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Microscopy and genomic approaches provide detailed descriptions of the three-dimensional folding of chromosomes and nuclear organization. The fundamental question is how activity of molecules at the nanometer scale can lead to complex and orchestrated spatial organization at the scale of chromosomes and the whole nucleus. At least three key mechanisms can bridge across scales: (1) tethering of specific loci to nuclear landmarks leads to massive reorganization of the nucleus; (2) spatial compartmentalization of chromatin, which is driven by molecular affinities, results in spatial isolation of active and inactive chromatin; and (3) loop extrusion activity of SMC (structural maintenance of chromosome) complexes can explain many features of interphase chromatin folding and underlies key phenomena during mitosis. Interestingly, many features of chromosome organization ultimately result from collective action and the interplay between these mechanisms, and are further modulated by transcription and topological constraints. Finally, we highlight some outstanding questions that are critical for our understanding of nuclear organization and function. We believe many of these questions can be answered in the coming years.

SUMMARYCohesin-mediated loop extrusion folds interphase chromosomes at the ten to hundreds kilobases scale. This process produces structural features such as loops and topologically associating domains. We identify three types of cis-elements that define the chromatin folding landscape generated by loop extrusion. First, CTCF sites form boundaries by stalling extruding cohesin, as shown before. Second, transcription termination sites form boundaries by acting as cohesin unloading sites. RNA polymerase II contributes to boundary formation at transcription termination sites. Third, transcription start sites form boundaries that are mostly independent of cohesin, but are sites where cohesin can pause. Together with cohesin loading at enhancers, and possibly other cis-elements, these loci create a dynamic pattern of cohesin traffic along the genome that guides enhancer-promoter interactions. Disturbing this traffic pattern, by removing CTCF barriers, renders cells sensitive to knock-out of genes involved in transcription initiation, such as the SAGA and TFIID complexes, and RNA processing such DEAD-Box RNA helicases. In the absence of CTCF, several of these factors fail to be efficiently recruited to active promoters. We propose that the complex pattern of cohesin movement along chromatin contributes to appropriate promoter-enhancer interactions and localization of transcription and RNA processing factors to active genes.HIGHLIGHTSAt least three types of chromatin boundaries regulate a cohesin traffic pattern.The cohesin traffic pattern guides enhancer-promoter interactions.Removing CTCF renders cells sensitive to deletion of RNA processing and gene regulation genes.Depleting CTCF affects localization of RNA processing and gene regulatory proteins.

Skates are cartilaginous fish whose body plan features enlarged wing-like pectoral fins, enabling them to thrive in benthic environments1,2. However, the molecular underpinnings of this unique trait remain unclear. Here we investigate the origin of this phenotypic innovation by developing the little skate Leucoraja erinacea as a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins—including gene expression, chromatin occupancy and three-dimensional conformation—we find skate-specific genomic rearrangements that alter the three-dimensional regulatory landscape of genes that are involved in the planar cell polarity pathway. Functional inhibition of planar cell polarity signalling resulted in a reduction in anterior fin size, confirming that this pathway is a major contributor to batoid fin morphology. We also identified a fin-specific enhancer that interacts with several hoxa genes, consistent with the redeployment of hox gene expression in anterior pectoral fins, and confirmed its potential to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganization and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait.

Despite the well-established role of nuclear organization in the regulation of gene expression, little is known about the reverse: how transcription shapes the spatial organization of the genome. Owing to the small sizes of most previously studied genes and the limited resolution of microscopy, the structure and spatial arrangement of a single transcribed gene are still poorly understood. Here we study several long highly expressed genes and demonstrate that they form open-ended transcription loops with polymerases moving along the loops and carrying nascent RNAs. Transcription loops can span across micrometres, resembling lampbrush loops and polytene puffs. The extension and shape of transcription loops suggest their intrinsic stiffness, which we attribute to decoration with multiple voluminous nascent ribonucleoproteins. Our data contradict the model of transcription factories and suggest that although microscopically resolvable transcription loops are specific for long highly expressed genes, the mechanisms underlying their formation could represent a general aspect of eukaryotic transcription.

