Higher-order Chromatin Structure Research Articles

Phase Separation: Direct and Indirect Driving Force for High-Order Chromatin Organization.

The multi-level spatial chromatin organization in the nucleus is closely related to chromatin activity. The mechanism of chromatin organization and remodeling attract much attention. Phase separation describes the biomolecular condensation which is the basis for membraneless compartments in cells. Recent research shows that phase separation is a key aspect to drive high-order chromatin structure and remodeling. In addition, chromatin functional compartmentalization in the nucleus which is formed by phase separation also plays an important role in overall chromatin structure. In this review, we summarized the latest work about the role of phase separation in spatial chromatin organization, focusing on direct and indirect effects of phase separation on 3D chromatin organization and its impact on transcription regulation.

Unraveling the effects of ubiquitylation on nucleosome assembly and dynamics with coarse-grain simulations.

Ubiquitylation of histone proteins remodels and destabilizes the structure, assembly, and dynamics of the nucleosome due to the steric hindrance introduced by the bulky ubiquitin moiety. Histone proteins have multiple ubiquitylation sites, and each appears to have distinct effects on the structure and dynamics of the nucleosome. The spatial dependence of ubiquitin in regulating nucleosome dynamics remains poorly understood due to the non-specific interactions of dyes used in FRET experiments and the poor resolution of ubiquitylated nucleosomes in structural experiments. Using coarse-grain simulations along with Markov State Models, we have studied the reassembly of nucleosome intermediates with canonical and ubiquitylated histones. Results show that the reassembly of hexasomes with ubiquitylated H2A histones at lysine 119 have a destabilized tetramer/dimer interface when compared with the reassembly of hexasomes with canonical histones. Ubiquitin moieties undergo stochastic motions and only form transient interactions with the exit and entry DNA strands. These effects are altered by ubiquitylation at other histone sites, which show distinct effects on nucleosomal structures and dynamics. Together, these results provide a molecular-level understanding of the mechanism by which ubiquitylation location creates distinct effects on the structure and dynamics of the nucleosome and higher-order chromatin structures.

Deciphering Hierarchical Chromatin Domains and Preference of Genomic Position Forming Boundaries in Single Mouse Embryonic Stem Cells.

The exploration of single-cell 3D genome maps reveals that chromatin domains are indeed physical structures presenting in single cells, and domain boundaries vary from cell to cell. However, systematic analysis of the association between regulatory factor binding and elements and the formation of chromatin domains in single cells has not yet emerged. To this end, a hierarchical chromatin domain structure identification algorithm (named as HiCS) is first developed from individual single-cell Hi-C maps, with superior performance in both accuracy and efficiency. The results suggest that in addition to the known CTCF-cohesin complex, Polycomb, TrxG, pluripotent protein families, and other multiple factors also contribute to shaping chromatin domain boundaries in single embryonic stem cells. Different cooperation patterns of these regulatory factors drive genomic position categories with differential preferences forming boundaries, and the most extensive six types of retrotransposons are differentially distributed in these genomic position categories with preferential localization. The above results suggest that these different retrotransposons within genomic regions interplay with regulatory factors navigating the preference of genomic positions forming boundaries, driving the formation of higher-order chromatin structures, and thus regulating cell functions in single mouse embryonic stem cells.

PfSET2 Is Involved in Genome Organization of Var Gene Family in Plasmodium falciparum.

The three-dimensional (3D) genome structure of human malaria parasite Plasmodium falciparum is highly organized and plays important roles in regulating coordinated expression patterns of specific genes such as virulence genes which are involved in antigenic variation and immune escape. However, the molecular mechanisms that control 3D genome of the parasite remain elusive. Here, by analyzing genome organization of P. falciparum, we identify high-interacting regions (HIRs) with strong chromatin interactions at telomeres and virulence genes loci. Specifically, HIRs are highly enriched with repressive histone marks (H3K36me3 and H3K9me3) and form the transcriptional repressive center. Deletion of PfSET2, which controls H3K36me3 level, results in marked reduction of both intrachromosomal and interchromosomal interactions for HIRs. Importantly, such chromatin reorganization coordinates with dynamic changes in epigenetic feature in HIRs and transcriptional activation of var genes. Additionally, different cluster of var genes based on the pattern of chromatin interactions show distinct transcriptional activation potential after deletion of PfSET2. Our results uncover a fundamental mechanism that the epigenetic factor PfSET2 controls the 3D organization of heterochromatin to regulate the transcription activities of var genes family in P. falciparum. IMPORTANCE PfSET2 has been reported to play key role in silencing var genes in Plasmodium falciparum, while the underlying molecular mechanisms remain unclear. Here, we provide evidence that PfSET2 is essential to maintain 3D genome organization of heterochromatin region to keep var genes in transcription repressive state. These findings can contribute better understanding of the regulation of high-order chromatin structure in P. falciparum.

