3D Chromatin Organization Research Articles

Foxp3 orchestrates reorganization of chromatin architecture to establish regulatory T cell identity

Chromatin conformation reorganization is emerging as an important layer of regulation for gene expression and lineage specification. Yet, how lineage-specific transcription factors contribute to the establishment of cell type-specific 3D chromatin architecture in the immune cells remains unclear, especially for the late stages of T cell subset differentiation and maturation. Regulatory T cells (Treg) are mainly generated in the thymus as a subpopulation of T cells specializing in suppressing excessive immune responses. Here, by comprehensively mapping 3D chromatin organization during Treg cell differentiation, we show that Treg-specific chromatin structures were progressively established during its lineage specification, and highly associated with Treg signature gene expression. Additionally, the binding sites of Foxp3, a Treg lineage specifying transcription factor, were highly enriched at Treg-specific chromatin loop anchors. Further comparison of the chromatin interactions between wide-type Tregs versus Treg cells from Foxp3 knock-in/knockout or newly-generated Foxp3 domain-swap mutant mouse revealed that Foxp3 was essential for the establishment of Treg-specific 3D chromatin architecture, although it was not dependent on the formation of the Foxp3 domain-swapped dimer. These results highlighted an underappreciated role of Foxp3 in modulating Treg-specific 3D chromatin structure formation.

Perspective: “Current understanding of NADs dynamics and mechanisms of Disease”

Chromatin architecture is essential for gene regulation, and multiple levels of the 3D chromatin organization exhibit dynamic changes during organismal development and cell differentiation. Heterochromatin, termed compartment B in Hi-C datasets, is a phase-separating gene-silencing form of chromatin, preferentially located at the two nuclear sites, nuclear (lamina-associate chromatin domains, LADs) and nucleoli (nucleoli-associated chromatin domains, NADs) peripheries. LADs and NADs contain both interchangeable and location-specific chromatin domains. Recent studies suggest striking dynamics in LADs and NADs during the differentiation of embryonic stem cells into neural progenitors and neurons. Here we discuss recent advances in understanding NADs changes during neuronal differentiation and future questions on how NADs integrity can contribute to healthy neurodevelopment and neurodevelopment diseases.

HSFA1a: the quarterback of heat stress response and 3D-chromatin organization.

In eukaryotes, the highly complex chromatin exhibits structural dynamism to modulate cellular responses. Recently, Huang et al. have shown a novel role of the heat stress master regulator, HSFA1a, in transiently reorganizing the 3D chromatin to promote distal and proximal enhancer-promoter contacts to regulate stress-responsive genes in tomato.

Rewiring of the promoter-enhancer interactome and regulatory landscape in glioblastoma orchestrates gene expression underlying neurogliomal synaptic communication

Chromatin organization controls transcription by modulating 3D-interactions between enhancers and promoters in the nucleus. Alterations in epigenetic states and 3D-chromatin organization result in gene expression changes contributing to cancer. Here, we map the promoter-enhancer interactome and regulatory landscape of glioblastoma, the most aggressive primary brain tumour. Our data reveals profound rewiring of promoter-enhancer interactions, chromatin accessibility and redistribution of histone marks in glioblastoma. This leads to loss of long-range regulatory interactions and overall activation of promoters, which orchestrate changes in the expression of genes associated to glutamatergic synapses, axon guidance, axonogenesis and chromatin remodelling. SMAD3 and PITX1 emerge as major transcription factors controlling genes related to synapse organization and axon guidance. Inhibition of SMAD3 and neuronal activity stimulation cooperate to promote proliferation of glioblastoma cells in co-culture with glutamatergic neurons, and in mice bearing patient-derived xenografts. Our findings provide mechanistic insight into the regulatory networks that mediate neurogliomal synaptic communication.

