress. In the genome, the interplay of these processes creates complicated patterns of both positive (+) and negative (-) torsion. However, a challenge in generating an accurate torsion map is determining the zero-torsion baseline signal, which is conflated with chromatin accessibility. Here, we introduce a high-resolution method based on the intercalator trimethylpsoralen (TMP) to address this challenge. We describe a method to establish the zero-torsion baseline while preserving the chromatin state of the genome. This approach enables both high-resolution mapping of accessibility and torsional stress in chromatin in the cell. Our analysis reveals transcription-generated torsional domains consistent with the twin-supercoiled-domain model of transcription and suggests a role for torsional stress in recruiting topoisomerases and in regulating 3D genome architecture via cohesin. This new method provides a potential path forward for using TMP to measure torsional stress in the genome without the confounding contribution of accessibility in chromatin.

Single-cell transcriptome and chromatin accessibility mapping of upper lip and primary palate fusion.

Cleft lip and/or primary palate (CL/P) represent a prevalent congenital malformation, the aetiology of which is highly intricate. Although it is generally accepted that the condition arises from failed fusion between the upper lip and primary palate, the precise mechanism underlying this fusion process remains enigmatic. In this study, we utilized transposase-accessible chromatin sequencing (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) to interrogate lambdoidal junction tissue derived from C57BL/6J mouse embryos at critical stages of embryogenesis (10.5, 11.5 and 12.5 embryonic days). We successfully identified distinct subgroups of mesenchymal and ectodermal cells involved in the fusion process and characterized their unique transcriptional profiles. Furthermore, we conducted cell differentiation trajectory analysis, revealing a dynamic repertoire of genes that are sequentially activated or repressed during pseudotime, facilitating the transition of relevant cell types. Additionally, we employed scATAC data to identify key genes associated with the fusion process and demonstrated differential chromatin accessibility across major cell types. Finally, we constructed a dynamic intercellular communication network and predicted upstream transcriptional regulators of critical genes involved in important signalling pathways. Our findings provide a valuable resource for future studies on upper lip and primary palate development, as well as congenital defects.

Detailed mapping of chromatin accessibility and transcriptome of the healthy breast.

Detailed mapping of chromatin accessibility and transcriptome of the healthy breast.

Variants in tubule epithelial regulatory elements mediate most heritable differences in human kidney function.

Kidney disease is highly heritable; however, the causal genetic variants, the cell types in which these variants function, and the molecular mechanisms underlying kidney disease remain largely unknown. To identify genetic loci affecting kidney function, we performed a GWAS using multiple kidney function biomarkers and identified 462 loci. To begin to investigate how these loci affect kidney function, we generated single-cell chromatin accessibility (scATAC-seq) maps of the human kidney and identified candidate cis-regulatory elements (cCREs) for kidney podocytes, tubule epithelial cells, and kidney endothelial, stromal, and immune cells. Kidney tubule epithelial cCREs explained 58% of kidney function SNP-heritability and kidney podocyte cCREs explained an additional 6.5% of SNP-heritability. In contrast, little kidney function heritability was explained by kidney endothelial, stromal, or immune cell-specific cCREs. Through functionally informed fine-mapping, we identified putative causal kidney function variants and their corresponding cCREs. Using kidney scATAC-seq data, we created a deep learning model (which we named ChromKid) to predict kidney cell type-specific chromatin accessibility from sequence. ChromKid and allele specific kidney scATAC-seq revealed that many fine-mapped kidney function variants locally change chromatin accessibility in tubule epithelial cells. Enhancer assays confirmed that fine-mapped kidney function variants alter tubule epithelial regulatory element function. To map the genes which these regulatory elements control, we used CRISPR interference (CRISPRi) to target these regulatory elements in tubule epithelial cells and assessed changes in gene expression. CRISPRi of enhancers harboring kidney function variants regulated NDRG1 and RBPMS expression. Thus, inherited differences in tubule epithelial NDRG1 and RBPMS expression may predispose to kidney disease in humans. We conclude that genetic variants affecting tubule epithelial regulatory element function account for most SNP-heritability of human kidney function. This work provides an experimental approach to identify the variants, regulatory elements, and genes involved in polygenic disease.

