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The occurrence and formation of genomic structural variants (SVs) is known to be influenced by the 3D chromatin architecture, but the extent and magnitude have been challenging to study. Here, we apply Hi-C to study chromatin organization before and after induction of chromothripsis in human cells. We use Hi-C to manually assemble the derivative chromosomes following the occurrence of massive complex rearrangements, which allows us to study the sources of SV formation and their consequences on gene regulation. We observe an action–reaction interplay whereby the 3D chromatin architecture directly impacts the location and formation of SVs. In turn, the SVs reshape the chromatin organization to alter the local topologies, replication timing, and gene regulation in cis. We show that SVs have a strong tendency to occur between similar chromatin compartments and replication timing regions. Moreover, we find that SVs frequently occur at 3D loop anchors, that SVs can cause a switch in chromatin compartments and replication timing, and that this is a major source of SV-mediated effects on nearby gene expression changes. Finally, we provide evidence for a general mechanistic bias of the 3D chromatin on SV occurrence using data from more than 2700 patient-derived cancer genomes.

Chromatin architecture mapping in 3D formats has increased our understanding of how regulatory sequences and gene expression are connected and regulated in a genome. The 3D chromatin genome shows extensive remodeling during embryonic development, and although the cleavage-stage embryos of most species lack structure before zygotic genome activation (pre-ZGA), zebrafish has been reported to have structure. Here, we aimed to determine the chromosomal architecture in paternal/sperm zebrafish gamete cells to discern whether it either resembles or informs early pre-ZGA zebrafish embryo chromatin architecture. First, we assessed the higher-order architecture through advanced low-cell in situ Hi-C. The structure of zebrafish sperm, packaged by histones, lacks topological associated domains and instead displays “hinge-like” domains of ∼150 kb that repeat every 1–2 Mbs, suggesting a condensed repeating structure resembling mitotic chromosomes. The pre-ZGA embryos lacked chromosomal structure, in contrast to prior work, and only developed structure post-ZGA. During post-ZGA, we find chromatin architecture beginning to form at small contact domains of a median length of ∼90 kb. These small contact domains are established at enhancers, including super-enhancers, and chemical inhibition of Ep300a (p300) and Crebbpa (CBP) activity, lowering histone H3K27ac, but not transcription inhibition, diminishes these contacts. Together, this study reveals hinge-like domains in histone-packaged zebrafish sperm chromatin and determines that the initial formation of high-order chromatin architecture in zebrafish embryos occurs after ZGA primarily at enhancers bearing high H3K27ac.

Made available in DSpace on 2021-06-25T10:15:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-08 Welch Foundation National Science Foundation We introduce the Nucleome Data Bank (NDB), a web-based platform to simulate and analyze the three-dimensional (3D) organization of genomes. The NDB enables physics-based simulation of chromosomal structural dynamics through the MEGABASE + MiChroM computational pipeline. The input of the pipeline consists of epigenetic information sourced from the Encode database; the output consists of the trajectories of chromosomal motions that accurately predict Hi-C and fluorescence insitu hybridization data, as well as multiple observations of chromosomal dynamics in vivo. As an intermediate step, users can also generate chromosomal sub-compartment annotations directly from the same epigenetic input, without the use of any DNA-DNA proximity ligation data. Additionally, the NDB freely hosts both experimental and computational structural genomics data. Besides being able to perform their own genome simulations and download the hosted data, users can also analyze and visualize the same data through custom-designed web-based tools. In particular, the one-dimensional genetic and epigenetic data can be overlaid onto accurate 3D structures of chromosomes, to study the spatial distribution of genetic and epigenetic features. The NDB aims to be a shared resource to biologists, biophysicists and all genome scientists. The NDB is available at https://ndb.rice.edu. Center for Theoretical Biological Physics Rice University Brazilian Biorenewables National Laboratory-LNBR Brazilian Center for Research in Energy and Materials-CNPEM Department of Physics São Paulo State University (UNESP) Institute of Biosciences Humanities and Exact Sciences Department of Computer Science University of Houston Theoretical and Computational Physics Laboratory University of Costa Rica Chemical Engineering Department Military Institute of Engineering The Center for Genome Architecture Department of Molecular and Human Genetics Baylor College of Medicine Department of Physics and Astronomy Rice University Department of Chemistry Rice University Department of Biosciences Rice University Department of Physics Northeastern University Department of Physics São Paulo State University (UNESP) Institute of Biosciences Humanities and Exact Sciences Welch Foundation: C-0016 Welch Foundation: C-1792 National Science Foundation: CHE-1614101 National Science Foundation: PHY-2019745

Chromatin loops are a major component of 3D nuclear organization, visually apparent as intense point-to-point interactions in Hi-C maps. Identification of these loops is a critical part of most Hi-C analyses. However, current methods often miss visually evident CTCF loops in Hi-C data sets from mammals, and they completely fail to identify high intensity loops in other organisms. We present SIP, Significant Interaction Peak caller, and SIPMeta, which are platform independent programs to identify and characterize these loops in a time- and memory-efficient manner. We show that SIP is resistant to noise and sequencing depth, and can be used to detect loops that were previously missed in human cells as well as loops in other organisms. SIPMeta corrects for a common visualization artifact by accounting for Manhattan distance to create average plots of Hi-C and HiChIP data. We then demonstrate that the use of SIP and SIPMeta can lead to biological insights by characterizing the contribution of several transcription factors to CTCF loop stability in human cells. We also annotate loops associated with the SMC component of the dosage compensation complex (DCC) in Caenorhabditis elegans and demonstrate that loop anchors represent bidirectional blocks for symmetrical loop extrusion. This is in contrast to the asymmetrical extrusion until unidirectional blockage by CTCF that is presumed to occur in mammals. Using HiChIP and multiway ligation events, we then show that DCC loops form a network of strong interactions that may contribute to X Chromosome–wide condensation in C. elegans hermaphrodites.

The dramatic change in morphology of chromosomal DNAs between interphase and mitosis is one of the defining features of the eukaryotic cell cycle. Two types of enzymes, namely cohesin and condensin confer the topology of chromosomal DNA by extruding DNA loops. While condensin normally configures chromosomes exclusively during mitosis, cohesin does so during interphase. The processivity of cohesin's loop extrusion during interphase is limited by a regulatory factor called WAPL, which induces cohesin to dissociate from chromosomes via a mechanism that requires dissociation of its kleisin from the neck of SMC3. We show here that a related mechanism may be responsible for blocking condensin II from acting during interphase. Cells derived from patients affected by microcephaly caused by mutations in the MCPH1 gene undergo premature chromosome condensation but it has never been established for certain whether MCPH1 regulates condensin II directly. We show that deletion of Mcph1 in mouse embryonic stem cells unleashes an activity of condensin II that triggers formation of compact chromosomes in G1 and G2 phases, which is accompanied by enhanced mixing of A and B chromatin compartments, and that this occurs even in the absence of CDK1 activity. Crucially, inhibition of condensin II by MCPH1 depends on the binding of a short linear motif within MCPH1 to condensin II's NCAPG2 subunit. We show that the activities of both Cohesin and Condensin II may be restricted during interphase by similar types of mechanisms as MCPH1's ability to block condensin II's association with chromatin is abrogated by the fusion of SMC2 with NCAPH2. Remarkably, in the absence of both WAPL and MCPH1, cohesin and condensin II transform chromosomal DNAs of G2 cells into chromosomes with a solenoidal axis showing that both cohesin and condensin must be tightly regulated to adjust the structure of chromatids for their successful segregation.