High-speed AFM imaging reveals DNA capture and loop extrusion dynamics by cohesin-NIPBL

3D chromatin organization plays a critical role in regulating gene expression, DNA replication, recombination, and repair. While initially discovered for its role in sister chromatid cohesion, emerging evidence suggests that the cohesin complex (SMC1, SMC3, RAD21, and SA1/SA2), facilitated by NIPBL, mediates topologically associating domains and chromatin loops through DNA loop extrusion. However, information on how conformational changes of cohesin-NIPBL drive its loading onto DNA, initiation, and growth of DNA loops is still lacking. In this study, high-speed atomic force microscopy imaging reveals that cohesin-NIPBL captures DNA through arm extension, assisted by feet (shorter protrusions), and followed by transfer of DNA to its lower compartment (SMC heads, RAD21, SA1, and NIPBL). While binding at the lower compartment, arm extension leads to the capture of a second DNA segment and the initiation of a DNA loop that is independent of ATP hydrolysis. The feet are likely contributed by the C-terminal domains of SA1 and NIPBL and can transiently bind to DNA to facilitate the loading of the cohesin complex onto DNA. Furthermore, high-speed atomic force microscopy imaging reveals distinct forward and reverse DNA loop extrusion steps by cohesin-NIPBL. These results advance our understanding of cohesin by establishing direct experimental evidence for a multistep DNA-binding mechanism mediated by dynamic protein conformational changes.

A chromEM-staining protocol optimized for cardiac tissue.

Three-dimensional (3D) chromatin organization has a key role in defining the transcription program of cells during development. Its alteration is the cause of gene expression changes responsible for several diseases. Thus, we need new tools to study this aspect of gene expression regulation. To this end, ChromEM was recently developed: this is an electron-microscopy staining technique that selectively marks nuclear DNA without altering its structure and, thus, allows better visualization of 3D chromatin conformation. However, despite increasingly frequent application of this staining technique on cells, it has not yet been applied to visualize chromatin ultrastructure in tissues. Here, we provide a protocol to carry out ChromEM on myocardial tissue harvested from the left ventricles of C57BL/6J mice and use this in combination with transmission electron microscopy (TEM) to measure some morphological parameters of peripheral heterochromatin in cardiomyocytes. This protocol could also be used, in combination with electron tomography, to study 3D chromatin organization in cardiomyocytes in different aspects of heart pathobiology (e.g., heart development, cardiac aging, and heart failure) as well as help to set-up ChromEM in other tissues.

Epigenomic analysis of Alzheimer's disease brains reveals diminished CTCF binding on genes involved in synaptic organization

Epigenetic aberrations are suggested to play an important role in transcriptional alterations in Alzheimer's disease (AD). One of the key mechanisms of epigenetic regulation of gene expression is through the dynamic organization of chromatin structure via the master genome architecture protein, CCCTC-binding factor (CTCF). By forming chromatin loops, CTCF can influence gene transcription in a complex manner. To find out whether genome-wide DNA binding sites for CTCF are altered in AD, we compared CTCF chromatin immunoprecipitation sequencing (ChIP-Seq) data from frontal cortex of human AD patients and normal controls (n = 9 pairs, all females). We have revealed that CTCF-binding affinity on many genes is significantly reduced in AD patients, and these genes are enriched in synaptic organization, cell adhesion, and actin cytoskeleton, including synaptic scaffolding molecules and receptors, such as SHANK2, HOMER1, NRXN1, CNTNAP2 and GRIN2A, and protocadherin (PCDH) and cadherin (CDH) family members. By comparing transcriptomic data from AD patients, we have discovered that many of the synaptic and adhesion genes with reduced CTCF binding in AD are significantly reduced in their mRNA expression. Moreover, a significant overlap of genes with the diminished CTCF binding and the reduced H3K27ac is identified in AD, with the common genes enriched in synaptic organization. These data suggest that the CTCF-controlled 3D chromatin organization is perturbed in AD, which may be linked to the diminished expression of target genes, probably through changes in histone modification.

Conserved H3K27me3-associated chromatin looping mediates physical interactions of gene clusters in plants.

