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The nucleus of mammalian cells displays a distinct spatial segregation of active euchromatic and inactive heterochromatic regions of the genome 1 , 2 . In conventional nuclei, microscopy shows that euchromatin is localized in the nuclear interior and heterochromatin at the nuclear periphery 1 , 2 . Genome-wide chromosome conformation capture (Hi-C) analyses show this segregation as a plaid pattern of contact enrichment within euchromatin and heterochromatin compartments 3 , and depletion between them. Many mechanisms for the formation of compartments have been proposed, such as attraction of heterochromatin to the nuclear lamina 2 , 4 , preferential attraction of similar chromatin to each other 1 , 4 – 12 , higher levels of chromatin mobility in active chromatin 13 – 15 and transcription-related clustering of euchromatin 16 , 17 . However, these hypotheses have remained inconclusive, owing to the difficulty of disentangling intra-chromatin and chromatin–lamina interactions in conventional nuclei 18 . The marked reorganization of interphase chromosomes in the inverted nuclei of rods in nocturnal mammals 19 , 20 provides an opportunity to elucidate the mechanisms that underlie spatial compartmentalization. Here we combine Hi-C analysis of inverted rod nuclei with microscopy and polymer simulations. We find that attractions between heterochromatic regions are crucial for establishing both compartmentalization and the concentric shells of pericentromeric heterochromatin, facultative heterochromatin and euchromatin in the inverted nucleus. When interactions between heterochromatin and the lamina are added, the same model recreates the conventional nuclear organization. In addition, our models allow us to rule out mechanisms of compartmentalization that involve strong euchromatin interactions. Together, our experiments and modelling suggest that attractions between heterochromatic regions are essential for the phase separation of the active and inactive genome in inverted and conventional nuclei, whereas interactions of the chromatin with the lamina are necessary to build the conventional architecture from these segregated phases. Attractions between heterochromatic regions are essential for phase separation of the active and inactive genome in inverted and conventional nuclei, whereas chromatin–lamina interactions are necessary to build the conventional genomic architecture from these segregated phases.

Lamins (A/C and B) are major constituents of the nuclear lamina (NL). Structurally conserved lamina-associated domains (LADs) are formed by genomic regions that contact the NL. Lamins are also found in the nucleoplasm, with a yet unknown function. Here we map the genome-wide localization of lamin B1 in an euchromatin-enriched fraction of the mouse genome and follow its dynamics during the epithelial-to-mesenchymal transition (EMT). Lamin B1 associates with actively expressed and open euchromatin regions, forming dynamic euchromatin lamin B1-associated domains (eLADs) of about 0.3 Mb. Hi-C data link eLADs to the 3D organization of the mouse genome during EMT and correlate lamin B1 enrichment at topologically associating domain (TAD) borders with increased border strength. Having reduced levels of lamin B1 alters the EMT transcriptional signature and compromises the acquisition of mesenchymal traits. Thus, during EMT, the process of genome reorganization in mouse involves dynamic changes in eLADs. Lamina-associated domains (LADs) contact lamins in the nuclear lamina, and lamin B1 was thought to bind heterochromatic regions at the nuclear envelope. Here, the authors show lamin B1 associates with actively expressed euchromatin regions, creating dynamic euchromatin lamina-associated domains (eLADs) during epithelial-to-mesenchymal transition.

In eukaryotes, DNA is packed inside the cell nucleus in the form of chromatin, which consists of DNA, proteins such as histones, and RNA. Euchromatin, which is permissive for transcription, is spatially organized into transcriptionally inactive domains interspersed with pockets of transcriptional activity. While transcription and RNA have been implicated in euchromatin organization, it remains unclear how their interplay forms and maintains transcription pockets. Here we combine theory and experiment to analyze the dynamics of euchromatin organization as pluripotent zebrafish cells exit mitosis and begin transcription. We show that accumulation of RNA induces formation of transcription pockets which displace transcriptionally inactive chromatin. We propose that the accumulating RNA recruits RNA-binding proteins that together tend to separate from transcriptionally inactive euchromatin. Full phase separation is prevented because RNA remains tethered to transcribed euchromatin through RNA polymerases. Instead, smaller scale microphases emerge that do not grow further and form the typical pattern of euchromatin organization. How euchromatin organisation and transcription are related is unclear. Here, the authors observe the dynamics of euchromatin organization showing that accumulating RNA recruits RNA-binding proteins that together with transcribed euchromatin separate from non-transcribed euchromatin, forming microphases.