Megabase-scale intervals of active, gene-rich and inactive, gene-poor chromatin are known to segregate, forming the A and B compartments. Fine mapping of the contents of these A and B compartments has been hitherto impossible, owing to the extraordinary sequencing depths required to distinguish between the long-range contact patterns of individual loci, and to the computational complexity of the associated calculations. Here, we generate the largest published in situ Hi-C map to date, spanning 33 billion contacts. We also develop a computational method, dubbed PCA of Sparse, SUper Massive Matrices (POSSUMM), that is capable of efficiently calculating eigenvectors for sparse matrices with millions of rows and columns. Applying POSSUMM to our Hi-C dataset makes it possible to assign loci to the A and B compartment at 500 bp resolution. We find that loci frequently alternate between compartments as one moves along the contour of the genome, such that the median compartment interval is only 12.5 kb long. Contrary to the findings in coarse-resolution compartment profiles, we find that individual genes are not uniformly positioned in either the A compartment or the B compartment. Instead, essentially all (95%) active gene promoters localize in the A compartment, but the likelihood of localizing in the A compartment declines along the body of active genes, such that the transcriptional termini of long genes (>60 kb) tend to localize in the B compartment. Similarly, nearly all active enhancers elements (95%) localize in the A compartment, even when the flanking sequences are comprised entirely of inactive chromatin and localize in the B compartment. These results are consistent with a model in which DNA-bound regulatory complexes give rise to phase separation at the scale of individual DNA elements.

Made available in DSpace on 2022-04-29T08:31:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-08-12 The human genome is organized within a nucleus where chromosomes fold into an ensemble of different conformations. Chromosome conformation capture techniques such as Hi-C provide information about the genome architecture by creating a 2D heat map. Initially, Hi-C map experiments were performed in human interphase cell lines. Recently, efforts were expanded to several different organisms, cell lines, tissues, and cell cycle phases where obtaining high-quality maps is challenging. Poor sampled Hi-C maps present high sparse matrices where compartments located far from the main diagonal are difficult to observe. Aided by recently developed models for chromatin folding and dynamics investigation, we introduce a framework to enhance the compartments' information far from the diagonal observed in experimental sparse matrices. The simulations were performed using the Open-MiChroM platform aided by new trained parameters in the minimal chromatin model (MiChroM) energy function. The simulations optimized on a downsampled experimental map (10% of the original data) allow the prediction of a contact frequency similar to that of the complete (100%) experimental Hi-C. The modeling results open a discussion on how simulations and modeling can increase the statistics and help fill in some Hi-C regions not captured by poor sampling experiments. Open-MiChroM simulations allow us to explore the 3D genome organization of different organisms, cell lines, and cell phases that often do not produce high-quality Hi-C maps. Center for Theoretical Biological Physics Department of Physics & Astronomy Rice University Instituto de Biociências Letras e Ciências Exatas UNESP Univ. Estadual Paulista Departamento de Física The Center for Genome Architecture Department of Molecular and Human Genetics Baylor College of Medicine Instituto de Biociências Letras e Ciências Exatas UNESP Univ. Estadual Paulista Departamento de Física

The dramatic change in morphology of chromosomal DNAs between interphase and mitosis is one of the defining features of the eukaryotic cell cycle. Two types of enzymes, namely cohesin and condensin confer the topology of chromosomal DNA by extruding DNA loops. While condensin normally configures chromosomes exclusively during mitosis, cohesin does so during interphase. The processivity of cohesin’s LE during interphase is limited by a regulatory factor called WAPL, which induces cohesin to dissociate from chromosomes via a mechanism that requires dissociation of its kleisin from the neck of SMC3. We show here that a related mechanism may be responsible for blocking condensin II from acting during interphase. Cells from patients carrying mutations in the Mcph1 gene undergo premature chromosome condensation but it has never been established for certain whether MCPH1 regulates condensin II directly. We show that deletion of Mcph1 in mouse embryonic stem cells unleashes an activity of condensin II that triggers formation of compact chromosomes in G1 and G2 phases, which is accompanied by enhanced mixing of A and B chromatin compartments, and that this occurs even in the absence of CDK1 activity. Crucially, inhibition of condensin II by MCPH1 depends on the binding of a short linear motif within MCPH1 to condensin II’s NCAPG2 subunit. We show that the activities of both Cohesin and Condensin II may be restricted during interphase by similar types of mechanisms as MCPH1’s ability to block condensin II’s association with chromatin is abrogated by the fusion of SMC2 with NCAPH2. Remarkably, in the absence of both WAPL and MCPH1, cohesin and condensin II transform chromosomal DNAs of G2 cells into chromosomes with a solenoidal axis showing that both SMC complexes must be tightly regulated to adjust both the chromatid’s structure and their segregation.

Chromatin looping plays an important role in genome regulation. However, because ChIP-seq and loop-resolution Hi-C (DNA-DNA proximity ligation) are extremely challenging in mammalian early embryos, the developmental stage at which cohesin-mediated loops form remains unknown. Here, we study early development in medaka (the Japanese killifish, Oryzias latipes) at 12 time points before, during, and after gastrulation (the onset of cell differentiation) and characterize transcription, protein binding, and genome architecture. We find that gastrulation is associated with drastic changes in genome architecture, including the formation of the first loops between sites bound by the insulator protein CTCF and a large increase in the size of contact domains. In contrast, the binding of the CTCF is fixed throughout embryogenesis. Loops form long after genome-wide transcriptional activation, and long after domain formation seen in mouse embryos. These results suggest that, although loops may play a role in differentiation, they are not required for zygotic transcription. When we repeated our experiments in zebrafish, loops did not emerge until gastrulation, that is, well after zygotic genome activation. We observe that loop positions are highly conserved in synteny blocks of medaka and zebrafish, indicating that the 3D genome architecture has been maintained for >110–200 million years of evolution.

