Chromatin Domains Research Articles

HTAD: a human-in-the-loop framework for supervised chromatin domain detection

Topologically associating domains (TADs) are essential units of genome architecture, influencing transcriptional regulation and diseases. Despite numerous methods proposed for TAD identification, it remains challenging due to complex background and nested TAD structures. We introduce HTAD, a human-in-the-loop TAD caller that combines machine learning with human supervision to achieve high accuracy. HTAD begins with feature extraction for potential TAD border pairs, followed by an interactive labeling process through active learning. Performance assessments using public curation and synthetic datasets demonstrate HTAD’s superiority over other state-of-the-art methods and reveal highly hierarchical TAD structures, offering a human-in-the-loop solution for detecting complex genomic patterns.

ChromEMT: visualizing and reconstructing chromatin ultrastructure and 3D organization in situ.

Structure determines function. The discovery of the DNA double-helix structure revealed how genetic information is stored and copied. In the mammalian cell nucleus, up to two meters of DNA is compacted by histones to form nucleosome/DNA particle chains that form euchromatin and heterochromatin domains, chromosome territories and mitotic chromosomes upon cell division. A critical question is what are the structures, interactions and 3D organization of DNA as chromatin in the nucleus and how do they determine DNA replication timing, gene expression and ultimately cell fate. To visualize genomic DNA across these different length scales in the nucleus, we developed ChromEMT, a method that selectively enhances the electron density and contrast of DNA and interacting nucleosome particles, which enables nucleosome chains, chromatin domains, chromatin ultrastructure and 3D organization to be imaged and reconstructed by using multi-tilt electron microscopy tomography (EMT). ChromEMT exploits a membrane-permeable, fluorescent DNA-binding dye, DRAQ5, which upon excitation drives the photo-oxidation and precipitation of diaminobenzidine polymers on the surface of DNA/nucleosome particles that are visible in the electron microscope when stained with osmium. Here, we describe a detailed protocol for ChromEMT, including DRAQ5 staining, photo-oxidation, sample preparation and multi-tilt EMT that can be applied broadly to reconstruct genomic DNA structure and 3D interactions in cells and tissues and different kingdoms of life. The entire procedure takes ~9 days and requires expertise in electron microscopy sample sectioning and acquisition of multi-tilt EMT data sets.

TopoLoop: A new tool for chromatin loop detection in live cells via single-particle tracking.

We present a novel method for identifying topological features of chromatin domains in live cells using single-particle tracking and topological data analysis (TDA). By applying TDA to particle trajectories, we can effectively detect complex spatial patterns, such as loops, that are often missed by traditional time series analysis. Using simulations of polymer bead-spring chains, we have validated the accuracy of our method and determined its limitations for detecting loops. Our approach offers a promising avenue for exploring the topological complexity of chromatin in living cells using TDA techniques.

TAD-dependent sub-TAD is required for enhancer-promoter interaction enabling the β-globin transcription.

Topologically associating domains (TADs) are chromatin domains in the eukaryotic genome. TADs often comprise several sub-TADs. The boundaries of TADs and sub-TADs are enriched in CTCF, an architectural protein. Deletion of CTCF-binding motifs at one boundary disrupts the domains, often resulting in a transcriptional decrease in genes inside the domains. However, it is not clear how TAD and sub-TAD affect each other in the domain formation. Unaffected gene transcription was observed in the β-globin locus when one boundary of TAD or sub-TAD was destroyed. Here, we disrupted β-globin TAD and sub-TAD by deleting CTCF motifs at both boundaries in MEL/ch11 cells. Disruption of TAD impaired sub-TAD, but sub-TAD disruption did not affect TAD. Both TAD and sub-TAD disruption compromised the β-globin transcription, accompanied by the loss of enhancer-promoter interactions. However, histone H3 occupancy and H3K27ac were largely maintained across the β-globin locus. Genome-wide analysis showed that putative enhancer-promoter interactions and gene transcription were decreased by the disruption of CTCF-mediated topological domains in neural progenitor cells. Collectively, our results indicate that there is unequal relationship between TAD and sub-TAD formation. TAD is likely not sufficient for gene transcription, and, therefore, sub-TAD appears to be required. TAD-dependently formed sub-TADs are considered to provide chromatin environments for enhancer-promoter interactions enabling gene transcription.