Involvement of linker histone variant H1a in the regulation of early preimplantation development in mice.

Linker histone variants regulate higher-order chromatin structure and various cellular processes. It has been suggested that linker histone variant H1a loosens chromatin structure and activates transcription. However, its role in early mouse development remains to be elucidated. We investigated the functions of H1a during preimplantation development using H1a gene-deleted mice. Although H1a homozygous knockout (KO) mice were born without any abnormalities, the number of offspring were reduced when the mothers but not fathers were homozygous KO animals. Maternal H1a KO compromised development during the morula and blastocyst stages, but not differentiation of the inner cell mass or trophectoderm. Thus, maternal linker histone H1a is important in early development.

Structural variants drive context-dependent oncogene activation in cancer.

Higher-order chromatin structure is important for the regulation of genes by distal regulatory sequences1,2. Structural variants (SVs) that alter three-dimensional (3D) genome organization can lead to enhancer-promoter rewiring and human disease, particularly in the context of cancer3. However, only a small minority of SVs are associated with altered gene expression4,5, and it remains unclear why certain SVs lead to changes in distal gene expression and others do not. To address these questions, we used a combination of genomic profiling and genome engineering to identify sites of recurrent changes in 3D genome structure in cancer and determine the effects of specific rearrangements on oncogene activation. By analysing Hi-C data from 92 cancer cell lines and patient samples, we identified loci affected by recurrent alterations to 3D genome structure, including oncogenes such as MYC, TERT and CCND1. By using CRISPR-Cas9 genome engineering to generate de novo SVs, we show that oncogene activity can be predicted by using 'activity-by-contact' models that consider partner region chromatin contacts and enhancer activity. However, activity-by-contact models are only predictive of specific subsets of genes in the genome, suggesting that different classes of genes engage in distinct modes of regulation by distal regulatory elements. These results indicate that SVs that alter 3D genome organization are widespread in cancer genomes and begin to illustrate predictive rules for the consequences of SVs on oncogene activation.

Genomic innovation and regulatory rewiring during evolution of the cotton genus Gossypium.

Phenotypic diversity and evolutionary innovation ultimately trace to variation in genomic sequence and rewiring of regulatory networks. Here, we constructed a pan-genome of the Gossypium genus using ten representative diploid genomes. We document the genomic evolutionary history and the impact of lineage-specific transposon amplification on differential genome composition. The pan-3D genome reveals evolutionary connections between transposon-driven genome size variation and both higher-order chromatin structure reorganization and the rewiring of chromatin interactome. We linked changes in chromatin structures to phenotypic differences in cotton fiber and identified regulatory variations that decode the genetic basis of fiber length, the latter enabled by sequencing 1,005 transcriptomes during fiber development. We showcase how pan-genomic, pan-3D genomic and genetic regulatory data serve as a resource for delineating the evolutionary basis of spinnable cotton fiber. Our work provides insights into the evolution of genome organization and regulation and will inform cotton improvement by enabling regulome-based approaches.

The Nodewalk assay to quantitate chromatin fiber interactomes in very small cell populations.

The chromosome conformation capture method and its derivatives, such as circularized chromosome conformation capture, carbon copy chromosome conformation capture, high-throughput chromosome conformation capture and capture high-throughput chromosome conformation capture, have pioneered our understanding of the principles of chromosome folding in the nucleus. These technical advances, however, cannot precisely quantitate interaction frequency in very small input samples. Here we describe a protocol for the Nodewalk assay, which is based on converting chromosome conformation capture DNA samples to RNA and subsequently to cDNA using strategically placed primers. This pipeline enables the quantitative analyses of chromatin fiber interactions without compromising its sensitivity down to <300 cells, making it suitable for MiSeq analyses of higher-order chromatin structures in biopsies, circulating tumor cells and transitional cell states, for example. Importantly, the quality of the Nodewalk sample can be assessed before sequencing to avoid unnecessary costs. Moreover, it enables analyses from hundreds of different restriction enzyme fragment viewpoints within the same initial small input sample to uncover complex, genome-wide networks. Following optimization of the different steps, the entire protocol can be completed within 2 weeks.

