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.

/pdf/comprehensive-mapping-of-long-range-interactions-reveals-15wp5ww9ic.pdf

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

Drought resiliency strategies combine developmental, physiological, cellular, and molecular mechanisms. Here, we compare drought responses in two resilient spring wheat (Triticum aestivum) genotypes: a well-studied drought-resilient Drysdale and a resilient genotype from the US Pacific North-West Hollis. While both genotypes utilize higher water use efficiency through the reduction of stomatal conductance, other mechanisms differ. First, Hollis deploys the drought escape mechanism to a greater extent than Drysdale by accelerating the flowering time and reducing root growth. Second, Drysdale uses physiological mechanisms such as non-photochemical quenching (NPQ) to dissipate the excess of harvested light energy and sustain higher Fv/Fm and ϕPSII, whereas Hollis maintains constant NPQ but lower Fv/Fm and ϕPSII values. Furthermore, more electron donors of the electron transport chain are in the oxidized state in Hollis than in Drysdale. Third, many ROS homeostasis parameters, including peroxisome abundance, transcription of peroxisome biogenesis genes PEX11 and CAT, catalase protein level, and enzymatic activity, are higher in Hollis than in Drysdale. Fourth, transcription of autophagy flux marker ATG8.4 is upregulated to a greater degree in Hollis than in Drysdale under drought, whereas relative ATG8 protein abundance under drought stress is lower in Hollis than in Drysdale. These data demonstrate the activation of autophagy in both genotypes and a greater autophagic flux in Hollis. In conclusion, wheat varieties utilize different drought tolerance mechanisms. Combining these mechanisms within one genotype offers a promising strategy to advance crop resiliency.

/pdf/drought-tolerance-strategies-and-autophagy-in-resilient-qm6suk31.pdf

Drought Tolerance Strategies and Autophagy in Resilient Wheat Genotypes

Chromosome conformation capture and its variants interrogate population-average chromatin structure at a higher resolution and throughput than microscopic methods. Capture Hi-C is a variant tailored for the simultaneous assessment of all interactions with thousands of specific bait sequences, so is particularly suited to genome-wide studies of promoter interactions with distal regulatory elements, such as enhancers. We present the principles and methods for Promoter Capture Hi-C (PCHi-C), from experimental design to data analysis.

Assessment of 3D Interactions Between Promoters and Distal Regulatory Elements with Promoter Capture Hi-C (PCHi-C).

X chromosome dosage compensation in Drosophila requires chromosome-wide coordination of gene activation. The male-specific-lethal dosage compensation complex (DCC) identifies X chromosomal High Affinity Sites (HAS) from which it boosts transcription. A sub-class of HAS, PionX sites, represent first contacts on the X. Here, we explored the chromosomal interactions of representative PionX sites by high-resolution 4C and determined the global chromosome conformation by Hi-C in sex-sorted embryos. Male and female X chromosomes display similar nuclear architecture, concordant with clustered, constitutively active genes. PionX sites, like HAS, are evenly distributed in the active compartment and engage in short- and long-range interactions beyond compartment boundaries. By de novo induction of DCC in female cells, we monitored the extent of activation surrounding PionX sites. This revealed a remarkable range of DCC action not only in linear proximity, but also at megabase distance if close in space, suggesting that DCC profits from pre-existing chromosome folding to activate genes.

/pdf/the-drosophila-dosage-compensation-complex-activates-target-2mvazmc5sn.pdf

The Drosophila Dosage Compensation Complex activates target genes by chromosome looping within the active compartment

Dissecting Gene Repression: Not Just Location, Location, Location

Abstract The action of cis-regulatory elements with either activation or repression functions underpins the precise regulation of gene expression during normal development and cell differentiation. Gene activation by the combined activities of promoters and distal enhancers has been extensively studied in normal and pathological contexts. In sharp contrast, gene repression by cis-acting silencers, defined as genetic elements that negatively regulate gene transcription in a position-independent fashion, is less well understood. Here, we repurpose the STARR-seq approach as a novel high-throughput reporter strategy to quantitatively assess silencer activity in mammals. We assessed silencer activity from DNase hypersensitive I sites in a mouse T cell line. Identified silencers were associated with either repressive or active chromatin marks and enriched for binding motifs of known transcriptional repressors. CRISPR-mediated genomic deletions validated the repressive function of distinct silencers involved in the repression of non-T cell genes and genes regulated during T cell differentiation. Finally, we unravel an association of silencer activity with short tandem repeats, highlighting the role of repetitive elements in silencer activity. Our results provide a general strategy for genome-wide identification and characterization of silencer elements.

/pdf/short-tandem-repeats-are-important-contributors-to-silencer-2kldjyuz.pdf

Tom Sexton

Papers

Drought Tolerance Strategies and Autophagy in Resilient Wheat Genotypes

Assessment of 3D Interactions Between Promoters and Distal Regulatory Elements with Promoter Capture Hi-C (PCHi-C).

The Drosophila Dosage Compensation Complex activates target genes by chromosome looping within the active compartment

Dissecting Gene Repression: Not Just Location, Location, Location

Short tandem repeats are important contributors to silencer elements in T cells