Single-nucleus chromatin landscape dataset of mouse brain development and aging

The development and aging of the brain constitute a lifelong dynamic process, marked by structural and functional changes that entail highly coordinated cellular differentiation and epigenetic regulatory mechanisms. Chromatin accessibility serves as the foundational basis for genetic activity. However, the holistic and dynamic chromatin landscape that spans various brain regions throughout development and ageing remains predominantly unexplored. In this study, we employed single-nucleus ATAC-seq to generate comprehensive chromatin accessibility maps, incorporating data from 69,178 cells obtained from four distinct brain regions – namely, the olfactory bulb (OB), cerebellum (CB), prefrontal cortex (PFC), and hippocampus (HP) – across key developmental time points at 7 P, 3 M, 12 M, and 18 M. We delineated the distribution of cell types across different age stages and brain regions, providing insight into chromatin accessible regions and key transcription factors specific to different cell types. Our data contribute to understanding the epigenetic basis of the formation of different brain regions, providing a dynamic landscape and comprehensive resource for revealing gene regulatory programs during brain development and aging.

The single-cell chromatin landscape in gonadal cell lineage specification

Gonad development includes sex determination and divergent maturation of the testes and ovaries. Recent advances in measuring gene expression in single cells are providing new insights into this complex process. However, the underlying epigenetic regulatory mechanisms remain unclear. Here, we profiled chromatin accessibility in mouse gonadal cells of both sexes from embryonic day 11.5 to 14.5 using single-cell assay for transposase accessible chromatin by sequencing (scATAC-seq). Our results showed that individual cell types can be inferred by the chromatin landscape, and that cells can be temporally ordered along developmental trajectories. Integrative analysis of transcriptomic and chromatin-accessibility maps identified multiple putative regulatory elements proximal to key gonadal genes Nr5a1, Sox9 and Wt1. We also uncover cell type-specific regulatory factors underlying cell type specification. Overall, our results provide a better understanding of the epigenetic landscape associated with the progressive restriction of cell fates in the gonad.

The super-pangenome of Populus unveils genomic facets for its adaptation and diversification in widespread forest trees

Understanding the underlying mechanisms and links between genome evolution and adaptive innovations stands as a key goal in evolutionary studies. Poplars, among the world’s most widely distributed and cultivated trees, exhibit extensive phenotypic diversity and environmental adaptability. In this study, we present a genus-level super-pangenome comprising 19 Populus genomes, revealing the likely pivotal role of private genes in facilitating local environmental and climate adaptation. Through the integration of pangenomes with transcriptomes, methylomes, and chromatin accessibility mapping, we unveil that the evolutionary trajectories of pangenes and duplicated genes are closely linked to local genomic landscapes of regulatory and epigenetic architectures, notably CG methylation in gene-body regions. Further comparative genomic analyses have enabled the identification of 142 202 structural variants across species that intersect with a significant number of genes and contribute substantially to both phenotypic and adaptive divergence. We have experimentally validated a ∼180-bp presence/absence variant affecting the expression of the CUC2 gene, crucial for leaf serration formation. Finally, we developed a user-friendly web-based tool encompassing the multi-omics resources associated with the Populus super-pangenome (http://www.populus-superpangenome.com). Together, the present pioneering super-pangenome resource in forest trees not only aids in the advancement of breeding efforts of this globally important tree genus but also offers valuable insights into potential avenues for comprehending tree biology.