PRC1 and CTCF-Mediated Transition from Poised to Active Chromatin Loops Drives Bivalent Gene Activation.

Polycomb Repressive Complex 1 (PRC1) and CCCTC-binding factor (CTCF) are critical regulators of 3D chromatin architecture that influence cellular transcriptional programs. Spatial chromatin structures comprise conserved compartments, topologically associating domains (TADs), and dynamic, cell-type-specific chromatin loops. Although the role of CTCF in chromatin organization is well-known, the involvement of PRC1 is less understood. In this study, we identified an unexpected, essential role for the canonical Pcgf2-containing PRC1 complex (cPRC1.2), a known transcriptional repressor, in activating bivalent genes during differentiation. Our Hi-C analysis revealed that cPRC1.2 forms chromatin loops at bivalent promoters, rendering them silent yet poised for activation. Using mouse embryonic stem cells (ESCs) with CRISPR/Cas9-mediated gene editing, we found that the loss of Pcgf2, though not affecting the global level of H2AK119ub1, disrupts these cPRC1.2 loops in ESCs and impairs the transcriptional induction of crucial target genes necessary for neuronal differentiation. Furthermore, we identified CTCF enrichment at cPRC1.2 loop anchors and at Polycomb group (PcG) bodies, nuclear foci with concentrated PRC1 and its tethered chromatin domains, suggesting that PRC1 and CTCF cooperatively shape chromatin loop structures. Through virtual 4C and other genomic analyses, we discovered that establishing neuronal progenitor cell (NPC) identity involves a switch from cPRC1.2-mediated chromatin loops to CTCF-mediated active loops, enabling the expression of critical lineage-specific factors. This study uncovers a novel mechanism by which pre-formed PRC1 and CTCF loops at lineage-specific genes maintain a poised state for subsequent gene activation, advancing our understanding of the role of chromatin architecture in controlling cell fate transitions.

Read-write mechanisms of H2A ubiquitination by Polycomb repressive complex 1.

Epigenetic inheritance of silent chromatin domains is fundamental to cellular memory during embryogenesis, but it must overcome the dilution of repressive histone modifications during DNA replication1. One such modification, histone H2A lysine 119 monoubiquitination (H2AK119Ub), needs to be re-established by the Polycomb repressive complex 1 (PRC1) E3 ligase to restore the silent Polycomb domain2,3. However, the exact mechanism behind this restoration remains unknown. Here, combining cryo-electron microscopy (cryo-EM) and functional approaches, we characterize the read-write mechanism of the non-canonical PRC1-containing RYBP (ncPRC1RYBP). This mechanism, which functions as a positive-feedback loop in epigenetic regulation4,5, emphasizes the pivotal role of ncPRC1RYBP in restoring H2AK119Ub. We observe an asymmetrical binding of ncPRC1RYBP to H2AK119Ub nucleosomes, guided in part by the N-terminal zinc-finger domain of RYBP binding to residual H2AK119Ub on nascent chromatin. This recognition positions the RING domains of RING1B and BMI1 on the unmodified nucleosome side, enabling recruitment of the E2 enzyme to ubiquitinate H2AK119 within the same nucleosome (intra-nucleosome read-write) or across nucleosomes (inter-nucleosome read-write). Collectively, our findings provide key structural and mechanistic insights into the dynamic interplay of epigenetic regulation, highlighting the significance of ncPRC1RYBP in H2AK119Ub restoration to sustain repressive chromatin domains.

Correlative light and soft X-ray tomography of in situ mesoscale heterochromatin structure in intact cells

Heterochromatin organization is critical to many genome-related programs including transcriptional silencing and DNA repair. While super-resolution imaging, electron microscopy, and multiomics methods have provided indirect insights into the heterochromatin organization, a direct measurement of mesoscale heterochromatin ultrastructure is still missing. We use a combination of correlative light microscopy and cryo-soft X-ray tomography (CLXT) to analyze heterochromatin organization in the intact hydrated state of human mammary fibroblast cells. Our analysis reveals that the heterochromatin ultra-structure has a typical mean domain size of approximately 80 nm and a mean separation of approximately 120 nm between domains. Functional perturbations yield further insights into the molecular density and alterations in the mesoscale organization of the heterochromatin regions. Furthermore, our polymer simulations provide a mechanistic basis for the experimentally observed size and separation distributions of the mesoscale chromatin domains. Collectively, our results provide direct, label-free observation of heterochromatin organization in the intact hydrated state of cells.