Mammalian sperm exhibit an unusual and heavily-compacted genomic packaging state. In addition to its role in organizing the compact and hydrodynamic sperm head, it has been proposed that sperm chromatin architecture helps to program gene expression in the early embryo. Scores of genome-wide surveys in sperm have reported patterns of chromatin accessibility, histone localization, histone modification, and chromosome folding. Here, we revisit these studies in light of our recent finding that sperm obtained from the mouse epididymis are contaminated with low levels of cell-free chromatin. In the absence of proper sperm lysis we readily recapitulate multiple prominent genomewide surveys of sperm chromatin, suggesting that these profiles primarily reflect contaminating cell-free chromatin. Removal of cell-free DNA, along with appropriate lysis conditions, are required to reveal a sperm chromatin state distinct from any yet reported. Using ATAC-Seq to explore relatively accessible genomic loci, we identify a landscape of open loci associated with genes expressed during late spermiogenesis. Histone modification and chromosome folding studies also strongly support the hypothesis that prior studies suffer from contamination, but technical challenges associated with reliably preserving the architecture of the compacted sperm head prevent us from confidently assaying true localization patterns for these epigenetic marks. Together, our studies strongly argue that our knowledge of mammalian chromosome packaging remains largely incomplete, and motivate future efforts to more accurately characterize genome organization in mature sperm.

During the rapid and reductive cleavage divisions of early embryogenesis, subcellular structures such as the nucleus and mitotic spindle scale to decreasing cell size. Mitotic chromosomes also decrease in size during development, presumably to coordinately scale with mitotic spindles, but underlying mechanisms are unclear. Here we combinein vivoandin vitroapproaches using eggs and embryos from the frogXenopus laevisto show that mitotic chromosome scaling is mechanistically distinct from other forms of subcellular scaling. We found that mitotic chromosomes scale continuously with cell, spindle and nuclear sizein vivo. However, unlike for spindles and nuclei, mitotic chromosome size cannot be re-set by cytoplasmic factors from earlier developmental stages.In vitro, increasing nucleo-cytoplasmic (N/C) ratio is sufficient to recapitulate mitotic chromosome scaling, but not nuclear or spindle scaling, through differential loading of maternal factors during interphase. An additional pathway involving importin α scales mitotic chromosomes to cell surface area/volume (SA/V) during metaphase. Finally, single-chromosome immunofluorescence and analysis of Hi-C data suggest that mitotic chromosomes scale through decreased recruitment of condensin I, resulting in major rearrangements of DNA loop architecture to accommodate the same amount of DNA on a shorter axis. Together, our findings demonstrate how mitotic chromosome size is set by spatially and temporally distinct developmental cues in the early embryo.

SUMMARYThe topological state of chromosomes is an important factor controlling their mechanical properties, dynamics and function. Recent work has shown that interphase chromosomes are largely free of entanglements. How cycling cells establish this state from a dense mitotic chromosome and maintain it through interphase in the presence of Topoisomerase II activity remains mysterious. Using multi-contact 3C, Hi-C, and polymer simulations, we find that mitotic chromosomes are intrachromosomally entangled, while interphase chromosomes are not, pointing to cell cycle-dependent control of chromosome topology. The majority of mitotic entanglements are removed during anaphase/telophase, with most of the remaining ones removed during early G1, in a process that requires Topoisomerase II. Polymer simulations show that swelling and decondensation of mitotic chromosomes during mitotic exit produce entropic forces that bias Topoisomerase II activity towards decatenation of condensin loops. This is followed by prevention of formation of new entanglements by lower Topoisomerase II activity in G1, and, in heterochromatic B compartments, by interplay between cohesin and Topoisomerase II. Our results show how cells control chromosome topology during mitotic exit and G1 employing both biophysical and molecular mechanisms to modulate Topoisomerase II activity and directionality.