The chromatin landscape of the euryarchaeon Haloferax volcanii

BackgroundArchaea, together with Bacteria, represent the two main divisions of life on Earth, with many of the defining characteristics of the more complex eukaryotes tracing their origin to evolutionary innovations first made in their archaeal ancestors. One of the most notable such features is nucleosomal chromatin, although archaeal histones and chromatin differ significantly from those of eukaryotes, not all archaea possess histones and it is not clear if histones are a main packaging component for all that do. Despite increased interest in archaeal chromatin in recent years, its properties have been little studied using genomic tools.ResultsHere, we adapt the ATAC-seq assay to archaea and use it to map the accessible landscape of the genome of the euryarchaeote Haloferax volcanii. We integrate the resulting datasets with genome-wide maps of active transcription and single-stranded DNA (ssDNA) and find that while H. volcanii promoters exist in a preferentially accessible state, unlike most eukaryotes, modulation of transcriptional activity is not associated with changes in promoter accessibility. Applying orthogonal single-molecule footprinting methods, we quantify the absolute levels of physical protection of H. volcanii and find that Haloferax chromatin is similarly or only slightly more accessible, in aggregate, than that of eukaryotes. We also evaluate the degree of coordination of transcription within archaeal operons and make the unexpected observation that some CRISPR arrays are associated with highly prevalent ssDNA structures.ConclusionsOur results provide the first comprehensive maps of chromatin accessibility and active transcription in Haloferax across conditions and thus a foundation for future functional studies of archaeal chromatin.

Epigenomic analysis of formalin-fixed paraffin-embedded samples by CUT&Tag

For more than a century, formalin-fixed paraffin-embedded (FFPE) sample preparation has been the preferred method for long-term preservation of biological material. However, the use of FFPE samples for epigenomic studies has been difficult because of chromatin damage from long exposure to high concentrations of formaldehyde. Previously, we introduced Cleavage Under Targeted Accessible Chromatin (CUTAC), an antibody-targeted chromatin accessibility mapping protocol based on CUT&Tag. Here we show that simple modifications of our CUTAC protocol either in single tubes or directly on slides produce high-resolution maps of paused RNA Polymerase II at enhancers and promoters using FFPE samples. We find that transcriptional regulatory element differences produced by FFPE-CUTAC distinguish between mouse brain tumors and identify and map regulatory element markers with high confidence and precision, including microRNAs not detectable by RNA-seq. Our simple workflows make possible affordable epigenomic profiling of archived biological samples for biomarker identification, clinical applications and retrospective studies.

Dynamic changes in whole genome DNA methylation, chromatin and gene expression during mouse lens differentiation

BackgroundCellular differentiation is marked by temporally and spatially coordinated gene expression regulated at multiple levels. DNA methylation represents a universal mechanism to control chromatin organization and its accessibility. Cytosine methylation of CpG dinucleotides regulates binding of methylation-sensitive DNA-binding transcription factors within regulatory regions of transcription, including promoters and distal enhancers. Ocular lens differentiation represents an advantageous model system to examine these processes as lens comprises only two cell types, the proliferating lens epithelium and postmitotic lens fiber cells all originating from the epithelium.ResultsUsing whole genome bisulfite sequencing (WGBS) and microdissected lenses, we investigated dynamics of DNA methylation and chromatin changes during mouse lens fiber and epithelium differentiation between embryos (E14.5) and newborns (P0.5). Histone H3.3 variant chromatin landscapes were also generated for both P0.5 lens epithelium and fibers by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Tissue-specific features of DNA methylation patterns are demonstrated via comparative studies with embryonic stem (ES) cells and neural progenitor cells (NPCs) at Nanog, Pou5f1, Sox2, Pax6 and Six3 loci. Comparisons with ATAC-seq and RNA-seq data demonstrate that reduced methylation is associated with increased expression of fiber cell abundant genes, including crystallins, intermediate filament (Bfsp1 and Bfsp2) and gap junction proteins (Gja3 and Gja8), marked by high levels of histone H3.3 within their transcribed regions. Interestingly, Pax6-binding sites exhibited predominantly DNA hypomethylation in lens chromatin. In vitro binding of Pax6 proteins showed Pax6’s ability to interact with sites containing one or two methylated CpG dinucleotides.ConclusionsOur study has generated the first data on methylation changes between two different stages of mammalian lens development and linked these data with chromatin accessibility maps, presence of histone H3.3 and gene expression. Reduced DNA methylation correlates with expression of important genes involved in lens morphogenesis and lens fiber cell differentiation.

