We describe Hi-C, a method that probes the three-dimensional architecture of whole genomes by coupling proximity-based ligation with massively parallel sequencing. We constructed spatial proximity maps of the human genome with Hi-C at a resolution of 1 megabase. These maps confirm the presence of chromosome territories and the spatial proximity of small, gene-rich chromosomes. We identified an additional level of genome organization that is characterized by the spatial segregation of open and closed chromatin to form two genome-wide compartments. At the megabase scale, the chromatin conformation is consistent with a fractal globule, a knot-free, polymer conformation that enables maximally dense packing while preserving the ability to easily fold and unfold any genomic locus. The fractal globule is distinct from the more commonly used globular equilibrium model. Our results demonstrate the power of Hi-C to map the dynamic conformations of whole genomes.

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Comprehensive mapping of long-range interactions reveals folding principles of the human genome.

A 3D Map of the Human Genome at Kilobase Resolution Reveals Principles of Chromatin Looping

The spatial organization of the genome is intimately linked to its biological function, yet our understanding of higher order genomic structure is coarse, fragmented and incomplete. In the nucleus of eukaryotic cells, interphase chromosomes occupy distinct chromosome territories, and numerous models have been proposed for how chromosomes fold within chromosome territories. These models, however, provide only few mechanistic details about the relationship between higher order chromatin structure and genome function. Recent advances in genomic technologies have led to rapid advances in the study of three-dimensional genome organization. In particular, Hi-C has been introduced as a method for identifying higher order chromatin interactions genome wide. Here we investigate the three-dimensional organization of the human and mouse genomes in embryonic stem cells and terminally differentiated cell types at unprecedented resolution. We identify large, megabase-sized local chromatin interaction domains, which we term 'topological domains', as a pervasive structural feature of the genome organization. These domains correlate with regions of the genome that constrain the spread of heterochromatin. The domains are stable across different cell types and highly conserved across species, indicating that topological domains are an inherent property of mammalian genomes. Finally, we find that the boundaries of topological domains are enriched for the insulator binding protein CTCF, housekeeping genes, transfer RNAs and short interspersed element (SINE) retrotransposons, indicating that these factors may have a role in establishing the topological domain structure of the genome.

/pdf/topological-domains-in-mammalian-genomes-identified-by-3n3egsejz2.pdf

Topological domains in mammalian genomes identified by analysis of chromatin interactions

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.

Integrative analysis of 111 reference human epigenomes

We developed the Genomic Regions Enrichment of Annotations Tool (GREAT) to analyze the functional significance of cis-regulatory regions identified by localized measurements of DNA binding events across an entire genome. Whereas previous methods took into account only binding proximal to genes, GREAT is able to properly incorporate distal binding sites and control for false positives using a binomial test over the input genomic regions. GREAT incorporates annotations from 20 ontologies and is available as a web application. Applying GREAT to data sets from chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-seq) of multiple transcription-associated factors, including SRF, NRSF, GABP, Stat3 and p300 in different developmental contexts, we recover many functions of these factors that are missed by existing gene-based tools, and we generate testable hypotheses. The utility of GREAT is not limited to ChIP-seq, as it could also be applied to open chromatin, localized epigenomic markers and similar functional data sets, as well as comparative genomics sets.

/pdf/great-improves-functional-interpretation-of-cis-regulatory-47gi1yqk9r.pdf

GREAT improves functional interpretation of cis-regulatory regions

Despite a growing understanding of how epigenetic marks such as histone modifications locally modify the activity of the chromatin with which they are associated, we know little about how marked regions on different parts of the genome are able to intercommunicate to effect regulation of gene expression programs. Recent advances in methods that systematically map pairwise chromatin interactions have uncovered important principles of chromosome folding, which are tightly linked to the epigenetic mark profiles and, hence, functional state of the underlying chromatin fiber.

/pdf/chromosome-folding-driver-or-passenger-of-epigenetic-state-44pch9qkxl.pdf

Chromosome folding: driver or passenger of epigenetic state?

Chromosomes must be tightly folded within cell nuclei but in a manner ensuring accessibility to the machinery regulating transcription, replication, and repair. Recent studies have identified the hierarchical organization principles of chromosome folding, which impact on transcriptional regulation of gene programs. Differentiation of pluripotent stem cells is characterized by a transition from generally open and transcriptionally permissive chromatin to a more polarized state, whereby lineage-specific genes are maintained in an “open” configuration and genes in other lineages are shut down within repressive, more compacted chromatin. Despite such an apparent difference between pluripotent and differentiated chromatin states, the underlying architectural organization principles are very similar. Here, we provide an overview on our understanding of how chromosome folding is linked to cell fate control via transcriptional regulation, focusing on the similarities and differences between pluripotent and somatic cells and special cases (e.g., mitosis) where chromatin folding appears very different.

Chromatin architecture and topology in pluripotent stem cells

ABSTRACT The histone variant macroH2A1.1 plays a role in cancer development and metastasis. To determine the underlying molecular mechanisms, we mapped the genome-wide localization of endogenous macroH2A1.1 in the human breast cancer cell line MDA-MB-231. We demonstrate that macroH2A1.1 specifically binds to active promoters and enhancers in addition to facultative heterochromatin. Selective knock down of macroH2A1.1 deregulates the expression of hundreds of highly active genes. Depending on the chromatin landscape, macroH2A1.1 acts through two distinct molecular mechanisms. The first mitigates excessive transcription by binding over domains including the promoter and the gene body. The second stimulates expression of RNA polymerase II (Pol II)-paused genes, including genes regulating mammary tumor cell migration. In contrast to the first mechanism, macroH2A1.1 specifically associates with the transcription start site of Pol II-paused genes. These processes occur in a predefined local 3D genome landscape, but do not require rewiring of enhancer-promoter contacts. We thus propose that macroH2A1.1 serves as a transcriptional modulator with a potential role in assisting the conversion of promoter-locked Pol II into a productive, elongating Pol II.

/pdf/the-histone-variant-macroh2a1-1-regulates-rna-polymerase-ii-2wqm6tpa.pdf

The histone variant macroH2A1.1 regulates RNA polymerase II-paused genes within defined chromatin interaction landscapes

Chromosome conformation capture and its variants have allowed chromatin topology to be interrogated at a superior resolution and throughput than by microscopic methods. Among the method derivatives, 4C-seq (circular chromosome conformation capture, coupled to high-throughput sequencing) is a versatile, cost-effective means of assessing all chromatin interactions with a specific genomic region of interest, making it particularly suitable for interrogating chromatin looping events. We present the principles and procedures for designing and implementing successful 4C-seq experiments.

Tom Sexton

Papers

Chromosome folding: driver or passenger of epigenetic state?

Chromatin architecture and topology in pluripotent stem cells

The histone variant macroH2A1.1 regulates RNA polymerase II-paused genes within defined chromatin interaction landscapes

4C-Seq: Interrogating Chromatin Looping with Circular Chromosome Conformation Capture.

Precise measurements of chromatin diffusion dynamics by modeling using Gaussian processes (Inter-Mito)