Insertions and deletions (indels) are common sources of structural variation, and insertions originating from spontaneous DNA lesions are frequent in cancer. We developed a highly sensitive assay in human cells (Indel-Seq) to monitor rearrangements at the TRIM37 acceptor locus which reports indels stemming from experimentally-induced and spontaneous genome instability. Templated insertions derive from sequences genome-wide and are enriched within 100 kb of donor regions flanking a DSB. Insertions require contact between donor and acceptor loci as well as DNA-PK catalytic activity. Notably, these templated insertions originate from actively transcribed loci, underscoring transcription as a critical source of spontaneous genome instability. Transcription-coupled insertions involve a DNA/RNA hybrid intermediate and are stimulated by DNA end-processing. Using engineered Cas9 breaks, we establish that ssDNA overhangs at the acceptor site greatly stimulate insertions. Indel-Seq revels that insertions are generated via at least three distinct pathways. Our studies indicate that insertions result from movement and subsequent contact between acceptor and donor loci followed invasion or annealing, then by non-homologous end-joining at the acceptor site.

Nuclear actin-based movements have been shown to orchestrate clustering of DNA double-strand breaks (DSBs) into homology-directed repair domains. Here we describe multiscale three-dimensional genome reorganization following DNA damage and analyze the contribution of the nuclear WASP-ARP2/3-actin pathway toward chromatin topology alterations and pathologic repair. Hi-C analysis reveals genome-wide, DNA damage-induced chromatin compartment flips facilitated by ARP2/3 that enrich for open, A compartments. Damage promotes interactions between DSBs, which in turn facilitate aberrant, actin-dependent intra- and inter-chromosomal rearrangements. Our work establishes that clustering of resected DSBs into repair domains by nuclear actin assembly is coordinated with multiscale alterations in genome architecture that enable homology-directed repair while also increasing nonhomologous end-joining-dependent translocation frequency.

Nuclear compartmentalization of active and inactive chromatin is thought to occur through microphase separation mediated by interactions between loci of similar type. The nature and dynamics of these interactions are not known. We developed liquid chromatin Hi-C to map the stability of associations between loci. Before fixation and Hi-C, chromosomes are fragmented, which removes strong polymeric constraint, enabling detection of intrinsic locus–locus interaction stabilities. Compartmentalization is stable when fragments are larger than 10–25 kb. Fragmentation of chromatin into pieces smaller than 6 kb leads to gradual loss of genome organization. Lamin-associated domains are most stable, whereas interactions for speckle- and polycomb-associated loci are more dynamic. Cohesin-mediated loops dissolve after fragmentation. Liquid chromatin Hi-C provides a genome-wide view of chromosome interaction dynamics. Liquid chromatin Hi-C maps the intrinsic stability of associations between loci. Lamin-associated domains are most stable, whereas interactions for speckle- and polycomb-associated loci are more dynamic.

The relationships between chromosomal compartmentalization, chromatin state and function are poorly understood. Here by profiling long-range contact frequencies in HCT116 colon cancer cells, we distinguish three silent chromatin states, comprising two types of heterochromatin and a state enriched for H3K9me2 and H2A.Z that exhibits neutral three-dimensional interaction preferences and which, to our knowledge, has not previously been characterized. We find that heterochromatin marked by H3K9me3, HP1α and HP1β correlates with strong compartmentalization. We demonstrate that disruption of DNA methyltransferase activity greatly remodels genome compartmentalization whereby domains lose H3K9me3-HP1α/β binding and acquire the neutrally interacting state while retaining late replication timing. Furthermore, we show that H3K9me3-HP1α/β heterochromatin is permissive to loop extrusion by cohesin but refractory to CTCF binding. Together, our work reveals a dynamic structural and organizational diversity of the silent portion of the genome and establishes connections between the regulation of chromatin state and chromosome organization, including an interplay between DNA methylation, compartmentalization and loop extrusion.