Background: The human genome folds in 3 dimensions to form thousands of chromatin loops inside the nucleus, encasing genes and cis -regulatory elements for accurate gene expression control. Physical tethers of loops are anchored by the DNA-binding protein CTCF and the cohesin ring complex. Because heart failure is characterized by hallmark gene expression changes, it was recently reported that substantial CTCF-related chromatin reorganization underpins the myocardial stress–gene response, paralleled by chromatin domain boundary changes observed in CTCF knockout. Methods: We undertook an independent and orthogonal analysis of chromatin organization with mouse pressure-overload model of myocardial stress (transverse aortic constriction) and cardiomyocyte-specific knockout of Ctcf . We also downloaded published data sets of similar cardiac mouse models and subjected them to independent reanalysis. Results: We found that the cardiomyocyte chromatin architecture remains broadly stable in transverse aortic constriction hearts, whereas Ctcf knockout resulted in ≈99% abolition of global chromatin loops. Disease gene expression changes correlated instead with differential histone H3K27-acetylation enrichment at their respective proximal and distal interacting genomic enhancers confined within these static chromatin structures. Moreover, coregulated genes were mapped out as interconnected gene sets on the basis of their multigene 3D interactions. Conclusions: This work reveals a more stable genome-wide chromatin framework than previously described. Myocardial stress–gene transcription responds instead through H3K27-acetylation enhancer enrichment dynamics and gene networks of coregulation. Robust and intact CTCF looping is required for the induction of a rapid and accurate stress response.

Vertebrate genomes are partitioned into contact domains defined by enhanced internal contact frequency and formed by two principal mechanisms: compartmentalization of transcriptionally active and inactive domains, and stalling of chromosomal loop-extruding cohesin by CTCF bound at domain boundaries. While Drosophila has widespread contact domains and CTCF, it is currently unclear whether CTCF-dependent domains exist in flies. We genetically ablate CTCF in Drosophila and examine impacts on genome folding and transcriptional regulation in the central nervous system. We find that CTCF is required to form a small fraction of all domain boundaries, while critically controlling expression patterns of certain genes and supporting nervous system function. We also find that CTCF recruits the pervasive boundary-associated factor Cp190 to CTCF-occupied boundaries and co-regulates a subset of genes near boundaries together with Cp190. These results highlight a profound difference in CTCF-requirement for genome folding in flies and vertebrates, in which a large fraction of boundaries are CTCF-dependent and suggest that CTCF has played mutable roles in genome architecture and direct gene expression control during metazoan evolution., Although the Drosophila genome has widespread contact domains and CTCF, it remains unclear whether CTCF-dependent domains exist in flies. Here, the authors ablate CTCF in Drosophila and find that CTCF is required to form a small fraction of all domain boundaries, suggesting differences in the role of CTCF for genome folding in flies and vertebrates.

The mechanisms underlying gene repression and silencers are poorly understood. Here we investigate the hypothesis that H3K27me3-rich regions of the genome, defined from clusters of H3K27me3 peaks, may be used to identify silencers that can regulate gene expression via proximity or looping. We find that H3K27me3-rich regions are associated with chromatin interactions and interact preferentially with each other. H3K27me3-rich regions component removal at interaction anchors by CRISPR leads to upregulation of interacting target genes, altered H3K27me3 and H3K27ac levels at interacting regions, and altered chromatin interactions. Chromatin interactions did not change at regions with high H3K27me3, but regions with low H3K27me3 and high H3K27ac levels showed changes in chromatin interactions. Cells with H3K27me3-rich regions knockout also show changes in phenotype associated with cell identity, and altered xenograft tumor growth. Finally, we observe that H3K27me3-rich regions-associated genes and long-range chromatin interactions are susceptible to H3K27me3 depletion. Our results characterize H3K27me3-rich regions and their mechanisms of functioning via looping., Mechanisms underlying gene repression and silencers remain poorly understood. Here the authors investigate the role of H3K27me3-rich regions in the genome, as defined from clusters of H3K27me3 peaks, in regulating gene expression via looping.

Cohesin plays an essential role in chromatin loop extrusion, but its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is less well understood. Using live-cell and super-resolved 3D microscopy, here we find that cohesin depletion in a human colon cancer derived cell line results in endomitosis and a single multilobulated nucleus with chromosome territories pervaded by interchromatin channels. Chromosome territories contain chromatin domain clusters with a zonal organization of repressed chromatin domains in the interior and transcriptionally competent domains located at the periphery. These clusters form microscopically defined, active and inactive compartments, which likely correspond to A/B compartments, which are detected with ensemble Hi-C. Splicing speckles are observed nearby within the lining channel system. We further observe that the multilobulated nuclei, despite continuous absence of cohesin, pass through S-phase with typical spatio-temporal patterns of replication domains. Evidence for structural changes of these domains compared to controls suggests that cohesin is required for their full integrity., The role of cohesin in organizing a functional nuclear architecture remains poorly understood. Here the authors show that cohesin depleted cells pass through endomitosis forming a multilobulated nucleus able to proceed through S-phase with typical features of active and inactive nuclear compartments and spatio-temporal patterns of replication domains.

We study the structural ensembles of human chromosomes across different cell types. Using computer simulations, we generate cell-specific 3D chromosomal structures and compare them to recently published chromatin structures obtained through microscopy. We demonstrate using a combination of machine learning and polymer physics simulations that epigenetic information can be used to predict the structural ensembles of multiple human cell lines. The chromosomal structures obtained in silico are quantitatively consistent with those obtained through microscopy as well as DNA-DNA proximity ligation assays. Theory predicts that chromosome structures are fluid and can only be described by an ensemble, which is consistent with the observation that chromosomes exhibit no unique fold. Nevertheless, our analysis of both structures from simulation and microscopy reveals that short segments of chromatin make transitions between a closed conformation and an open dumbbell conformation. This conformational transition appears to be consistent with a two-state process with an effective free energy cost of about four times the effective information theoretic temperature. Finally, we study the conformational changes associated with the switching of genomic compartments observed in human cell lines. Genetically identical but epigenetically distinct cell types appear to rearrange their respective structural ensembles to expose segments of transcriptionally active chromatin, belonging to the A genomic compartment, towards the surface of the chromosome, while inactive segments, belonging to the B compartment, move to the interior. The formation of genomic compartments resembles hydrophobic collapse in protein folding, with the aggregation of denser and predominantly inactive chromatin driving the positioning of active chromatin toward the surface of individual chromosomal territories.

Higher-order chromatin structure and DNA methylation are implicated in multiple developmental processes, but their relationship to cell state is unknown. Here, we find that large (>7.3 kb) DNA methylation nadirs (termed "grand canyons") can form long loops connecting anchor loci that may be dozens of megabases (Mb) apart, as well as inter-chromosomal links. The interacting loci cover a total of ∼3.5 Mb of the human genome. The strongest interactions are associated with repressive marks made by the Polycomb complex and are diminished upon EZH2 inhibitor treatment. The data are suggestive of the formation of these loops by interactions between repressive elements in the loci, forming a genomic subcompartment, rather than by cohesion/CTCF-mediated extrusion. Interestingly, unlike previously characterized subcompartments, these interactions are present only in particular cell types, such as stem and progenitor cells. Our work reveals that H3K27me3-marked large DNA methylation grand canyons represent a set of very-long-range loops associated with cellular identity.