A chromosome-scale epigenetic map of the Hydra genome reveals conserved regulators of cell state.

The epithelial and interstitial stem cells of the freshwater polyp Hydra are the best-characterized stem cell systems in any cnidarian, providing valuable insight into cell type evolution and the origin of stemness in animals. However, little is known about the transcriptional regulatory mechanisms that determine how these stem cells are maintained and how they give rise to their diverse differentiated progeny. To address such questions, a thorough understanding of transcriptional regulation in Hydra is needed. To this end, we generated extensive new resources for characterizing transcriptional regulation in Hydra, including new genome assemblies for Hydra oligactis and the AEP strain of Hydra vulgaris, an updated whole-animal single-cell RNA-seq atlas, and genome-wide maps of chromatin interactions, chromatin accessibility, sequence conservation, and histone modifications. These data revealed the existence of large kilobase-scale chromatin interaction domains in the Hydra genome that contain transcriptionally coregulated genes. We also uncovered the transcriptomic profiles of two previously molecularly uncharacterized cell types: isorhiza-type nematocytes and somatic gonad ectoderm. Finally, we identified novel candidate regulators of cell type-specific transcription, several of which have likely been conserved at least since the divergence of Hydra and the jellyfish Clytia hemisphaerica more than 400 million years ago.

Integrative Single-Cell Analysis Reveals Transcriptional and Epigenetic Regulatory Features of Clear Cell Renal Cell Carcinoma.

A comprehensive analysis of gene expression and DNA regulation in ccRCC using scATAC-seq and scRNA-seq reveals the DNA regulatory programs of ccRCC at the single-cell level.

Integrated chromatin accessibility and DNA methylation analysis to reveal the critical epigenetic modification and regulatory mechanism in gonadal differentiation of the sequentially hermaphroditic fish, Monopterus albus

BackgroundMonopterus albus is a hermaphroditic and economically farmed fish that undergoes sex reversal from ovary to testis via ovotestis during gonadal development. The epigenetic changes that are associated with gonadal development in this species remain unclear.MethodsWe produced DNA methylome, transcriptome, and chromatin accessibility maps of the key stages of gonad development: ovary, ovotestis, and testis. The expression of the key candidate genes was detected using qRT-PCR and in situ hybridization and the methylation levels were analysed using bisulphite sequencing PCR. Promoter activity and regulation were assessed using dual-luciferase reporter assays.ResultsGonadal development exhibits highly dynamic transcriptomic, DNA methylation, and chromatin accessibility changes. We found that DNA methylation status, especially of the transcription start site, was significantly negatively correlated with gene expression while chromatin accessibility exhibited no correlation with gene expression during gonadal development. The epigenetic signatures revealed many novel regulatory elements and genes involved in sex reversal, which were validated. DNA methylation detection and site mutation of plastin-2 promoter, as a candidate gene, revealed that DNA methylation could impact the binding of transcription factor dmrt1 and foxl2 through methylation and demethylation to regulate plastin-2 expression during gonadal development.ConclusionsThese data provide novel insights into epigenetic modification and help elucidate the potential molecular mechanism by which dynamic modification of DNA methylation plays a crucial role in gonadal development.

Integrative single-cell analysis of cardiogenesis identifies developmental trajectories and non-coding mutations in congenital heart disease

To define the multi-cellular epigenomic and transcriptional landscape of cardiac cellular development, we generated single-cell chromatin accessibility maps of human fetal heart tissues. We identified eight major differentiation trajectories involving primary cardiac cell types, each associated with dynamic transcription factor (TF) activity signatures. We contrasted regulatory landscapes of iPSC-derived cardiac cell types and their invivo counterparts, which enabled optimization of invitro differentiation of epicardial cells. Further, we interpreted sequence based deep learning models of cell-type-resolved chromatin accessibility profiles to decipher underlying TF motif lexicons. De novo mutations predicted to affect chromatin accessibility in arterial endothelium were enriched in congenital heart disease (CHD) cases vs. controls. Invitro studies in iPSCs validated the functional impact of identified variation on the predicted developmental cell types. This work thus defines the cell-type-resolved cis-regulatory sequence determinants of heart development and identifies disruption of cell type-specific regulatory elements in CHD.