A new chromatin flavor to cap chromosomes: Where structure, function, and evolution meet

Soman, A., Wong, S.Y., etal. find that telomeric DNA assembles into a new high-order chromatin structure resembling a columnar stack of nucleosomes with dynamic properties. This raises new questions on telomere biology mechanisms and chromatin evolution.

TGM2-mediated histone transglutamination is dictated by steric accessibility

Recent studies have identified serotonylation of glutamine-5 on histone H3 (H3Q5ser) as a novel posttranslational modification (PTM) associated with active transcription. While H3Q5ser is known to be installed by tissue transglutaminase 2 (TGM2), the substrate characteristics affecting deposition of the mark, at the level of both chromatin and individual nucleosomes, remain poorly understood. Here, we show that histone serotonylation is excluded from constitutive heterochromatic regions in mammalian cells. Biochemical studies reveal that the formation of higher-order chromatin structures associated with heterochromatin impose a steric barrier that is refractory to TGM2-mediated histone monoaminylation. A series of structure-activity relationship studies, including the use of DNA-barcoded nucleosome libraries, shows that steric hindrance also steers TGM2 activity at the nucleosome level, restricting monoaminylation to accessible sites within histone tails. Collectively, our data indicate that the activity of TGM2 on chromatin is dictated by substrate accessibility rather than by primary sequence determinants or by the existence of preexisting PTMs, as is the case for many other histone-modifying enzymes.

Modulation of the high-order chromatin structure by Polycomb complexes.

The multi-subunit Polycomb Repressive Complex (PRC) 1 and 2 act, either independently or synergistically, to maintain and enforce a repressive state of the target chromatin, thereby regulating the processes of cell lineage specification and organismal development. In recent years, deep sequencing-based and imaging-based technologies, especially those tailored for mapping three-dimensional (3D) chromatin organization and structure, have allowed a better understanding of the PRC complex-mediated long-range chromatin contacts and DNA looping. In this review, we review current advances as for how Polycomb complexes function to modulate and help define the high-order chromatin structure and topology, highlighting the multi-faceted roles of Polycomb proteins in gene and genome regulation.

Structure of the NuA4 acetyltransferase complex bound to the nucleosome.

Deoxyribonucleic acid in eukaryotes wraps around the histone octamer to form nucleosomes1, the fundamental unit of chromatin. The N termini of histone H4 interact with nearby nucleosomes and play an important role in the formation of high-order chromatin structure and heterochromatin silencing2-4. NuA4 in yeast and its homologue Tip60 complex in mammalian cells are the key enzymes that catalyse H4 acetylation, which in turn regulates chromatin packaging and function in transcription activation and DNA repair5-10. Here we report the cryo-electron microscopy structure of NuA4 from Saccharomycescerevisiae bound to the nucleosome. NuA4 comprises two major modules: the catalytic histone acetyltransferase (HAT) module and the transcription activator-binding (TRA) module. The nucleosome is mainly bound by the HAT module and is positioned close to a polybasic surface of the TRA module, which is important for the optimal activity of NuA4. The nucleosomal linker DNA carrying the upstream activation sequence is oriented towards the conserved, transcription activator-binding surface of the Tra1 subunit, which suggests a potential mechanism of NuA4 to act as a transcription co-activator. The HAT module recognizes the disk face of the nucleosome through the H2A-H2B acidic patch and nucleosomal DNA, projecting the catalytic pocket of Esa1 to the N-terminal tail of H4 and supporting its function in selective acetylation of H4. Together, our findings illustrate how NuA4 is assembled and provide mechanistic insights into nucleosome recognition and transcription co-activation by a HAT.

3D chromatin remodeling potentiates transcriptional programs driving cell invasion

The contribution of deregulated chromatin architecture, including topologically associated domains (TADs), to cancer progression remains ambiguous. CCCTC-binding factor (CTCF) is a central regulator of higher-order chromatin structure that undergoes copy number loss in over half of all breast cancers, but the impact of this defect on epigenetic programming and chromatin architecture remains unclear. We find that under physiological conditions, CTCF organizes subTADs to limit the expression of oncogenic pathways, including phosphatidylinositol 3-kinase (PI3K) and cell adhesion networks. Loss of a single CTCF allele potentiates cell invasion through compromised chromatin insulation and a reorganization of chromatin architecture and histone programming that facilitates de novo promoter-enhancer contacts. However, this change in the higher-order chromatin landscape leads to a vulnerability to inhibitors of mTOR. These data support a model whereby subTAD reorganization drives both modification of histones at de novo enhancer-promoter contacts and transcriptional up-regulation of oncogenic transcriptional networks.