Eukaryotic genomes are folded into loops. It is thought that these are formed by cohesin complexes via extrusion, either until loop expansion is arrested by CTCF or until cohesin is removed from DNA by WAPL. Although WAPL limits cohesin’s chromatin residence time to minutes, it has been reported that some loops exist for hours. How these loops can persist is unknown. We show that during G1-phase, mammalian cells contain acetylated cohesinSTAG1 which binds chromatin for hours, whereas cohesinSTAG2 binds chromatin for minutes. Our results indicate that CTCF and the acetyltransferase ESCO1 protect a subset of cohesinSTAG1 complexes from WAPL, thereby enable formation of long and presumably long-lived loops, and that ESCO1, like CTCF, contributes to boundary formation in chromatin looping. Our data are consistent with a model of nested loop extrusion, in which acetylated cohesinSTAG1 forms stable loops between CTCF sites, demarcating the boundaries of more transient cohesinSTAG2 extrusion activity.

Chromatin loops are a major component of 3D nuclear organization, visually apparent as intense point-to-point interactions in Hi-C maps. Identification of these loops is a critical part of most Hi-C analyses. However, current methods often miss visually evident CTCF loops in Hi-C data sets from mammals, and they completely fail to identify high intensity loops in other organisms. We present SIP, Significant Interaction Peak caller, and SIPMeta, which are platform independent programs to identify and characterize these loops in a time- and memory-efficient manner. We show that SIP is resistant to noise and sequencing depth, and can be used to detect loops that were previously missed in human cells as well as loops in other organisms. SIPMeta corrects for a common visualization artifact by accounting for Manhattan distance to create average plots of Hi-C and HiChIP data. We then demonstrate that the use of SIP and SIPMeta can lead to biological insights by characterizing the contribution of several transcription factors to CTCF loop stability in human cells. We also annotate loops associated with the SMC component of the dosage compensation complex (DCC) in Caenorhabditis elegans and demonstrate that loop anchors represent bidirectional blocks for symmetrical loop extrusion. This is in contrast to the asymmetrical extrusion until unidirectional blockage by CTCF that is presumed to occur in mammals. Using HiChIP and multiway ligation events, we then show that DCC loops form a network of strong interactions that may contribute to X Chromosome–wide condensation in C. elegans hermaphrodites.

Eukaryotic genomes are folded into loops. It is thought that these are formed by cohesin complexes via extrusion, either until loop expansion is arrested by CTCF or until cohesin is removed from DNA by WAPL. Although WAPL limits cohesin’s chromatin residence time to minutes, it has been reported that some loops exist for hours. How these loops can persist is unknown. We show that during G1-phase, mammalian cells contain acetylated cohesinSTAG1 which binds chromatin for hours, whereas cohesinSTAG2 binds chromatin for minutes. Our results indicate that CTCF and the acetyltransferase ESCO1 protect a subset of cohesinSTAG1 complexes from WAPL, thereby enable formation of long and presumably long-lived loops, and that ESCO1, like CTCF, contributes to boundary formation in chromatin looping. Our data are consistent with a model of nested loop extrusion, in which acetylated cohesinSTAG1 forms stable loops between CTCF sites, demarcating the boundaries of more transient cohesinSTAG2 extrusion activity.

The RAG endonuclease initiates Igh V(D)J assembly in B cell progenitors by joining D segments to JH segments, before joining upstream VH segments to DJH intermediates1. In mouse progenitor B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain2 at the 3' end of Igh contains an internal subdomain that spans the 5' CBE anchor (IGCR1)3, the DH segments, and a RAG-bound recombination centre (RC)4. The RC comprises the JH-proximal D segment (DQ52), four JH segments, and the intronic enhancer (iEμ)5. Robust RAG-mediated cleavage is restricted to paired V(D)J segments flanked by complementary recombination signal sequences (12RSS and 23RSS)6. D segments are flanked downstream and upstream by 12RSSs that mediate deletional joining with convergently oriented JH-23RSSs and VH-23RSSs, respectively6. Despite 12/23 compatibility, inversional D-to-JH joining via upstream D-12RSSs is rare7,8. Plasmid-based assays have attributed the lack of inversional D-to-JH joining to sequence-based preference for downstream D-12RSSs9, as opposed to putative linear scanning mechanisms10,11. As RAG linearly scans convergent CBE-anchored chromatin loops4,12-14, potentially formed by cohesin-mediated loop extrusion15-18, we revisited its scanning role. Here we show that the chromosomal orientation of JH-23RSS programs RC-bound RAG to linearly scan upstream chromatin in the 3' Igh subdomain for convergently oriented D-12RSSs and, thereby, to mediate deletional joining of all D segments except RC-based DQ52, which joins by a diffusion-related mechanism. In a DQ52-based RC, formed in the absence of JH segments, RAG bound by the downstream DQ52-RSS scans the downstream constant region exon-containing 3' Igh subdomain, in which scanning can be impeded by targeted binding of nuclease-dead Cas9, by transcription through repetitive Igh switch sequences, and by the 3' Igh CBE-based loop anchor. Each scanning impediment focally increases RAG activity on potential substrate sequences within the impeded region. High-resolution mapping of chromatin interactions in the RC reveals that such focal RAG targeting is associated with corresponding impediments to the loop extrusion process that drives chromatin past RC-bound RAG.

Mammalian genomes harbor millions of noncoding elements called enhancers that quantitatively regulate gene expression, but it remains unclear which enhancers regulate which genes. Here we describe an experimental approach, based on CRISPR interference, RNA FISH, and flow cytometry (CRISPRi-FlowFISH), to perturb enhancers in the genome, and apply it to test >3,000 potential regulatory enhancer-gene connections across multiple genomic loci. A simple equation based on a mechanistic model for enhancer function performed remarkably well at predicting the complex patterns of regulatory connections we observe in our CRISPR dataset. This Activity-by-Contact (ABC) model involves multiplying measures of enhancer activity and enhancer-promoter 3D contacts, and can predict enhancer-gene connections in a given cell type based on chromatin state maps. Together, CRISPRi-FlowFISH and the ABC model provide a systematic approach to map and predict which enhancers regulate which genes, and will help to interpret the functions of the thousands of disease risk variants in the noncoding genome.

Enhancer elements in the human genome control how genes are expressed in specific cell types and harbor thousands of genetic variants that influence risk for common diseases1-4. Yet, we still do not know how enhancers regulate specific genes, and we lack general rules to predict enhancer-gene connections across cell types5,6. We developed an experimental approach, CRISPRi-FlowFISH, to perturb enhancers in the genome, and we applied it to test >3,500 potential enhancer-gene connections for 30 genes. We found that a simple activity-by-contact model substantially outperformed previous methods at predicting the complex connections in our CRISPR dataset. This activity-by-contact model allows us to construct genome-wide maps of enhancer-gene connections in a given cell type, on the basis of chromatin state measurements. Together, CRISPRi-FlowFISH and the activity-by-contact model provide a systematic approach to map and predict which enhancers regulate which genes, and will help to interpret the functions of the thousands of disease risk variants in the noncoding genome.