FFPE-ATAC: A Highly Sensitive Method for Profiling Chromatin Accessibility in Formalin-Fixed Paraffin-Embedded Samples.

In basic and translational cancer research, the majority of biopsies are stored in formalin-fixed paraffin-embedded (FFPE) samples. Chromatin accessibility reflects the degree to which nuclear macromolecules can physically interact with chromatinized DNA and plays a key role in gene regulation in different physiological conditions. As such, the profiling of chromatin accessibility in archived FFPE tissue can be critical to understanding gene regulation in health and disease. Due to the high degree of DNA damage in FFPE samples, accurate mapping of chromatin accessibility in these specimens is extremely difficult. To address this issue, we recently established FFPE-ATAC, a highly sensitive method based on T7-Tn5-mediated transposition followed by in vitro transcription (IVT), to generate high-quality chromatin accessibility profiles with 500-50,000 nuclei from a single FFPE tissue section. In FFPE-ATAC, which we describe here, the T7-Tn5 adaptors are inserted into the genome after FFPE sample preparation and are unlikely to sustain the DNA breakage that occurs during reverse cross-linking of these samples. It should, therefore, remain at the ends of broken accessible chromatin sites after reverse cross-linking. IVT is then used to convert the two ends of the broken DNA fragments to RNA molecules before making sequencing libraries from the IVT RNAs and further decoding Tn5 adaptor insertion sites in the genome. Through this strategy, users can decode the flanking sequences of the accessible chromatin even if there are breaks between adjacent pairs of T7-T5 adaptor insertion sites. This method is applicable to dissecting chromatin profiles of a small section of the tissue sample, characterizing stage and region-specific gene regulation and disease-associated chromatin regulation in FFPE tissues. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Nuclei isolation from FFPE tissue samples Basic Protocol 2: T7-Tn5 transposase tagmentation, reverse-crosslinking, and in vitro transcription Basic Protocol 3: Preparation of libraries for high-throughput sequencing.

Abstract 2362: Pax3-foxo1 coordinates enhancer architecture, eRNA transcription, and RNA polymerase pause release at select gene targets