Higher-order chromatin organization is essential for transcriptional regulation, genome stability maintenance, and other genome functions. Increasing evidence has revealed significant differences in 3D chromatin organization between plants and animals. However, the extent, pattern, and rules of chromatin organization in plants are still unclear. In this study, we systematically identified and characterized long-range chromatin loops in the Arabidopsis 3D genome. We identified hundreds of long-range cis chromatin loops and found their anchor regions are closely associated with H3K27me3 epigenetic modifications. Furthermore, we demonstrated that these chromatin loops are dependent on Polycomb group (PcG) proteins, suggesting that the Polycomb repressive complex 2 (PRC2) complex is essential for establishing and maintaining these novel loops. Although most of these PcG-medicated chromatin loops are stable, many of these loops are tissue-specific or dynamically regulated by different treatments. Interestingly, tandemly arrayed gene clusters and metabolic gene clusters are enriched in anchor regions. Long-range H3K27me3-marked chromatin interactions are associated with the coregulation of specific gene clusters. Finally, we also identified H3K27me3-associated chromatin loops associated with gene clusters in Oryza sativa and Glycine max, indicating that these long-range chromatin loops are conserved in plants. Our results provide novel insights into genome evolution and transcriptional coregulation in plants.

Chromatin alternates between A and B compartments at kilobase scale for subgenic organization

Nuclear compartments are prominent features of 3D chromatin organization, but sequencing depth limitations have impeded investigation at ultra fine-scale. CTCF loops are generally studied at a finer scale, but the impact of looping on proximal interactions remains enigmatic. Here, we critically examine nuclear compartments and CTCF loop-proximal interactions using a combination of in situ Hi-C at unparalleled depth, algorithm development, and biophysical modeling. Producing a large Hi-C map with 33 billion contacts in conjunction with an algorithm for performing principal component analysis on sparse, super massive matrices (POSSUMM), we resolve compartments to 500 bp. Our results demonstrate that essentially all active promoters and distal enhancers localize in the A compartment, even when flanking sequences do not. Furthermore, we find that the TSS and TTS of paused genes are often segregated into separate compartments. We then identify diffuse interactions that radiate from CTCF loop anchors, which correlate with strong enhancer-promoter interactions and proximal transcription. We also find that these diffuse interactions depend on CTCF’s RNA binding domains. In this work, we demonstrate features of fine-scale chromatin organization consistent with a revised model in which compartments are more precise than commonly thought while CTCF loops are more protracted.

3D genome organization and epigenetic regulation in autoimmune diseases.

Three-dimensional (3D) genomics is an emerging field of research that investigates the relationship between gene regulatory function and the spatial structure of chromatin. Chromatin folding can be studied using chromosome conformation capture (3C) technology and 3C-based derivative sequencing technologies, including chromosome conformation capture-on-chip (4C), chromosome conformation capture carbon copy (5C), and high-throughput chromosome conformation capture (Hi-C), which allow scientists to capture 3D conformations from a single site to the entire genome. A comprehensive analysis of the relationships between various regulatory components and gene function also requires the integration of multi-omics data such as genomics, transcriptomics, and epigenomics. 3D genome folding is involved in immune cell differentiation, activation, and dysfunction and participates in a wide range of diseases, including autoimmune diseases. We describe hierarchical 3D chromatin organization in this review and conclude with characteristics of C-techniques and multi-omics applications of the 3D genome. In addition, we describe the relationship between 3D genome structure and the differentiation and maturation of immune cells and address how changes in chromosome folding contribute to autoimmune diseases.

Multiomic analysis of cohesin reveals that ZBTB transcription factors contribute to chromatin interactions.