SUMMARY Somatic hypermutation (SHM) introduces point mutations into immunoglobulin (Ig) genes but also causes mutations in other parts of the genome. We have used lentiviral SHM reporter vectors to identify regions of the genome that are susceptible (“hot”) and resistant (“cold”) to SHM, revealing that SHM susceptibility and resistance are often properties of entire topologically associated domains (TADs). Comparison of hot and cold TADs reveals that while levels of transcription are equivalent, hot TADs are enriched for the cohesin loader NIPBL, super-enhancers, markers of paused/stalled RNA polymerase 2, and multiple important B cell transcription factors. We demonstrate that at least some hot TADs contain enhancers that possess SHM targeting activity and that insertion of a strong Ig SHM-targeting element into a cold TAD renders it hot. Our findings lead to a model for SHM susceptibility involving the cooperative action of cis-acting SHM targeting elements and the dynamic and architectural properties of TADs., Graphical Abstract, In Brief Senigl et al. show that genome susceptibility to somatic hypermutation (SHM) is confined within topologically associated domains (TADs) and is linked to markers of strong enhancers and stalled transcription and high levels of the cohesin loader NIPBL. Insertion of an ectopic SHM targeting element renders an entire TAD susceptible to SHM.

In vivo, the human genome folds into a characteristic ensemble of 3D structures. The mechanism driving the folding process remains unknown. We report a theoretical model for chromatin (Minimal Chromatin Model) that explains the folding of interphase chromosomes and generates chromosome conformations consistent with experimental data. The energy landscape of the model was derived by using the maximum entropy principle and relies on two experimentally derived inputs: a classification of loci into chromatin types and a catalog of the positions of chromatin loops. First, we trained our energy function using the Hi-C contact map of chromosome 10 from human GM12878 lymphoblastoid cells. Then, we used the model to perform molecular dynamics simulations producing an ensemble of 3D structures for all GM12878 autosomes. Finally, we used these 3D structures to generate contact maps. We found that simulated contact maps closely agree with experimental results for all GM12878 autosomes. The ensemble of structures resulting from these simulations exhibited unknotted chromosomes, phase separation of chromatin types, and a tendency for open chromatin to lie at the periphery of chromosome territories.

Hi-Culfite, a protocol combining Hi-C and whole-genome bisulfite sequencing (WGBS), determines chromatin contacts and DNA methylation simultaneously. Hi-Culfite also reveals relationships that cannot be seen when the two assays are performed separately. For instance, we show that loci associated with open chromatin exhibit context-sensitive methylation: when their spatial neighbors lie in closed chromatin, they are much more likely to be methylated.

SUMMARYEndoC-βH1 is emerging as a critical human beta cell model to study the genetic and environmental etiologies of beta cell function, especially in the context of diabetes. Comprehensive knowledge of its molecular landscape is lacking yet required to fully take advantage of this model. Here, we report extensive chromosomal (spectral karyotyping), genetic (genotyping), epigenetic (ChIP-seq, ATAC-seq), chromatin interaction (Hi-C, Pol2 ChIA-PET), and transcriptomic (RNA-seq, miRNA-seq) maps of this cell model. Integrated analyses of these maps define known (e.g.,PDX1, ISL1) and putative (e.g.,PCSK1, mir-375) beta cell-specific chromatin interactions and transcriptionalcis-regulatory networks, and identify allelic effects oncis-regulatory element use and expression.Importantly, comparative analyses with maps generated in primary human islets/beta cells indicate substantial preservation of chromatin looping, but also highlight chromosomal heterogeneity and fetal genomic signatures in EndoC-βH1. Together, these maps, and an interactive web application we have created for their exploration, provide important tools for the broad community in the design and success of experiments to probe and manipulate the genetic programs governing beta cell identity and (dys)function in diabetes.

Over 90% of genetic variants associated with complex human traits map to non-coding regions, but little is understood about how they modulate gene regulation in health and disease. One possible mechanism is that genetic variants affect the activity of one or more cis-regulatory elements leading to gene expression variation in specific cell types. To identify such cases, we analyzed ATAC-seq and RNA-seq profiles from stimulated primary CD4 + T cells in up to 105 healthy donors. We found that regions of accessible chromatin (ATAC-peaks) are co-accessible at kilobase and megabase resolution, consistent with the three-dimensional chromatin organization measured by in situ Hi-C in T cells. Fifteen percent of genetic variants located within ATAC-peaks affected the accessibility of the corresponding peak (local-ATAC-QTLs). Local-ATAC-QTLs have the largest effects on co-accessible peaks, are associated with gene expression and are enriched for autoimmune disease variants. Our results provide insights into how natural genetic variants modulate cis-regulatory elements, in isolation or in concert, to influence gene expression.

Higher order chromatin structure and DNA methylation are implicated in multiple developmental processes, but their relationship to cell state is unknown. Here, we found that large (~10kb) DNA methylation nadirs can form long loops connecting anchor loci that may be dozens of megabases apart, as well as interchromosomal links. The interacting loci comprise ~3.5Mb of the human genome. The data are more consistent with the formation of these loops by phase separation of the interacting loci to form a genomic subcompartment, rather than with CTCF-mediated extrusion. Interestingly, unlike previously characterized genomic subcompartments, this subcompartment is only present in particular cell types, such as stem and progenitor cells. Further, we identify one particular loop anchor that is functionally associated with maintenance of the hematopoietic stem cell state. Our work reveals that H3K27me3-marked large DNA methylation nadirs represent a novel set of very long-range loops and links associated with cellular identity.SummaryHi-C and DNA methylation analyses reveal novel chromatin loops between distant sites implicated in stem and progenitor cell function.

The locations of chromatin loops in Drosophila were determined by Hi-C (chemical cross-linking, restriction digestion, ligation, and high-throughput DNA sequencing). Whereas most loop boundaries or “anchors” are associated with CTCF protein in mammals, loop anchors in Drosophila were found most often in association with the polycomb group (PcG) protein Polycomb (Pc), a subunit of polycomb repressive complex 1 (PRC1). Loops were frequently located within domains of PcG-repressed chromatin. Promoters located at PRC1 loop anchors regulate some of the most important developmental genes and are less likely to be expressed than those not at PRC1 loop anchors. Although DNA looping has most commonly been associated with enhancer–promoter communication, our results indicate that loops are also associated with gene repression.