Skates are cartilaginous fish whose novel body plan features remarkably enlarged wing-like pectoral fins that allow them to thrive in benthic environments. The molecular underpinnings of this unique trait, however, remain elusive. Here we investigate the origin of this phenotypic innovation by developing the little skate Leucoraja erinacea as a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins (gene expression, chromatin occupancy and three-dimensional (3D) conformation) we find skate-specific genomic rearrangements that alter the 3D regulatory landscape of genes involved in the planar cell polarity (PCP) pathway. Functional inhibition of PCP signaling resulted in marked reduction of anterior fin size, confirming this pathway as a major contributor of batoid fin morphology. We also identified a fin-specific enhancer that interacts with 3' HOX genes, consistent with the redeployment of Hox gene expression in anterior pectoral fins, and confirmed the potential of this element to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganizations and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait.

The publication of the Caenorhabditis briggsae reference genome in 2003 enabled the first comparative genomics studies between C. elegans and C. briggsae, shedding light on the evolution of genome content and structure in the Caenorhabditis genus. However, despite being widely used, the currently available C. briggsae reference genome is substantially less complete and structurally accurate than the C. elegans reference genome. Here, we used high-coverage Oxford Nanopore long-read and chromosome-conformation capture data to generate chromosome-level reference genomes for two C. briggsae strains: QX1410, a new reference strain closely related to the laboratory AF16 strain, and VX34, a highly divergent strain isolated in China. We also sequenced 99 recombinant inbred lines generated from reciprocal crosses between QX1410 and VX34 to create a recombination map and identify chromosomal domains. Additionally, we used both short- and long-read RNA sequencing data to generate high-quality gene annotations. By comparing these new reference genomes to the current reference, we reveal that hyper-divergent haplotypes cover large portions of the C. briggsae genome, similar to recent reports in C. elegans and C. tropicalis. We also show that the genomes of selfing Caenorhabditis species have undergone more rearrangement than their outcrossing relatives, which has biased previous estimates of rearrangement rate in Caenorhabditis. These new genomes provide a substantially improved platform for comparative genomics in Caenorhabditis and narrow the gap between the quality of genomic resources available for C. elegans and C. briggsae.

Uveal Melanoma (UM) is a rare cancer resulting from transformation of melanocytes residing in the uveal tract. Integrative analysis has identified four molecular and clinical subsets in UM. To improve our molecular understanding of UM we performed extensive multi-omics characterization comparing two aggressive UM patient-derived xenograft models with normal choroidal melanocytes, including DNA optical mapping, specific histone modifications, and DNA topology analysis by Hi-C. Our gene expression and cytogenetic analyses suggest that genomic instability is a hallmark of UM. We also identified a recurrent deletion in the BAP1 promoter resulting in loss of expression and associated with high risk of metastases in UM patients. Hi-C revealed chromatin topology changes associated with up-regulation of PRAME, an independent prognostic biomarker in UM and potential therapeutic target. Our findings illustrate how multi-omics approaches can improve the understanding of tumorigenesis and reveals two novel mechanisms of gene expression dysregulation in UM.

Chromosome segregation requires both compaction and disentanglement of sister chromatids. We describe SisterC, a chromosome conformation capture assay that distinguishes interactions between and along identical sister chromatids. SisterC employs 5-bromo-2′-deoxyuridine (BrdU) incorporation during S-phase to label newly replicated strands, followed by Hi-C and then the destruction of 5-bromodeoxyuridine-containing strands via Hoechst/ultraviolet treatment. After sequencing of the remaining intact strands, this allows assignment of Hi-C products as inter- and intra-sister interactions based on the strands that reads are mapped to. We performed SisterC on mitotic Saccharomyces cerevisiae cells. We find precise alignment of sister chromatids at centromeres. Along arms, sister chromatids are less precisely aligned, with inter-sister connections every ~35 kilobase (kb). Inter-sister interactions occur between cohesin binding sites that are often offset by 5 to 25 kb. Along sister chromatids, cohesin results in the formation of loops of up to 50 kb. SisterC allows study of the complex interplay between sister chromatid compaction and their segregation during mitosis. It remains impossible using conventional Hi-C to differentiate interactions between and along sister chromatids. SisterC relies on selective destruction of nascent DNA and in combination with Hi-C offers a means to study intra- and inter-sister interactions independently.