DNA double-strand breaks are repaired by multiple pathways, including non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ). The balance of these pathways is dependent on the local chromatin context, but the underlying mechanisms are poorly understood. By combining knockout screening with a dual MMEJ:NHEJ reporter inserted in 19 different chromatin environments, we identified dozens of DNA repair proteins that modulate pathway balance dependent on the local chromatin state. Proteins that favor NHEJ mostly synergize with euchromatin, while proteins that favor MMEJ generally synergize with distinct types of heterochromatin. Examples of the former are BRCA2 and POLL, and of the latter the FANC complex and ATM. Moreover, in a diversity of human cancer types, loss of several of these proteins alters the distribution of pathway-specific mutations between heterochromatin and euchromatin. Together, these results uncover a complex network of proteins that regulate MMEJ:NHEJ balance in a chromatin context-dependent manner. DNA double-strand breaks are repaired by multiple pathways. The balance of these pathways depends on the local chromatin context, but the underlying mechanisms are poorly understood. Here the authors uncover a network of proteins that regulate pathway balance in a chromatin context-dependent manner.

Recent efforts have succeeded in surveying open chromatin at the single-cell level, but high-throughput, single-cell assessment of heterochromatin and its underlying genomic determinants remains challenging. We engineered a hybrid transposase including the chromodomain (CD) of the heterochromatin protein-1α (HP-1α), which is involved in heterochromatin assembly and maintenance through its binding to trimethylation of the lysine 9 on histone 3 (H3K9me3), and developed a single-cell method, single-cell genome and epigenome by transposases sequencing (scGET-seq), that, unlike single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq), comprehensively probes both open and closed chromatin and concomitantly records the underlying genomic sequences. We tested scGET-seq in cancer-derived organoids and human-derived xenograft (PDX) models and identified genetic events and plasticity-driven mechanisms contributing to cancer drug resistance. Next, building upon the differential enrichment of closed and open chromatin, we devised a method, Chromatin Velocity, that identifies the trajectories of epigenetic modifications at the single-cell level. Chromatin Velocity uncovered paths of epigenetic reorganization during stem cell reprogramming and identified key transcription factors driving these developmental processes. scGET-seq reveals the dynamics of genomic and epigenetic landscapes underlying any cellular processes. Single-cell mapping of heterochromatin and euchromatin using chromatin velocity defines trajectories of epigenetic modifications.

Membraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, our current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. We investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with the euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, we reveal a hierarchical organization of the PCH domains into distinct \"territories.\" Strikingly, H3K9me2-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. Our findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin.

Chromatin consists of a complex of DNA and histone proteins as its core components and plays an important role in both packaging DNA and regulating DNA metabolic pathways such as DNA replication, transcription, recombination, and chromosome segregation. Proper functioning of chromatin further involves a network of interactions among molecular complexes that modify chromatin structure and organization to affect the accessibility of DNA to transcription factors leading to the activation or repression of the transcription of target DNA loci. Based on its structure and compaction state, chromatin is categorized into euchromatin, heterochromatin, and centromeric chromatin. In this review, we discuss distinct chromatin factors and molecular complexes that constitute euchromatin—open chromatin structure associated with active transcription; heterochromatin—less accessible chromatin associated with silencing; centromeric chromatin—the site of spindle binding in chromosome segregation.