SUMMARYThe three-dimensional arrangement of the human genome comprises a complex network of structural and regulatory chromatin loops important for coordinating changes in transcription during human development. To better understand the mechanisms underlying context-specific 3D chromatin structure and transcription during cellular differentiation, we generated comprehensive in situ Hi-C maps of DNA loops during human monocyte-to-macrophage differentiation. We demonstrate that dynamic looping events are regulatory rather than structural in nature and uncover widespread coordination of dynamic enhancer activity at preformed and acquired DNA loops. Enhancer-bound loop formation and enhancer-activation of preformed loops represent two distinct modes of regulation that together form multi-loop activation hubs at key macrophage genes. Activation hubs connect 3.4 enhancers per promoter and exhibit a strong enrichment for Activator Protein 1 (AP-1) binding events, suggesting multi-loop activation hubs driven by cell-type specific transcription factors may represent an important class of regulatory chromatin structures for the spatiotemporal control of transcription.HIGHLIGHTSHigh resolution and high sensitivity of loop detection via deeply sequenced in situ Hi-C experiments during monocyte to macrophage differentiation (> 10 billion total reads)Multi-loop interaction communities identified surrounding key macrophage genes.Multi-loop communities connect dynamic enhancers through both static and newly acquired DNA loops, forming hubs of activationMacrophage activation hubs are enriched for AP-1 bound long-range enhancer interactions, suggesting cell-type specific TFs drive changes in 3D structure and transcription through regulatory DNA loops

The three-dimensional arrangement of the human genome comprises a complex network of structural and regulatory chromatin loops important for coordinating changes in transcription during human development. To better understand the mechanisms underlying context-specific 3D chromatin structure and transcription during cellular differentiation, we generated comprehensive in situ Hi-C maps of DNA loops in human monocytes and differentiated macrophages. We demonstrate that dynamic looping events are regulatory rather than structural in nature and uncover widespread coordination of dynamic enhancer activity at preformed and acquired DNA loops. Enhancer-bound loop formation and enhancer activation of preformed loops together form multi-loop activation hubs at key macrophage genes. Activation hubs connect 3.4 enhancers per promoter and exhibit a strong enrichment for activator protein 1 (AP-1)-binding events, suggesting that multi-loop activation hubs involving cell-type-specific transcription factors represent an important class of regulatory chromatin structures for the spatiotemporal control of transcription.

The locations of chromatin loops in Drosophila were determined by Hi-C (chemical cross-linking, restriction digestion, ligation, and high-throughput DNA sequencing). Whereas most loop boundaries or “anchors” are associated with CTCF protein in mammals, loop anchors in Drosophila were found most often in association with the polycomb group (PcG) protein Polycomb (Pc), a subunit of Polycomb Repressive Complex 1 (PRC1). Loops were frequently located within domains of PcG-repressed chromatin. Promoters located at PRC1 loop anchors regulate some of the most important developmental genes and are less likely to be expressed than those not at PRC1 loop anchors. Although DNA looping has most commonly been associated with enhancer-promoter communication, our results indicate that loops are also associated with gene repression.

Over 90% of genetic variants associated with complex human traits map to non-coding regions, but little is understood about how they modulate gene regulation in health and disease. One possible mechanism is that genetic variants affect the activity of one or more cis-regulatory elements leading to gene expression variation in specific cell types. To identify such cases, we analyzed Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) and RNA-seq profiles from activated CD4+ T cells of up to 105 healthy donors. We found that regions of accessible chromatin (ATAC-peaks) are co-accessible at kilobase and megabase resolution, in patterns consistent with the 3D organization of chromosomes measured by in situ Hi-C in T cells. 15% of genetic variants located within ATAC-peaks affected the accessibility of the corresponding peak through disrupting binding sites for transcription factors important for T cell differentiation and activation. These ATAC quantitative trait nucleotides (ATAC-QTNs) have the largest effects on co-accessible peaks, are associated with gene expression from the same aliquot of cells, are rarely affecting core binding motifs, and are enriched for autoimmune disease variants. Our results provide insights into how natural genetic variants modulate cis- regulatory elements, in isolation or in concert, to influence gene expression in primary immune cells that play a key role in many human diseases.

SUMMARY EndoC-βH1 is emerging as a critical human β cell model to study the genetic and environmental etiologies of β cell (dys)function and diabetes. Comprehensive knowledge of its molecular landscape is lacking, yet required, for effective use of this model. Here, we report chromosomal (spectral karyotyping), genetic (genotyping), epigenomic (ChIP-seq and ATAC-seq), chromatin interaction (Hi-C and Pol2 ChIA-PET), and transcriptomic (RNA-seq and miRNA-seq) maps of EndoC-βH1. Analyses of these maps define known (e.g., PDX1 and ISL1) and putative (e.g., PCSK1 and mir-375) β cell-specific transcriptional cis-regulatory networks and identify allelic effects on cis-regulatory element use. Importantly, comparison with maps generated in primary human islets and/or β cells indicates preservation of chromatin looping but also highlights chromosomal aberrations and fetal genomic signatures in EndoC-βH1. Together, these maps, and a web application we created for their exploration, provide important tools for the design of experiments to probe and manipulate the genetic programs governing β cell identity and (dys)function in diabetes., In Brief EndoC-βH1 is becoming an important cellular model to study genes and pathways governing human β cell identity and function, but its (epi)genomic similarity to primary human islets is unknown. Lawlor et al. complete and compare extensive EndoC and primary human islet multiomic maps to identify shared and distinct genomic circuitry., Graphical Abstract

During interphase, the inactive X chromosome (Xi) is largely transcriptionally silent and adopts an unusual 3D configuration known as the “Barr body.” Despite the importance of X chromosome inactivation, little is known about this 3D conformation. We recently showed that in humans the Xi chromosome exhibits three structural features, two of which are not shared by other chromosomes. First, like the chromosomes of many species, Xi forms compartments. Second, Xi is partitioned into two huge intervals, called “superdomains,” such that pairs of loci in the same superdomain tend to colocalize. The boundary between the superdomains lies near DXZ4, a macrosatellite repeat whose Xi allele extensively binds the protein CCCTC-binding factor. Third, Xi exhibits extremely large loops, up to 77 megabases long, called “superloops.” DXZ4 lies at the anchor of several superloops. Here, we combine 3D mapping, microscopy, and genome editing to study the structure of Xi, focusing on the role of DXZ4. We show that superloops and superdomains are conserved across eutherian mammals. By analyzing ligation events involving three or more loci, we demonstrate that DXZ4 and other superloop anchors tend to colocate simultaneously. Finally, we show that deleting DXZ4 on Xi leads to the disappearance of superdomains and superloops, changes in compartmentalization patterns, and changes in the distribution of chromatin marks. Thus, DXZ4 is essential for proper Xi packaging.

In vivo, the human genome folds into a characteristic ensemble of three-dimensional structures. The mechanism driving the folding process remains unknown. We report a theoretical model for chromatin (Minimal Chromatin Model) that explains the folding of interphase chromosomes and generates chromosome conformations consistent with experimental data. The energy landscape of the model was derived by using the maximum entropy principle and relies on two experimentally derived inputs: a classification of loci into chromatin types and a catalog of the positions of chromatin loops. First, we trained our energy function using the Hi-C contact map of chromosome 10 from human GM12878 lymphoblastoid cells. Then we used the model to perform molecular dynamics simulations producing an ensemble of 3D structures for all GM12878 autosomes. Finally, we used these 3D structures to generate contact maps. We found that simulated contact maps closely agree with experimental results for all GM12878 autosomes.The ensemble of structures resulting from these simulations exhibited unknotted chromosomes, phase separation of chromatin types, and a tendency for open chromatin to lie at the periphery of chromosome territories.One Sentence Summary:We report a model for chromatin that explains and accurately reproduces the three-dimensional structure of chromosomes in interphase.