During interphase, the eukaryotic genome is organized into chromosome territories that are spatially segregated into compartment domains. The extent to which interacting domains or chromosomes are entangled is not known. We analyze series of co-occurring chromatin interactions using multi-contact 3C (MC-3C) in human cells to provide insights into the topological entanglement of chromatin. Multi-contact interactions represent percolation paths (C-walks) through three-dimensional (3D) chromatin space. We find that the order of interactions within C-walks that occur across interfaces where chromosomes or compartment domains interact is not random. Polymer simulations show that such C-walks are consistent with distal domains being topologically insulated, that is, not catenated. Simulations show that even low levels of random strand passage, for example by topoisomerase II, would result in entanglements, increased mixing at domain interfaces and an order of interactions within C-walks not consistent with experimental MC-3C data. Our results indicate that, during interphase, entanglements between chromosomes and chromosomal domains are rare. Analysis of co-occurring chromatin interactions using multi-contact 3C and polymer simulations shows that, during interphase, entanglements between chromosomes and chromosomal domains are rare.

The publication of the Caenorhabditis briggsae reference genome in 2003 enabled the first comparative genomics studies between C. elegans and C. briggsae, shedding light on the evolution of genome content and structure in the Caenorhabditis genus. However, despite being widely used, the currently available C. briggsae reference genome is substantially less complete and structurally accurate than the C. elegans reference genome. Here, we used high-coverage Oxford Nanopore long-read and chromosome conformation capture data to generate chromosomally resolved reference genomes for two C. briggsae strains: QX1410, a new reference strain closely related to the laboratory AF16 strain, and VX34, a highly divergent strain isolated in China. We also sequenced 99 recombinant inbred lines (RILs) generated from reciprocal crosses between QX1410 and VX34 to create a recombination map and identify chromosomal domains. Additionally, we used both short- and long-read RNA sequencing (RNA-seq) data to generate high-quality gene annotations. By comparing these new reference genomes to the current reference, we reveal that hyper-divergent haplotypes cover large portions of the C. briggsae genome, similar to recent reports in C. elegans and C. tropicalis. We also show that the genomes of selfing Caenorhabditis species have undergone more rearrangement than their outcrossing relatives, which has biased previous estimates of rearrangement rate in Caenorhabditis. These new genomes provide a substantially improved platform for comparative genomics in Caenorhabditis and narrow the gap between the quality of genomic resources available for C. elegans and C. briggsae.

Precise control of gene expression during differentiation relies on the interplay of chromatin and nuclear structure. Despite an established contribution of nuclear membrane proteins to developmental gene regulation, little is known regarding the role of inner nuclear proteins. Here we demonstrate that loss of the nuclear scaffolding protein Matrin-3 (Matr3) in erythroid cells leads to morphological and gene expression changes characteristic of accelerated maturation, as well as broad alterations in chromatin organization similar to those accompanying differentiation. Matr3 protein interacts with CTCF and the cohesin complex, and its loss perturbs their occupancy at a subset of sites. Destabilization of CTCF and cohesin binding correlates with altered transcription and accelerated differentiation. This association is conserved in embryonic stem cells. Our findings indicate Matr3 negatively affects cell fate transitions and demonstrate that a critical inner nuclear protein impacts occupancy of architectural factors, culminating in broad effects on chromatin organization and cell differentiation., Interactions between chromatin and nuclear components and whether these interactions affect development is not well understood. Here the authors show inner nuclear protein Matrin-3 (Matr3) loss leads to accelerated erythroid maturation, and that Matr3 is involved in chromosomal structure organization and compartmentalization to negatively regulate cell differentiation.