One bottleneck in understanding the principles of 3D chromatin structures is caused by the paucity of known regulators. Cohesin is essential for 3D chromatin organization, and its interacting partners are candidate regulators. Here, we performed proteomic profiling of the cohesin in chromatin and identified transcription factors, RNA-binding proteins and chromatin regulators associated with cohesin. Acute protein degradation followed by time-series genomic binding quantitation and BAT Hi-C analysis were conducted, and the results showed that the transcription factor ZBTB21 contributes to cohesin chromatin binding, 3D chromatin interactions and transcriptional repression. Strikingly, multiomic analyses revealed that the other four ZBTB factors interacted with cohesin, and double degradation of ZBTB21 and ZBTB7B led to a further decrease in cohesin chromatin occupancy. We propose that multiple ZBTB transcription factors orchestrate the chromatin binding of cohesin to regulate chromatin interactions, and we provide a catalog of many additional proteins associated with cohesin that warrant further investigation.

Impact of Chromatin 3D-Organization on Promoter–Superenhancer Interactions in Embryonic Stem vs Cancer Cells

The interaction of enhancers and superenhancers (SE) with promoters is functionally significant for the regulation of gene expression. Pattern of these interactions plays a key role in various processes, such as differentiation, malignant transformation, etc. In order to quantify the relationship between 3D chromatin organization and promoter–SE contacts, a computational analysis of chromatin conformations near the murine Nanog pluripotency gene was performed for normal embryonic stem (mESC) and lymphoma (CH12LX) cells. Using biophysical modeling approach, the following parameters of the promoter–SE interactions were identified: the distribution of distances between the Nanog promoter and the SEs, the frequency of contacts with one and several SEs simultaneously. In normal mESC expressing Nanog, the frequency of contacts of promoters with SEs is higher than in cancer cells, and complex contacts with two or more SEs are more frequent. The modelling reveals a small subpopulation of cancer cells, where the promoter contacts simultaneously three SEs. The predicted subpopulation of cancer cells with multiple promoter–SE contacts may be predisposed to increased stemness and hypothetically be considered as a reservoir for generation of cancer stem cells.

Impact of the 3D Chromatin Organization on Promoter–Super-Enhancer Interactions in Embryonic Stem Cells and Cancer Cells

Impact of the 3D Chromatin Organization on Promoter–Super-Enhancer Interactions in Embryonic Stem Cells and Cancer Cells

Landscape and perturbation of enhancer-driven core transcription regulatory circuits in cancer

Landscape and perturbation of enhancer-driven core transcription regulatory circuits in cancer

Binding by the Polycomb complex component BMI1 and H2A monoubiquitination shape local and long-range interactions in the Arabidopsis genome.

Three-dimensional (3D) chromatin organization is highly dynamic during development and seems to play a crucial role in regulating gene expression. Self-interacting domains, commonly called topologically associating domains (TADs) or compartment domains (CDs), have been proposed as the basic structural units of chromatin organization. Surprisingly, although these units have been found in several plant species, they escaped detection in Arabidopsis (Arabidopsis thaliana). Here, we show that the Arabidopsis genome is partitioned into contiguous CDs with different epigenetic features, which are required to maintain appropriate intra-CD and long-range interactions. Consistent with this notion, the histone-modifying Polycomb group machinery is involved in 3D chromatin organization. Yet, while it is clear that Polycomb repressive complex 2 (PRC2)-mediated trimethylation of histone H3 on lysine 27 (H3K27me3) helps establish local and long-range chromatin interactions in plants, the implications of PRC1-mediated histone H2A monoubiquitination on lysine 121 (H2AK121ub) are unclear. We found that PRC1, together with PRC2, maintains intra-CD interactions, but it also hinders the formation of H3K4me3-enriched local chromatin loops when acting independently of PRC2. Moreover, the loss of PRC1 or PRC2 activity differentially affects long-range chromatin interactions, and these 3D changes differentially affect gene expression. Our results suggest that H2AK121ub helps prevent the formation of transposable element/H3K27me1-rich long loops and serves as a docking point for H3K27me3 incorporation.