We recently used in situ Hi-C to create kilobase-resolution 3D maps of mammalian genomes. Here, we combine these maps with new Hi-C, microscopy, and genome-editing experiments to study the physical structure of chromatin fibers, domains, and loops. We find that the observed contact domains are inconsistent with the equilibrium state for an ordinary condensed polymer. Combining Hi-C data and novel mathematical theorems, we show that contact domains are also not consistent with a fractal globule. Instead, we use physical simulations to study two models of genome folding. In one, intermonomer attraction during polymer condensation leads to formation of an anisotropic "tension globule." In the other, CCCTC-binding factor (CTCF) and cohesin act together to extrude unknotted loops during interphase. Both models are consistent with the observed contact domains and with the observation that contact domains tend to form inside loops. However, the extrusion model explains a far wider array of observations, such as why loops tend not to overlap and why the CTCF-binding motifs at pairs of loop anchors lie in the convergent orientation. Finally, we perform 13 genome-editing experiments examining the effect of altering CTCF-binding sites on chromatin folding. The convergent rule correctly predicts the affected loops in every case. Moreover, the extrusion model accurately predicts in silico the 3D maps resulting from each experiment using only the location of CTCF-binding sites in the WT. Thus, we show that it is possible to disrupt, restore, and move loops and domains using targeted mutations as small as a single base pair.

Higher order chromatin structure and DNA methylation are implicated in multiple developmental processes, but their relationship to cell state is unknown. In order to understand how the DNA methylation is connected with nuclear architecture and can vary between cell types and during cell differentiation, we began to explore the 3D architecture of human hematopoietic stem and progenitor cells (HSPCs) by performing in situ Hi-C experiments at 5kb resolution. We found that large (~10kb) DNA methylation canyons can form long loops connecting anchor loci that may be dozens of megabases apart. These canyons also can form interchromosomal links (Fig.1a and 1b). We further confirmed these long-range interactions by performing 3D-FISH using two color fluorescent labeled probes that spanned the HOXA locus loop anchor (green) and the SP8 locus loop anchor (red), which are ~7MB apart (Fig. 1c). In order to begin to investigate mechanisms that may regulate these long loops and how they relate to commonly studied loops that are mediated by CTCF-extrusion, we examined their properties systematically. Interestingly, the anchors of long loops exhibited minimal enrichment for CTCF (1.04-fold), and, even when CTCF was bound, they did not obey the convergent rule. The data suggest these loops are formed by phase separation of the interacting loci to form a genomic subcompartment, rather than by CTCF-mediated extrusion. Next, we sought to determine whether other features correlated with these long loops. By aligning DNA methylation profiles with the Hi-C data, we observed that anchors often corresponded to regions of very low DNA methylation, and thus sought to analyze the relationship in detail. We found that the anchor position of the long loops had lower average DNA methylation levels than standard loop anchors and very often overlapped with DNA methylation canyons. Canyons are typically decorated with either active or repressive histone marks. We considered whether a particular group of canyons was associated with the long loops. Our findings further indicate that repressed regions marked by the polycomb-mediated histone modification H3K27me3 at DNA methylation canyons generally mediate the formation of canyon loops. Next, we considered whether the long loops associated with repressive grand canyons that we had annotated in HSPCs were present in other cell types. Using Aggregate Peak Analysis (APA), a computational strategy in which the Hi-C submatrices from the vicinity of multiple putative loops are superimposed, we examined 19 human cell types and 10 murine cell types in which loop-resolution Hi-C maps are available. Interestingly, unlike previously characterized genomic subcompartments, these long-range loops are only present in stem and progenitor cells, but not in differentiated cell types, such as T cells and erythroid progenitors (Fig. 1d). Further, we identified one particular loop anchor that lay at the anchor of a long loop and contained no apparent genes ("geneless" canyon, or "GLS"). The GLS harboring this anchor is 17 kb long, lies 1.4 Mb upstream of the HOXA1 gene, and forms long loops with a 28 kb grand canyon in the HOXA region. In order to understand the role of the GLS region in hematopoietic stem cells (HSCs), we deleted the GLS in HSPCs using Cas9-mediated editing and assayed the edited cells for their ability to form colonies. Strikingly, after deleting the GLS, the number of colonies and their size was greatly reduced in edited cells compared to control experiments using either random guide RNAs or electroporation only (Fig. 1e). After ex vivo culture, the overwhelming majority of GLS-knock out HSPCs acquired the marker CD38, indicating that they were differentiating. Similarly, HOXA gene expression, an indicator of HSPC function, was greatly diminished after GLS deletion compared to control cells. These data indicate that the GLS identified in our study is functionally associated with maintenance of the HSC state. Overall, our work reveals long-range interactions between H3K27me3-marked DNA methylation canyons comprising a novel microcompartment associated with cellular identity. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Inside the cell nucleus, genomes fold into organized structures that are characteristic of cell type. Here, we show that this chromatin architecture can be predicted de novo using epigenetic data derived from chromatin immunoprecipitation-sequencing (ChIP-Seq). We exploit the idea that chromosomes encode a 1D sequence of chromatin structural types. Interactions between these chromatin types determine the 3D structural ensemble of chromosomes through a process similar to phase separation. First, a neural network is used to infer the relation between the epigenetic marks present at a locus, as assayed by ChIP-Seq, and the genomic compartment in which those loci reside, as measured by DNA-DNA proximity ligation (Hi-C). Next, types inferred from this neural network are used as an input to an energy landscape model for chromatin organization [Minimal Chromatin Model (MiChroM)] to generate an ensemble of 3D chromosome conformations at a resolution of 50 kilobases (kb). After training the model, dubbed Maximum Entropy Genomic Annotation from Biomarkers Associated to Structural Ensembles (MEGABASE), on odd-numbered chromosomes, we predict the sequences of chromatin types and the subsequent 3D conformational ensembles for the even chromosomes. We validate these structural ensembles by using ChIP-Seq tracks alone to predict Hi-C maps, as well as distances measured using 3D fluorescence in situ hybridization (FISH) experiments. Both sets of experiments support the hypothesis of phase separation being the driving process behind compartmentalization. These findings strongly suggest that epigenetic marking patterns encode sufficient information to determine the global architecture of chromosomes and that de novo structure prediction for whole genomes may be increasingly possible.

Chromosomal Mapping The conformation of the genome in the nucleus and contacts between both proximal and distal loci influence gene expression. In order to map genomic contacts, Lieberman-Aiden et al. (p. 289 , see the cover) developed a technique to allow the detection of all interactions between genomic loci in the eukaryotic nucleus followed by deep sequencing. This technology was used to map the organization of the human genome and to examine the spatial proximity of chromosomal loci at one megabase resolution. The map suggests that the genome is partitioned into two spatial compartments that are related to local chromatin state and whose remodeling correlates with changes in the chromatin state.