PU.1 eviction at lymphocyte-specific chromatin domains mediates glucocorticoid response in acute lymphoblastic leukemia

The epigenetic landscape plays a critical role in cancer progression, yet its therapeutic potential remains underexplored. Glucocorticoids are essential components of treatments for lymphoid cancers, but resistance, driven in part by epigenetic changes at glucocorticoid-response elements, poses a major challenge to effective therapies. Here we show that glucocorticoid treatment induces distinct patterns of chromosomal organization in glucocorticoid-sensitive and resistant acute lymphoblastic leukemia xenograft models. These glucocorticoid-response elements are primed by the pioneer transcription factor PU.1, which interacts with the glucocorticoid receptor. Eviction of PU.1 promotes receptor binding, increasing the expression of genes involved in apoptosis and facilitating a stronger therapeutic response. Treatment with a PU.1 inhibitor enhances glucocorticoid sensitivity, demonstrating the clinical potential of targeting this pathway. This study uncovers a mechanism involving PU.1 and the glucocorticoid receptor, linking transcription factor activity with drug response, and suggesting potential therapeutic strategies for overcoming resistance.

An integrated view of the structure and function of the human 4D nucleome.

The dynamic three-dimensional (3D) organization of the human genome (the "4D Nucleome") is closely linked to genome function. Here, we integrate a wide variety of genomic data generated by the 4D Nucleome Project to provide a detailed view of human 3D genome organization in widely used embryonic stem cells (H1-hESCs) and immortalized fibroblasts (HFFc6). We provide extensive benchmarking of 3D genome mapping assays and integrate these diverse datasets to annotate spatial genomic features across scales. The data reveal a rich complexity of chromatin domains and their sub-nuclear positions, and over one hundred thousand structural loops and promoter-enhancer interactions. We developed 3D models of population-based and individual cell-to-cell variation in genome structure, establishing connections between chromosome folding, nuclear organization, chromatin looping, gene transcription, and DNA replication. We demonstrate the use of computational methods to predict genome folding from DNA sequence, uncovering potential effects of genetic variants on genome structure and function. Together, this comprehensive analysis contributes insights into human genome organization and enhances our understanding of connections between the regulation of genome function and 3D genome organization in general.

Chromatin condensed domains revealed by AFM, and their transformation in mechanically deformed normal and malignant cell nuclei

It has been generally accepted that heterochromatin is represented by a regular, dense and closed structure, while euchromatin is open and sparse. Recent evidence indicates that chromatin is comprised of irregular nucleosome clutches compacted within the nucleus. Transcriptional events transform the chromatin architecture, resulting in appearance of 100–300 nm nucleosomal aggregates. Meanwhile, the current paradigm of chromatin architecture is largely fragmented. In this communication, we unraveled chromatin ultrastructure of normal and malignant cell nuclei through mechanical deformation of the nuclei and Atomic Force Microscopy (AFM) analysis of the resulting landscape. In human skin fibroblasts cell nuclei, nanodomains of about 16.5–33.5 nm were revealed. Hierarchical folding of the chromatin of normal nuclei was observed: the nanodomains formed irregular fiber-like structures that coalesced into the macroscale chromatin compartments. In fibrosarcoma cell nuclei DNA supercoiling domains (SDs) of about 66.3–113.0 nm, uniformly distributed within the nuclei, were revealed. Transformation of the morphology of the condensed chromatin domains through up- and downregulation of supercoiling was demonstrated.