Polycomb Repressive Complexes (PRCs) are known for chemically modifying histones to compact chromatin structure and repress transcription. Broadly speaking, PRC1 monoubiquitinates histone 2A at lysine 119 (H2AK119ub), and PRC2 methylates histone H3 lysine 27 (H3K27me3, H3K27me2 and H3K27me1), but the scope and functions of these activities are complicated by a multiplicity of factors involving distinct cellular contexts and compositions of both complexes. Because epigenetic dysregulation is associated with neurodevelopmental disorders, but little is known about normal PRC activities in neurons, we used CUT&RUN to map PRC-dependent histone modifications in the mouse cerebellum at two postnatal timepoints (day 12 and 3 months). We find that H2AK119ub appears within both heterochromatin and euchromatin as the cerebellum matures, becoming enriched within active enhancers and promoters while being depleted from heterochromatin. Unexpectedly, the PRC1 product H2AK119ub appeared frequently without the accompaniment of the PRC2 product H3K27me3; leading to a much more dynamic chromatin state than when these two marks colocalized. Deposition of H2AK119ub at loci with the chromatin signature of active cis-regulatory elements tended to also gain the euchromatin-associated modifications H3K4me3 and H3K27ac during neurodevelopment. Importantly, deposition of H2AK119ub within both bivalent and H3K4me3-only promoters reduced transcription of downstream genes. The pattern of H2AK119ub deposition was specific to the cerebellum compared to liver and kidney. We then show that the PRC2 product H3K27me1 formed euchromatic zones that alternated with heterochromatic zones dominated by H3K27me3. Between the early and late timepoints H3K27me1 became enriched within a subset of expressed gene bodies and depleted from most other genes while remaining uncorrelated with the abundance of the corresponding mRNAs. Our data lead us to propose that deposition of H2AK119ub and H3K27me1 during cerebellar development likely fine-tunes the activity of cis-regulatory elements and transcription, respectively, and that PRC1 and PRC2 activities become uncoupled in the mature brain.

The eukaryotic genome is packaged into chromatin, which is composed of a nucleosomal filament that coils up to form more compact structures. Chromatin exists in two main forms: euchromatin, which is relatively decondensed and enriched in transcriptionally active genes, and heterochromatin, which is condensed and transcriptionally repressed. It is widely accepted that chromatin architecture modulates DNA accessibility, restricting the access of sequence-specific, gene-regulatory, transcription factors to the genome. However, the evidence for this model derives primarily from experiments with isolated nuclei, in which chromatin remodeling has ceased, resulting in a static chromatin structure. Here, using a DNA methyltransferase to measure accessibility in vivo, we show that both euchromatin and heterochromatin are fully accessible in living human cells, whereas centromeric α-satellite chromatin is partly inaccessible. We conclude that all nucleosomes in euchromatin and heterochromatin are highly dynamic in living cells, except for nucleosomes in centromeric chromatin. Chromatin structure is thought to regulate transcription factor access by modulating DNA accessibility. Here, the authors show that in living human cells, euchromatin and heterochromatin are fully accessible, while centromeric chromatin remains partially inaccessible.

The execution of developmental programs of gene expression requires an accurate partitioning of the genome into subnuclear compartments, with active euchromatin enriched centrally and silent heterochromatin at the nuclear periphery 1 . The existence of degenerative diseases linked to lamin A mutations suggests that perinuclear binding of chromatin contributes to cell-type integrity 2 , 3 . The methylation of lysine 9 of histone H3 (H3K9me) characterizes heterochromatin and mediates both transcriptional repression and chromatin anchoring at the inner nuclear membrane 4 . In Caenorhabditis elegans embryos, chromodomain protein CEC-4 bound to the inner nuclear membrane tethers heterochromatin through H3K9me 3 , 5 , whereas in differentiated tissues, a second heterochromatin-sequestering pathway is induced. Here we use an RNA interference screen in the cec-4 background and identify MRG-1 as a broadly expressed factor that is necessary for this second chromatin anchor in intestinal cells. However, MRG-1 is exclusively bound to euchromatin, suggesting that it acts indirectly. Heterochromatin detachment in double mrg-1; cec-4 mutants is rescued by depleting the histone acetyltransferase CBP-1/p300 or the transcription factor ATF-8, a member of the bZIP family (which is known to recruit CBP/p300). Overexpression of CBP-1 in cec-4 mutants is sufficient to delocalize heterochromatin in an ATF-8-dependent manner. CBP-1 and H3K27ac levels increase in heterochromatin upon mrg-1 knockdown, coincident with delocalization. This suggests that the spatial organization of chromatin in C. elegans is regulated both by the direct perinuclear attachment of silent chromatin, and by an active retention of CBP-1/p300 in euchromatin. The two pathways contribute differentially in embryos and larval tissues, with CBP-1 sequestration by MRG-1 having a major role in differentiated cells. MRG-1 indirectly promotes anchoring of chromatin in differentiated intestinal cells in Caenorhabditis elegans by sequestering the histone acetyltransferase CBP-1/p300.