We recently used in situ Hi-C to create kilobase-resolution 3D maps of mammalian genomes. Here, we combine these maps with new Hi-C, microscopy, and genome-editing experiments to study the physical structure of chromatin fibers, domains, and loops. We find that the observed contact domains are inconsistent with the equilibrium state for an ordinary condensed polymer. Combining Hi-C data and novel mathematical theorems, we show that contact domains are also not consistent with a fractal globule. Instead, we use physical simulations to study two models of genome folding. In one, intermonomer attraction during polymer condensation leads to formation of an anisotropic "tension globule." In the other, CCCTC-binding factor (CTCF) and cohesin act together to extrude unknotted loops during interphase. Both models are consistent with the observed contact domains and with the observation that contact domains tend to form inside loops. However, the extrusion model explains a far wider array of observations, such as why loops tend not to overlap and why the CTCF-binding motifs at pairs of loop anchors lie in the convergent orientation. Finally, we perform 13 genome-editing experiments examining the effect of altering CTCF-binding sites on chromatin folding. The convergent rule correctly predicts the affected loops in every case. Moreover, the extrusion model accurately predicts in silico the 3D maps resulting from each experiment using only the location of CTCF-binding sites in the WT. Thus, we show that it is possible to disrupt, restore, and move loops and domains using targeted mutations as small as a single base pair.

50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.

SummaryWe use in situ Hi-C to probe the 3D architecture of genomes, constructing haploid and diploid maps of nine cell types. The densest, in human lymphoblastoid cells, contains 4.9 billion contacts, achieving 1 kb resolution. We find that genomes are partitioned into contact domains (median length, 185 kb), which are associated with distinct patterns of histone marks and segregate into six subcompartments. We identify ∼10,000 loops. These loops frequently link promoters and enhancers, correlate with gene activation, and show conservation across cell types and species. Loop anchors typically occur at domain boundaries and bind CTCF. CTCF sites at loop anchors occur predominantly (>90%) in a convergent orientation, with the asymmetric motifs “facing” one another. The inactive X chromosome splits into two massive domains and contains large loops anchored at CTCF-binding repeats.PaperFlick

High order chromatin structure is implicated in multiple developmental processes and disease. However, a global picture of chromosomal looping interaction alterations during stem cell self-renewal and differentiation is lacking. Hematopoietic stem cell (HSCs) and their differentiated progenitors (HSPCs) offer a system in which to examine this. Of the key differentiated lineages, the erythroid lineage undergoes a unique nuclear condensation process during a well-characterized differentiation process which can be induced in vitro from CD34+ HSPCs. Thus erythroid differentiation offers an ideal model system to study differentiation-associated changes in high order chromatin structure. We have thus generated the in situ Hi-C contact map for human cord blood CD34+ CD38- HSPC (CD34+) and erythroid progenitors undergoing differentiation in vitro at day 7 from CD34+ HSPCs (EryD7). In our 5kb resolution map, we identified over 2000 chromosomal loop interactions in both CD34+ and Day 7 erythroid respectively . The EryD7 sample exhibited higher random intra-chromosomal interactions in comparison with CD34+, presumably due to nuclear condensation. By comparing the chromosomal loop interactions in the 2 cell types. We identified self-renewal and erythroid differentiation-specific looping patterns in the two cell types. Strikingly, we found that a gene depleted region (GDR) 2MB upstream of the HOXA cluster forms a strong chromosome loop with the HOXA cluster exclusively in the HSPCs (Fig1A). Within this GDR site, we identified two conserved CTCF sites, which are thought to organized chromosome looping. Utilizing the CRISPR-mediated deletion of each of the two CTCF sites, we found that deletion of either site reduce the colony forming ability of CD34+, indicating a loss of stem cell self-renewal. (Fig 1B) Gene expression analysis showed that HOXA9 expression was compromised the CTCF site deletion. These data suggest that the GDR is forming a distant regulatory loop which controls the expression of HOXA9 in HSPCs. Because the GDR is implicated in controlling HOXA9 expression, a key gene in leukemogenesis, we then tested the importance of this looping site in different leukemia cell lines that are dependent on HOXA9. Of those cell lines, we found the deletion of the CTCF sites inhibit the growth of DNMT3A and NPM1 mutated OCI-AML3 and promote the apoptosis. In contrast, growth of the MLL translocation cell line MV 4:11 was not abrogated by their deletion (Fig 1C). As a control cell line which doesn't express HOXA9, HL60 cells were not sensitive to the deletion of the GDR CTCF sites. Together, these data indicate leukemic cells may adopt different strategies to activate HOXA9. MLL translocation leukemias activates HOXA9 by the direct binding of the MLL fusion protein, while the NPM1 mutated leukemia is more likely to utilize the stem cell looping to activate HOXA9 expression. Among EryD7 specific interactions, we found the β-globin locus specifically forms chromatin loops at Day7 that are not evident in the CD34+ HSPCs. Detailed examination showed that Dnase I hypersensitivity sites HS5 and 3'HS1 both contains CTCF site and form chromosomal loops. Two other loop-forming CTCF sites, both on the telomeric and centromeric side of β-globin locus were also identified. Interestingly, we found a CTCF binding site adjacent to OR52A5 gene which forms a chromosomal loop with HS5 and is not well studied. To test the role of the chromosomal looping in the regulation of hemoglobin gene expression in β-globin locus, we deleted the OR52A5-CTCF site and the 3'HS1 CTCF site in K562 and adult CD34+ HSPCs. We found the deletion of OR52A5-CTCF resulted in a decrease of HBE and increase of HBB expression in K562 cells, which suggest the OR52A5 CTCF also plays a role in regulating hemoglobin gene expression in the β-globin locus (Fig 1D). Furthermore, we found the deletion of 3'HS1 CTCF resulted in a 4-fold increase of HBG2 expression in adult CD34+ HSPC during erythroid differentiation (Fig1 E). Thus this indicating the 3'HS1 and OR52A5-CTCF CTCF sites in β-globin locus are forming loops that regulate the β-globin locus gene expression. In summary, we have mapped the higher order chromatin structure alterations during stem cell differentiation and identified the critical looping interaction essential for the self-renewal and differentiation specific functions. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity (2-6). Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible (7-10). We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments. Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

We report the application of single-molecule-based sequencing technology for high-throughput profiling of histone modifications in mammalian cells. By obtaining over four billion bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of mouse embryonic stem cells, neural progenitor cells and embryonic fibroblasts. We find that lysine 4 and lysine 27 trimethylation effectively discriminates genes that are expressed, poised for expression, or stably repressed, and therefore reflect cell state and lineage potential. Lysine 36 trimethylation marks primary coding and non-coding transcripts, facilitating gene annotation. Trimethylation of lysine 9 and lysine 20 is detected at satellite, telomeric and active long-terminal repeats, and can spread into proximal unique sequences. Lysine 4 and lysine 9 trimethylation marks imprinting control regions. Finally, we show that chromatin state can be read in an allele-specific manner by using single nucleotide polymorphisms. This study provides a framework for the application of comprehensive chromatin profiling towards characterization of diverse mammalian cell populations.