Double-strand breaks in facultative heterochromatin require specific movements and chromatin changes for efficient repair

DNA double-strand breaks (DSBs) must be properly repaired within diverse chromatin domains to maintain genome stability. Whereas euchromatin has an open structure and is associated with transcription, facultative heterochromatin is essential to silence developmental genes and forms compact nuclear condensates, called polycomb bodies. Whether the specific chromatin properties of facultative heterochromatin require distinct DSB repair mechanisms remains unknown. Here, we integrate single DSB systems in euchromatin and facultative heterochromatin in Drosophila melanogaster and find that heterochromatic DSBs rapidly move outside polycomb bodies. These DSB movements coincide with a break-proximal reduction in the canonical heterochromatin mark histone H3 Lysine 27 trimethylation (H3K27me3). We demonstrate that DSB movement and loss of H3K27me3 at heterochromatic DSBs depend on the histone demethylase dUtx. Moreover, loss of dUtx specifically disrupts completion of homologous recombination at heterochromatic DSBs. We conclude that DSBs in facultative heterochromatin require dUtx-mediated loss of H3K27me3 to promote DSB movement and repair.

SATB1 prevents immune cell infiltration by regulating chromatin organization and gene expression of a chemokine gene cluster in T cells

SATB1, a key regulator of T cell development, governs lineage-specific transcriptional programs upon T cell activation. The absence of SATB1 has been linked to the initiation and progression of autoimmunity. However, its precise roles in this process remain incompletely understood. Here we show that conditional knockout of Satb1 in CD4+ T cells in mice led to T cell hyperactivation and inflammatory cell infiltration across multiple organs. Transcriptional profiling on activated T cells revealed that the loss of SATB1 led to aberrant upregulation of CC chemokines. Treating Satb1 conditional knockout mice with CC chemokine receptor inhibitor alleviated inflammatory cell infiltration. Intriguingly, SATB1’s transcriptional regulation of chemokine genes could not be attributed to its direct binding to chemokine promoters. Instead, SATB1 exerted its regulatory effects by controlling higher-order chromatin organization at a CC chemokine locus. The loss of SATB1 led to the emergence of a new chromatin domain encompassing the Ccl3, Ccl4, Ccl5, Ccl6, and Ccl9 genes and a distal enhancer, resulting in increased contacts between the enhancer and all five chemokine genes, thus inducing their upregulation. Collectively, these results demonstrate that SATB1 protects organs from immune cell infiltration by regulating chemokine expression, providing valuable insights into the development of autoimmunity-related phenotypes.

Structure and evolution of metapolycentromeres.

Metapolycentromeres consist of multiple sequential domains of centromeric chromatin associated with a centromere-specific variant of histone H3 (CENP-A), functioning collectively as a single centromere. To date, they have been revealed in nine flowering plant, five insect and six vertebrate species. In this paper, we focus on their structure and possible mechanisms of emergence and evolution. The metapolycentromeres may vary in the number of centromeric domains and in their genetic content and epigenetic modifications. However, these variations do not seem to affect their function. The emergence of metapolycentromeres has been attributed to multiple Robertsonian translocations and segmental duplications. Conditions of genomic instability, such as interspecific hybridization and malignant neoplasms, are suggested as triggers for the de novo emergence of metapolycentromeres. Addressing the "centromere paradox" - the rapid evolution of centromeric DNA and proteins despite their conserved cellular function - we explore the centromere drive hypothesis as a plausible explanation for the dynamic evolution of centromeres in general, and in particular the emergence of metapolycentromeres and holocentromeres. Apparently, metapolycentromeres are more common across different species than it was believed until recently. Indeed, a systematic review of the available cytogenetic publications allowed us to identify 27 candidate species with metapolycentromeres. Тhe list of the already established and newly revealed candidate species thus spans 27 species of flowering plants and eight species of gymnosperm plants, five species of insects, and seven species of vertebrates. This indicates an erratic phylogenetic distribution of the species with metapolycentromeres and may suggest an independent emergence of the metapolycentromeres in the course of evolution. However, the current catalog of species with identified and likely metapolycentromeres remains too short to draw reliable conclusions about their evolution, particularly in the absence of knowledge about related species without metapolycentromeres for comparative analysis. More studies are necessary to shed light on the mechanisms of metapolycentromere formation and evolution.