Abstract Background: Gene expression networks are perturbed in every human malignancy. One common way that this occurs is through chromosomal translocations such as the the t(2;13)(q35;q14), which creates the oncogenic transcription factor, PAX3-FOXO1, in alveolar rhabdomyosarcoma (aRMS). Identification of the direct transcriptional targets and mechanisms of control by translocated transcription factors is critical to our understanding of disease etiology and the development of targeted therapeutic approaches. However, defining the genes directly controlled by DNA binding proteins and the mechanisms of regulation at those direct target genes is challenging, because traditional genetic approaches to inactivate transcription factors cannot distinguish direct from indirect changes in gene expression. Methods: A CRISPR-based approach was used to engineer the endogenous PAX3-FOXO1 locus to incorporate the FKBP12F36V degron tag. This allowed degradation of PAX3-FOXO1 within 2 hr of treatment with the proteolysis targeting chimera, dTAG-47. We performed short time courses to analyze nascent transcription (precision nuclear run-on sequencing; PRO-seq), chromatin accessibility (ATAC-seq), and genome-wide mapping of transcriptional complexes (CUT&RUN). This identified a small cohort of high-confidence PAX3-FOXO1 gene targets and associated PAX3-FOXO1-regulated enhancer elements. In addition, we used CRISPR editing to add APEX2 and 3XFLAG tags to PAX3-FOXO1 to enable deep proteomics to identify PAX3-FOXO1-associated transcriptional complexes in aRMS cell lines. Results: Degradation of PAX3-FOXO1-FKBP12F36V led to growth inhibition, hallmarks of myogenic differentiation, and reduced growth in soft agar. PAX3-FOXO1 binding was detected at over 44,000 sites throughout the genome using CUT&RUN, and loss of chromatin bound PAX3-FOXO1 was observed within 2-4hr of PAX3-FOXO1 degradation. PRO-seq analysis defined a core transcriptional network that rapidly collapsed upon PAX3-FOXO1 degradation. Unexpectedly, loss of PAX3-FOXO1 impaired RNA polymerase pause release and transcription elongation at regulated gene targets. Deep proteomic analysis showed that PAX3-FOXO1 was in close proximity to many transcriptional regulatory complexes, but appeared to coordinate the activity of these complexes at only a few hundred sites. Moreover, the continued presence of PAX3-FOXO1 at these elements was required to maintain chromatin accessibility. Conclusion: This work demonstrates the utility of using rapid degradation of endogenous transcription factors to define their direct transcription targets and the mechanisms by which these factors control gene expression. Overall, this work provides a detailed mechanism by which PAX3-FOXO1 maintains an oncogenic transcriptional regulatory network, and emphasizes the utility of PAX3-FOXO1 as a therapeutic target in aRMS. Citation Format: Susu Zhang, Jing Wang, Qi Liu, Hayes McDonald, Monica Bomber, Hillary Layden, Jacob Ellis, Scott Borinstein, Scott Hiebert, Kristy Stengel. Pax3-foxo1 coordinates enhancer architecture, eRNA transcription, and RNA polymerase pause release at select gene targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2362.

229-LB: G6PC2, a GWAS Locus for Fasting Glucose and HbA1c, Regulates Glucose-Suppression of Glucagon Secretion in a-Cells

The G6PC2 locus, encoding the catalytic subunit of the islet-specific glucose-6-phosphatase enzyme, is an established risk locus for HbA1c and fasting glucose levels. A critical role of glycolytic flux in regulating glucagon secretion was established through manipulation of the activity and expression of the glycolytic enzyme glucokinase (GCK) which controls the setpoint for glucose-suppression of glucagon secretion (GSGS) . G6PC2 opposes the action of GCK by hydrolyzing glucose-6-phosphate back to glucose and creating a futile substrate cycle. Previous studies from germline and β-cell-specific G6PC2 null mice have demonstrated that G6PC2 ablation reduces glucose-6-phosphatase activity, glucose cycling, and glycolytic flux, resulting in a leftward shift of dose-response curve for glucose-stimulated insulin secretion. Through cell type specific chromatin accessibility maps of sorted human islet cells, we established that G6PC2 risk SNPs are in open chromatin regions in human α-cells, suggesting that they might not only affect β-cell but also α-cell expression of this critical gene. To test whether α-cell G6PC2 impacts glycemic control, we derived a new mouse line that induces ablation of the first three exons of G6PC2 after Cre activation – resulting in a null allele (G6pc2 loxP/loxP; Gcg-CreERT2; referred to as “α-G6PC2KO”) . Assessment of glucagonemia during glucose tolerance tests revealed a decrease in glucagon levels in α-G6PC2KO mice relative to controls. Intriguingly, fasting blood glucose levels were significantly lower in α-G6PC2KO mice. α-G6PC2KO mice recovered more slowly from hypoglycemia during an insulin tolerance test, and presented lower glucagon levels relative to control mice following the insulin bolus. α-G6PC2KO islets secreted less glucagon relative to control islets when incubated in low-glucose media, independent of alterations in insulin secretion or islet hormone content. Collectively, our data demonstrate that the G6PC2 locus impacts glycemic control via its action in α-cells. Disclosure V. Bahl: None. C. L. May: None. Human pancreas analysis program: n/a. K. H. Kaestner: None.