SummaryDNA end-resection and nuclear actin-based movements orchestrate clustering of double-strand breaks (DSBs) into homology-directed repair (HDR) domains. Here, we analyze how actin nucleation by ARP2/3 affects damage-dependent and -independent 3D genome reorganization and facilitates pathologic repair. We observe that DNA damage, followed by ARP2/3-dependent establishment of repair domains enhances local chromatin insulation at a set of damage-proximal boundaries and affects compartment organization genome-wide. Nuclear actin polymerization also promotes interactions between DSBs, which in turn facilitates aberrant intra- and inter-chromosomal rearrangements. Notably, BRCA1 deficiency, which decreases end-resection, DSB mobility, and subsequent HDR, nearly abrogates recurrent translocations between AsiSI DSBs. In contrast, loss of functional BRCA1 yields unique translocations genome-wide, reflecting a critical role in preventing spontaneous genome instability and subsequent rearrangements. Our work establishes that the assembly of DSB repair domains is coordinated with multiscale alterations in genome architecture that enable HDR despite increased risk of translocations with pathologic potential.

Background The coral-Symbiodiniaceae symbiosis is fundamental for the coral reef ecosystem. Corals provide various inorganic nutrients to their algal symbionts in exchange for the photosynthates to meet their metabolic demands. When becoming symbionts, Symbiodiniaceae cells show a reduced proliferation rate and a different life history. While it is generally believed that the animal hosts play critical roles in regulating these processes, far less is known about the molecular underpinnings that allow the corals to induce the changes in their symbionts. Results We tested symbiont cell proliferation and life stage changes in vitro in response to different nutrient-limiting conditions to determine the key nutrients and to compare the respective symbiont transcriptomic profiles to cells in hospite. We then examined the effects of nutrient repletion on symbiont proliferation in coral hosts and quantified life stage transitions in vitro using time-lapse confocal imaging. Here, we show that symbionts in hospite share gene expression and pathway activation profiles with free-living cells under nitrogen-limited conditions, strongly suggesting that symbiont proliferation in symbiosis is limited by nitrogen availability. Conclusions We demonstrate that nitrogen limitation not only suppresses cell proliferation but also life stage transition to maintain symbionts in the immobile coccoid stage. Nutrient repletion experiments in corals further confirmed that nitrogen availability is the major factor limiting symbiont density in hospite. Our study emphasizes the importance of nitrogen in coral-algae interactions and, more importantly, sheds light on the crucial role of nitrogen in symbiont life history regulation.

Layers of genome organization are becoming increasingly better characterized, but less is known about how these structures respond to perturbation or shape changes. Low-salt swelling of isolated chromatin fibers or nuclei has been used for decades to investigate the structural properties of chromatin. But, visible changes in chromatin appearance have not been linked to known building blocks of genome structure or features along the genome sequence. We combine low-salt swelling of isolated nuclei with genome-wide chromosome conformation capture (Hi-C) and imaging approaches to probe the effects of chromatin extension genome-wide. Photoconverted patterns on nuclei during expansion and contraction indicate that global genome structure is preserved after dramatic nuclear volume swelling, suggesting a highly elastic chromosome topology. Hi-C experiments before, during, and after nuclear swelling show changes in average contact probabilities at short length scales, reflecting the extension of the local chromatin fiber. But, surprisingly, during this large increase in nuclear volume, there is a striking maintenance of loops, TADs, active and inactive compartments, and chromosome territories. Subtle differences after expansion are observed, suggesting that the local chromatin state, protein interactions, and location in the nucleus can affect how strongly a given structure is maintained under stress. From these observations, we propose that genome topology is robust to extension of the chromatin fiber and isotropic shape change, and that this elasticity may be beneficial in physiological circumstances of changes in nuclear size and volume.