Integration of scHi-C and scRNA-seq data defines distinct 3D-regulated and biological-context dependent cell subpopulations

An integration of 3D chromatin structure and gene expression at single-cell resolution has yet been demonstrated. Here, we develop a computational method, a multiomic data integration (MUDI) algorithm, which integrates scHi-C and scRNA-seq data to precisely define the 3D-regulated and biological-context dependent cell subpopulations or topologically integrated subpopulations (TISPs). We demonstrate its algorithmic utility on the publicly available and newly generated scHi-C and scRNA-seq data. We then test and apply MUDI in a breast cancer cell model system to demonstrate its biological-context dependent utility. We find the newly defined topologically conserved associating domain (CAD) is the characteristic single-cell 3D chromatin structure and better characterizes chromatin domains in single-cell resolution. We further identify 20 TISPs uniquely characterizing 3D-regulated breast cancer cellular states. We reveal two of TISPs are remarkably resemble to high cycling breast cancer persister cells and chromatin modifying enzymes might be functional regulators to drive the alteration of the 3D chromatin structures. Our comprehensive integration of scHi-C and scRNA-seq data in cancer cells at single-cell resolution provides mechanistic insights into 3D-regulated heterogeneity of developing drug-tolerant cancer cells.

Cohesin mutations in acute myeloid leukemia.

The cohesin complex, encoded by SMC3, SMC1A, RAD21, and STAG2, is a critical regulator of DNA-looping and gene expression. Over a decade has passed since recurrent mutations affecting cohesin subunits were first identified in myeloid malignancies such as Acute Myeloid Leukemia (AML). Since that time there has been tremendous progress in our understanding of chromatin structure and cohesin biology, but critical questions remain because of the multiple critical functions the cohesin complex is responsible for. Recent findings have been particularly noteworthy with the identification of crosstalk between DNA-looping and chromatin domains, a deeper understanding of how cohesin establishes sister chromatid cohesion, a renewed interest in cohesin's role for DNA damage response, and work demonstrating cohesin's importance for Polycomb repression. Despite these exciting findings, the role of cohesin in normal hematopoiesis, and the precise mechanisms by which cohesin mutations promote cancer, remain poorly understood. This review discusses what is known about the role of cohesin in normal hematopoiesis, and how recent findings could shed light on the mechanisms through which cohesin mutations promote leukemic transformation. Important unanswered questions in the field, such as whether cohesin plays a role in HSC heterogeneity, and the mechanisms by which it regulates gene expression at a molecular level, will also be discussed. Particular attention will be given to the potential therapeutic vulnerabilities of leukemic cells with cohesin subunit mutations.

PICKLE-mediated nucleosome condensing drives H3K27me3 spreading for the inheritance of Polycomb memory during differentiation

Spreading of H3K27me3 is crucial for the maintenance of mitotically inheritable Polycomb-mediated chromatin silencing in animals and plants. However, how Polycomb repressive complex 2 (PRC2) accesses unmodified nucleosomes in spreading regions for spreading H3K27me3 remains unclear. Here, we show in Arabidopsis thaliana that the chromatin remodeler PICKLE (PKL) plays a specialized role in H3K27me3 spreading to safeguard cell identity during differentiation. PKL specifically localizes to H3K27me3 spreading regions but not to nucleation sites and physically associates with PRC2. Loss of PKL disrupts the occupancy of the PRC2 catalytic subunit CLF in spreading regions and leads to aberrant dedifferentiation. Nucleosome density increase endowed by the ATPase function of PKL ensures that unmodified nucleosomes are accessible to PRC2 catalytic activity for H3K27me3 spreading. Our findings demonstrate that PKL-dependent nucleosome compaction is critical for PRC2-mediated H3K27me3 read-and-write function in H3K27me3 spreading, thus revealing a mechanism by which repressive chromatin domains are established and propagated.