Abstract 5239: Integrative analysis of 3D chromatin organization at GWAS loci identifies RAPGEF1 as a melanoma susceptibility gene

Abstract Many GWAS loci occur in non-coding regions and often overlap with gene regulatory elements such as distant enhancers, making functional interpretation and target gene discovery challenging. The 3D chromatin organization brings enhancers in spatial proximity with a promoter to regulate target gene expression. Therefore, to map chromatin interactions between GWAS variants and target gene promoters we performed region-focused chromatin conformation capture assay (Capture-C) in primary human melanocytes. We baited the entire region of association for all 68 independent signals from the recent melanoma GWAS, and Capture-C interactions were called using CHiCAGO tool. Integrative analysis of Capture-C interactions with melanocyte- and melanoma-specific ATAC-sequencing, massive-parallel reporter assay (MPRA), ROADMAP chromatin imputed state model, and gene expression datasets helped prioritize target genes for functional follow-up. Capture-C assays identified physical chromatin interactions between fine-mapped risk variants and candidate causal gene (CCG) promoters at 90% of the GWAS loci; For 84% of the 68 loci, we observed at least one variant-to-gene promoter interaction longer than 100 kb, and for 20% of loci we found interactions beyond 1 Mb. For 76% of the 68 loci, the CCG-interacting variant was in annotated melanocyte or melanoma enhancer regions consistent with CCG regulation via an enhancer-promoter interaction. We observed at least one CCG-interacting variant in 63% and 51% of the 68 loci with distinct allele specific transcriptional activity in melanocyte and melanoma MPRA datasets respectively. A majority of the loci (60%) harbored CCG-linked risk variants in accessible chromatin regions in melanocytes and melanoma. Pathway enrichment analyses of Capture-C-nominated CCGs identified embryonic development, aryl hydrocarbon receptor signaling, and DNA repair pathways. Notably, we observed chromatin interactions between risk variants located near the 3’ of Rap Guanine Nucleotide Exchange Factor 1 (RAPGEF1) to the RAPGEF1 and UCK1 promoter regions. The risk allele of the lead variant (rs3780269) at this locus was associated with higher RAPGEF1 mRNA expression and was not associated with UCK1 expression in melanocytes. We performed a CRISPR knockout proliferation screen in immortalized melanocytes and identified RAPGEF1 as an essential gene for melanocyte growth or survival. Further, we validated the results of this screen by overexpressing RAPGEF1 in immortalized melanocytes and found that it leads to increased cellular growth. We are now characterizing its function in melanoma tumor incidence and progression in a zebrafish model. In summary, mapping GWAS loci chromatin interactions to target gene promoters and integrative analysis using cell-type specific datasets identified RAPGEF1 as a melanoma susceptibility gene. Citation Format: Rohit Thakur, Mai Xu, Alexandra Thornock, Hayley Sowards, Epring Long, Thomas Rheling, Karen Funderburk, Jinhu Yin, Rebecca Hennessey, Raj Chari, Tongwu Zhang, Lea Jessop, Timothy Myers, Matthew E. Johnson, Andrew D. Wells, Alessandra Chesi, Struan F. Grant, Mark I. Iles, Maria T. Landi, Matthew Law, Melanoma Meta-Analysis Consortium, Mitchell Machiela, Jiyeon Choi, Leonard I. Zon, Kevin M. Brown. Integrative analysis of 3D chromatin organization at GWAS loci identifies RAPGEF1 as a melanoma susceptibility gene. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5239.

NSD2 E1099K drives relapse in pediatric acute lymphoblastic leukemia by disrupting 3D chromatin organization