Chromatin structure undergoes global and local reorganization during murine dendritic cell development and activation

Classical dendritic cells (cDCs) are essential for immune responses and differentiate from hematopoietic stem cells via intermediate progenitors, such as monocyte-DC progenitors (MDPs) and common DC progenitors (CDPs). Upon infection, cDCs are activated and rapidly express host defense-related genes, such as those encoding cytokines and chemokines. Chromatin structures, including nuclear compartments and topologically associating domains (TADs), have been implicated in gene regulation. However, the extent and dynamics of their reorganization during cDC development and activation remain unknown. In this study, we comprehensively determined higher-order chromatin structures by Hi-C in DC progenitors and cDC subpopulations. During cDC differentiation, chromatin activation was initially induced at the MDP stage. Subsequently, a shift from inactive to active nuclear compartments occurred at the cDC gene loci in CDPs, which was followed by increased intra-TAD interactions and loop formation. Mechanistically, the transcription factor IRF8, indispensable for cDC differentiation, mediated chromatin activation and changes into the active compartments in DC progenitors, thereby possibly leading to cDC-specific gene induction. Using an infection model, we found that the chromatin structures of host defense-related gene loci were preestablished in unstimulated cDCs, indicating that the formation of higher-order chromatin structures prior to infection may contribute to the rapid responses to pathogens. Overall, these results suggest that chromatin structure reorganization is closely related to the establishment of cDC-specific gene expression and immune functions. This study advances the fundamental understanding of chromatin reorganization in cDC differentiation and activation.

Chromatin structure meets cryo-EM: Dynamic building blocks of the functional architecture

Chromatin is a dynamic molecular complex composed of DNA and proteins that package the DNA in the nucleus of eukaryotic cells. The basic structural unit of chromatin is the nucleosome core particle, composed of ~150 base pairs of genomic DNA wrapped around a histone octamer containing two copies each of four histones, H2A, H2B, H3, and H4. Individual nucleosome core particles are connected by short linker DNAs, forming a nucleosome array known as a beads-on-a-string fiber. Higher-order structures of chromatin are closely linked to nuclear events such as replication, transcription, recombination, and repair. Recently, a variety of chromatin structures have been determined by single-particle cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), and their structural details have provided clues about the chromatin architecture functions in the cell. In this review, we highlight recent cryo-EM structural studies of a fundamental chromatin unit to clarify the functions of chromatin.

Breaking boundaries: Pan BETi disrupt 3D chromatin structure, BD2-selective BETi are strictly epigenetic transcriptional regulators.

Bromodomain and extraterminal proteins (BETs) are more than just epigenetic regulators of transcription. Here we highlight a new role for the BET protein BRD4 in the maintenance of higher order chromatin structure at Topologically Associating Domain Boundaries (TADBs). BD2-selective and pan (non-selective) BET inhibitors (BETi) differentially support chromatin structure, selectively affecting transcription and cell viability. Using RNA-seq and BRD4 ChIP-seq, the differential effect of BETi treatment on the transcriptome and BRD4 chromatin occupancy of human aortic endothelial cells from diabetic patients (dHAECs) stimulated with TNFα was evaluated. Chromatin decondensation and DNA fragmentation was assessed by immunofluorescence imaging and quantification. Key dHAEC findings were verified in proliferating monocyte-like THP-1 cells using real time-PCR, BRD4 co-immunoprecipitation studies, western blots, proliferation and apoptosis assays. We discovered that 1) BRD4 co-localizes with Ying-Yang 1 (YY1) at TADBs, critical chromatin structure complexes proximal to many DNA repair genes. 2) BD2-selective BETi enrich BRD4/YY1 associations, while pan-BETi do not. 3) Failure to support chromatin structures through BRD4/YY1 enrichment inhibits DNA repair gene transcription, which induces DNA damage responses, and causes widespread chromatin decondensation, DNA fragmentation, and apoptosis. 4) BD2-selective BETi maintain high order chromatin structure and cell viability, while reducing deleterious pro-inflammatory transcription. BRD4 plays a previously unrecognized role at TADBs. BETi differentially impact TADB stability. Our results provide translational insight for the development of BETi as therapeutics for a range of diseases including CVD, chronic kidney disease, cancer, and COVID-19.

DbEmbryo multi-omics database for analyses of synergistic regulation in early mammalian embryo development.