Investigating chromatin accessibility during development and differentiation by ATAC-sequencing to guide the identification of cis-regulatory elements.

Mapping accessible chromatin across time scales can give insights into its dynamic nature, for example during cellular differentiation and tissue or organism development. Analysis of such data can be utilised to identify functional cis-regulatory elements (CRE) and transcription factor binding sites and, when combined with transcriptomics, can reveal gene regulatory networks (GRNs) of expressed genes. Chromatin accessibility mapping is a powerful approach and can be performed using ATAC-sequencing (ATAC-seq), whereby Tn5 transposase inserts sequencing adaptors into genomic DNA to identify differentially accessible regions of chromatin in different cell populations. It requires low sample input and can be performed and analysed relatively quickly compared with other methods. The data generated from ATAC-seq, along with other genomic approaches, can help uncover chromatin packaging and potential cis-regulatory elements that may be responsible for gene expression. Here, we describe the ATAC-seq approach and give examples from mainly vertebrate embryonic development, where such datasets have identified the highly dynamic nature of chromatin, with differing landscapes between cellular precursors for different lineages.

Systematic Integration of Epigenomic Landscapes in Human and Mouse Blood Cells to Predict Activity and Targets of Regulatory Elements

Thousands of genome‐wide epigenetic (epigenomic) datasets determined in the past decade have generated maps of chromatin accessibility, histone modifications, and binding by transcription factors and structural proteins in a large number of cell types in humans and model species. Such maps have revealed many informative associations between groups of epigenetic features and different classes of cis‐regulatory elements (CREs), and they enable a prediction of the locations of candidate CREs (cCREs) in many genomes. The actuation (opening of chromatin) and epigenetic state (combination of features such as histone modifications) acquired at specific cCREs has been associated with specific gene activation. However, despite the large number and rich variety of available epigenomic datasets, it is difficult for researchers to effectively utilize all the data relevant to their projects. Furthermore, comprehensive rules and catalogs have not been established for which CREs regulate specific target genes and how. Integrative analysis can help meet these needs, and our VISION project provides a ValIdated Systematic IntegratiONof epigenomic data in hematopoiesis in human and mouse blood cells. After careful curation and normalization of epigenomic data from many sources, we systematically integrated these data to generate a comprehensive view of the regulatory landscape (epigenetic states) and predicted cCREs in differentiating hematopoietic cell types jointly in both mouse and human. Multiple machine learning approaches reveal target genes predicted to be regulated by each element in different cell types. These approaches include predicting the regulatory impact of each element on targets by regression models, finding linkages between distinctive clusters of elements and potential target genes from self‐organizing maps, and visual overlays of chromatin interaction maps. Directed mutation and expression analysis around selected genes supports the utility of the assignments. These catalogs of highly annotated regulatory elements are useful for many applications, including interpretation of the phenotypic impact of genetic variants in noncoding genomic regions. Resources from the VISION project are available at our website usevision.org.

Regulation of chromatin accessibility by hypoxia and HIF.

Reduced oxygen availability (hypoxia) can act as a signalling cue in physiological processes such as development, but also in pathological conditions such as cancer or ischaemic disease. As such, understanding how cells and organisms respond to hypoxia is of great importance. The family of transcription factors called Hypoxia Inducible Factors (HIFs) co-ordinate a transcriptional programme required for survival and adaptation to hypoxia. However, the effects of HIF on chromatin accessibility are currently unclear. Here, using genome wide mapping of chromatin accessibility via ATAC-seq, we find hypoxia induces loci specific changes in chromatin accessibility are enriched at a subset hypoxia transcriptionally responsive genes, agreeing with previous data using other models. We show for the first time that hypoxia inducible changes in chromatin accessibility across the genome are predominantly HIF dependent, rapidly reversible upon reoxygenation and partially mimicked by HIF-α stabilisation independent of molecular dioxygenase inhibition. This work demonstrates that HIF is central to chromatin accessibility alterations in hypoxia, and has implications for our understanding of gene expression regulation by hypoxia and HIF.