BackgroundThe NSD2 p.E1099K (EK) mutation is shown to be enriched in patients with relapsed acute lymphoblastic leukemia (ALL), indicating a role in clonal evolution and drug resistance.ResultsTo uncover 3D chromatin architecture-related mechanisms underlying drug resistance, we perform Hi-C on three B-ALL cell lines heterozygous for NSD2 EK. The NSD2 mutation leads to widespread remodeling of the 3D genome, most dramatically in terms of compartment changes with a strong bias towards A compartment shifts. Systematic integration of the Hi-C data with previously published ATAC-seq, RNA-seq, and ChIP-seq data show an expansion in H3K36me2 and a shrinkage in H3K27me3 within A compartments as well as increased gene expression and chromatin accessibility. These results suggest that NSD2 EK plays a prominent role in chromatin decompaction through enrichment of H3K36me2. In contrast, we identify few changes in intra-topologically associating domain activity. While compartment changes vary across cell lines, a common core of decompacting loci are shared, driving the expression of genes/pathways previously implicated in drug resistance. We further perform RNA sequencing on a cohort of matched diagnosis/relapse ALL patients harboring the relapse-specific NSD2 EK mutation. Changes in patient gene expression upon relapse significantly correlate with core compartment changes, further implicating the role of NSD2 EK in genome decompaction.ConclusionsIn spite of cell-context-dependent changes mediated by EK, there appears to be a shared transcriptional program dependent on compartment shifts which could explain phenotypic differences across EK cell lines. This core program is an attractive target for therapeutic intervention.

Moderation of Structural DNA Properties by Coupled Dinucleotide Contents in Eukaryotes

Dinucleotides are known as determinants for various structural and physiochemical properties of DNA and for binding affinities of proteins to DNA. These properties (e.g., stiffness) and bound proteins (e.g., transcription factors) are known to influence important biological functions, such as transcription regulation and 3D chromatin organization. Accordingly, the question arises of how the considerable variations in dinucleotide contents of eukaryotic chromosomes could still provide consistent DNA properties resulting in similar functions and 3D conformations. In this work, we investigate the hypothesis that coupled dinucleotide contents influence DNA properties in opposite directions to moderate each other's influences. Analyzing all 2478 chromosomes of 155 eukaryotic species, considering bias from coding sequences and enhancers, we found sets of correlated and anti-correlated dinucleotide contents. Using computational models, we estimated changes of DNA properties resulting from this coupling. We found that especially pure A/T dinucleotides (AA, TT, AT, TA), known to influence histone positioning and AC/GT contents, are relevant moderators and that, e.g., the Roll property, which is known to influence histone affinity of DNA, is preferably moderated. We conclude that dinucleotide contents might indirectly influence transcription and chromatin 3D conformation, via regulation of histone occupancy and/or other mechanisms.

Transcription shapes 3D chromatin organization by interacting with loop extrusion

Cohesin folds mammalian interphase chromosomes by extruding the chromatin fiber into numerous loops. "Loop extrusion" can be impeded by chromatin-bound factors, such as CTCF, which generates characteristic and functional chromatin organization patterns. It has been proposed that transcription relocalizes or interferes with cohesin and that active promoters are cohesin loading sites. However, the effects of transcription on cohesin have not been reconciled with observations of active extrusion by cohesin. To determine how transcription modulates extrusion, we studied mouse cells in which we could alter cohesin abundance, dynamics, and localization by genetic "knockouts" of the cohesin regulators CTCF and Wapl. Through Hi-C experiments, we discovered intricate, cohesin-dependent contact patterns near active genes. Chromatin organization around active genes exhibited hallmarks of interactions between transcribing RNA polymerases (RNAPs) and extruding cohesins. These observations could be reproduced by polymer simulations in which RNAPs were moving barriers to extrusion that obstructed, slowed, and pushed cohesins. The simulations predicted that preferential loading of cohesin at promoters is inconsistent with our experimental data. Additional ChIP-seq experiments showed that the putative cohesin loader Nipbl is not predominantly enriched at promoters. Therefore, we propose that cohesin is not preferentially loaded at promoters and that the barrier function of RNAP accounts for cohesin accumulation at active promoters. Altogether, we find that RNAP is an extrusion barrier that is not stationary, but rather, translocates and relocalizes cohesin. Loop extrusion and transcription might interact to dynamically generate and maintain gene interactions with regulatory elements and shape functional genomic organization.

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.