During early mammalian embryo development, different epigenetic marks undergo reprogramming and play crucial roles in the mediation of gene expression. Currently, several databases provide multi-omics information on early embryos. However, how interconnected epigenetic markers function together to coordinate the expression of the genetic code in a spatiotemporal manner remains difficult to analyze, markedly limiting scientific and clinical research. Here, we present dbEmbryo, an integrated and interactive multi-omics database for human and mouse early embryos. dbEmbryo integrates data on gene expression, DNA methylation, histone modifications, chromatin accessibility, and higher-order chromatin structure profiles for human and mouse early embryos. It incorporates customized analysis tools, such as “multi-omics visualization,” “Gene&Peak annotation,” “ZGA gene cluster,” “cis-regulation,” “synergistic regulation,” “promoter signal enrichment,” and “3D genome.” Users can retrieve gene expression and epigenetic profile patterns to analyze synergistic changes across different early embryo developmental stages. We showed the uniqueness of dbEmbryo among extant databases containing data on early embryo development and provided an overview. Using dbEmbryo, we obtained a phase-separated model of transcriptional control during early embryo development. dbEmbryo offers web-based analytical tools and a comprehensive resource for biologists and clinicians to decipher molecular regulatory mechanisms of human and mouse early embryo development.

SmcHD1 underlies the formation of H3K9me3 blocks on the inactive X chromosome in mice.

Stable silencing of the inactive X chromosome (Xi) in female mammals is crucial for the development of embryos and their postnatal health. SmcHD1 is essential for stable silencing of the Xi, and its functional deficiency results in derepression of many X-inactivated genes. Although SmcHD1 has been suggested to play an important role in the formation of higher-order chromatin structure of the Xi, the underlying mechanism is largely unknown. Here, we explore the epigenetic state of the Xi in SmcHD1-deficient epiblast stem cells and mouse embryonic fibroblasts in comparison with their wild-type counterparts. The results suggest that SmcHD1 underlies the formation of H3K9me3-enriched blocks on the Xi, which, although the importance of H3K9me3 has been largely overlooked in mice, play a crucial role in the establishment of the stably silenced state. We propose that the H3K9me3 blocks formed on the Xi facilitate robust heterochromatin formation in combination with H3K27me3, and that the substantial loss of H3K9me3 caused by SmcHD1 deficiency leads to aberrant distribution of H3K27me3 on the Xi and derepression of X-inactivated genes.

An epigenome atlas of neural progenitors within the embryonic mouse forebrain

A comprehensive characterization of epigenomic organization in the embryonic mouse forebrain will enhance our understanding of neurodevelopment and provide insight into mechanisms of neurological disease. Here we collected single-cell chromatin accessibility profiles from four distinct neurogenic regions of the embryonic mouse forebrain using single nuclei ATAC-Seq (snATAC-Seq). We identified thousands of differentially accessible peaks, many restricted to distinct progenitor cell types or brain regions. We integrated snATAC-Seq and single cell transcriptome data to characterize changes of chromatin accessibility at enhancers and promoters with associated transcript abundance. Multi-modal integration of histone modifications (CUT&Tag and CUT&RUN), promoter-enhancer interactions (Capture-C) and high-order chromatin structure (Hi-C) extended these initial observations. This dataset reveals a diverse chromatin landscape with region-specific regulatory mechanisms and genomic interactions in distinct neurogenic regions of the embryonic mouse brain and represents an extensive public resource of a ‘ground truth’ epigenomic landscape at this critical stage of neurogenesis.

High enhancer activity is an epigenetic feature of HPV negative atypical head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease with significant mortality and frequent recurrence. Prior efforts to transcriptionally classify HNSCC into groups of varying prognoses have identified four accepted molecular subtypes of the disease: Atypical (AT), Basal (BA), Classical (CL), and Mesenchymal (MS). Here, we investigate the active enhancer landscapes of these subtypes using representative HNSCC cell lines and identify samples belonging to the AT subtype as having increased enhancer activity compared to the other 3 HNSCC subtypes. Cell lines belonging to the AT subtype are more resistant to enhancer-blocking bromodomain inhibitors (BETi). Examination of nascent transcripts reveals that both AT TCGA tumors and cell lines express higher levels of enhancer RNA (eRNA) transcripts for enhancers controlling BETi resistance pathways, such as lipid metabolism and MAPK signaling. Additionally, investigation of higher-order chromatin structure suggests more enhancer-promoter (E-P) contacts in the AT subtype, including on genes identified in the eRNA analysis. Consistently, known BETi resistance pathways are upregulated upon exposure to these inhibitors. Together, our results identify that the AT subtype of HNSCC is associated with higher enhancer activity, resistance to enhancer blockade, and increased signaling through pathways that could serve as future targets for sensitizing HNSCC to BET inhibition.