PML is a constitutive component of chromatin domains enriched in repetitive elements and duplicated gene clusters in cancer cells

Heterochromatin is a pivotal element in the functional organization of genomes. In our study, we delve into the heterochromatin pattern of association by the PML (promyelocytic leukemia) protein. By using PML chromatin immunoprecipitation and sequencing data and comparing computational methodologies to depict PML chromatin association, we describe PML-associated domains or PADs as large heterochromatic regions that exhibit similar genomic features across cancer cell lines. We show that PADs are specifically enriched in non-coding genes, duplicated gene clusters, and repetitive DNA elements. Moreover, we find enriched binding motifs of KZFPs, which are involved in orchestrating epigenetic repression at repetitive DNA elements. Hence, our findings suggest that PML conservatively associates to heterochromatic domains enriched in repetitive DNA elements and duplicated gene clusters in cancer. These findings contribute to a broader understanding of the complex regulatory framework of genome organization by heterochromatin in cancer.

Drosophila Protein Z4 Possesses ZAD Dimerization Domain.

The transcription factor Z4 (putzig) is one of the key proteins that determine the chromatin structure in Drosophila. Z4 is found at the boundaries of bands on polytene chromosomes, and the bands are currently thought to correlate with chromatin domains. Z4 is a component of a protein complex that additionally includes Chromator and BEAF-32, and a conserved domain is necessary to occur at the N end of Z4 to ensure its interaction with the two proteins. In this study, a zinc finger-associated domain (ZAD) domain was identified in Z4. The capability of dimerization was confirmed for the domain by biochemical methods. A dimer model of the domain was obtained using AlphaFold2, and the model structure was confirmed using small-angle X-ray scattering (SAXS). The dimer structure shows a fold typical of ZAD domains.

Members of an array of zinc finger proteins specify distinct Hox chromatin boundaries.

Partitioning of repressive from actively transcribed chromatin in mammalian cells fosters cell-type specific gene expression patterns. While this partitioning is reconstructed during differentiation, the chromatin occupancy of the key insulator, CTCF, is unchanged at the developmentally important Hox clusters. Thus, dynamic changes in chromatin boundaries must entail other activities. Given its requirement for chromatin loop formation, we examined cohesin-based chromatin occupancy without known insulators, CTCF and MAZ, and identified a family of zinc finger proteins (ZNFs), some of which exhibit tissue-specific expression. Two such ZNFs foster chromatin boundaries at the Hox clusters that are distinct from each other and from MAZ. PATZ1 was critical to the thoracolumbar boundary in differentiating motor neurons and mouse skeleton, while ZNF263 contributed to cervicothoracic boundaries. We propose that these insulating activities act with cohesin, alone or combinatorially, with or without CTCF, to implement precise positional identity and cell fate during development.

Structural perturbation of chromatin domains with multiple developmental regulators can severely impact gene regulation and development.

Chromatin domain boundaries delimited by CTCF motifs can restrict the range of enhancer action. However, disruption of domain structure often results in mild gene dysregulation and thus predicting the impact of boundary rearrangements on animal development remains challenging. Here, we tested whether structural perturbation of a chromatin domain with multiple developmental regulators can result in more acute gene dysregulation and severe developmental phenotypes. We targeted clusters of CTCF motifs in a domain of the mouse genome containing three FGF ligand genes - Fgf3, Fgf4, and Fgf15 - that regulate several developmental processes. Deletion of the 23.9kb cluster that defines the centromeric boundary of this domain resulted in ectopic interactions of the FGF genes with enhancers located across the deleted boundary that are active in the developing brain. This caused strong induction of FGF expression and perinatal lethality with encephalocele and orofacial cleft phenotypes. Heterozygous boundary deletion was sufficient to cause these fully penetrant phenotypes, and strikingly, loss of a single CTCF motif within the cluster also recapitulated ectopic FGF expression and caused encephalocele. However, such phenotypic sensitivity to perturbation of domain structure did not extend to all CTCF clusters of this domain, nor to all developmental processes controlled by these three FGF genes - for example, the ability to undergo lineage specification in the blastocyst and pre-implantation development were not affected. By tracing the impact of different chromosomal rearrangements throughout mouse development, we start to uncover the determinants of phenotypic robustness and sensitivity to perturbation of chromatin boundaries. Our data show how small sequence variants at certain domain boundaries can have a surprisingly outsized effect and must be considered as potential sources of gene dysregulation during development and disease.