The ring-like cohesin complex mediates sister chromatid cohesion by encircling pairs of sister chromatids. Cohesin also extrudes loops along chromatids. Whether the two activities involve similar mechanisms of DNA engagement is not known. We implemented an experimental approach based on isolated nuclei carrying engineered cleavable RAD21 proteins to precisely control cohesin ring integrity so that its role in chromatin looping could be studied under defined experimental conditions. This approach allowed us to identify cohesin complexes with distinct biochemical, and possibly structural properties, that mediate different sets of chromatin loops. When RAD21 is cleaved and the cohesin ring is opened, cohesin complexes at CTCF sites are released from DNA and loops at these elements are lost. In contrast, cohesin-dependent loops within chromatin domains and that are not anchored at CTCF sites are more resistant to RAD21 cleavage. The results show that the cohesin complex mediates loops in different ways depending on genomic context and suggests that it undergoes structural changes as it dynamically extrudes and encounters CTCF sites.

The ring-like cohesin complex mediates sister chromatid cohesion by encircling pairs of sister chromatids. Cohesin also extrudes loops along chromatids. Whether the two activities involve similar mechanisms of DNA engagement is not known. We implemented an experimental approach based on isolated nuclei carrying engineered cleavable RAD21 proteins to precisely control cohesin ring integrity so that its role in chromatin looping could be studied under defined experimental conditions. This approach allowed us to identify cohesin complexes with distinct biochemical, and possibly structural properties, that mediate different sets of chromatin loops. When RAD21 is cleaved and the cohesin ring is opened, cohesin complexes at CTCF sites are released from DNA and loops at these elements are lost. In contrast, cohesin-dependent loops within chromatin domains and that are not anchored at pairs of CTCF sites are more resistant to RAD21 cleavage. The results show that the cohesin complex mediates loops in different ways depending on genomic context and suggests that it undergoes structural changes as it dynamically extrudes and encounters CTCF sites.

Layers of genome organization are becoming increasingly better characterized, but less is known about how these structures respond to perturbation or shape changes. Low-salt swelling of isolated chromatin fibers or nuclei has been used for decades to investigate the structural properties of chromatin. But, visible changes in chromatin appearance have not been linked to known building blocks of genome structure or features along the genome sequence. We combine low-salt swelling of isolated nuclei with genome-wide chromosome conformation capture (Hi-C) and imaging approaches to probe the effects of chromatin extension genome-wide. Photoconverted patterns on nuclei during expansion and contraction indicate that global genome structure is preserved after dramatic nuclear volume swelling, suggesting a highly elastic chromosome topology. Hi-C experiments before, during, and after nuclear swelling show changes in average contact probabilities at short length scales, reflecting the extension of the local chromatin fiber. But, surprisingly, during this large increase in nuclear volume, there is a striking maintenance of loops, TADs, active and inactive compartments, and chromosome territories. Subtle differences after expansion are observed, suggesting that the local chromatin state, protein interactions, and location in the nucleus can affect how strongly a given structure is maintained under stress. From these observations, we propose that genome topology is robust to extension of the chromatin fiber and isotropic shape change, and that this elasticity may be beneficial in physiological circumstances of changes in nuclear size and volume.

Two dominant processes organizing chromosomes are loop extrusion and the compartmental segregation of active and inactive chromatin. The molecular players involved in loop extrusion during interphase, cohesin and CTCF, have been extensively studied and experimentally validated. However, neither the molecular determinants nor the functional roles of compartmentalization are well understood. Here, we distinguish three inactive chromatin states using contact frequency profiling, comprising two types of heterochromatin and a previously uncharacterized inactive state exhibiting a neutral interaction preference. We find that heterochromatin marked by long continuous stretches of H3K9me3, HP1α and HP1β correlates with a conserved signature of strong compartmentalization and is abundant in HCT116 colon cancer cells. We demonstrate that disruption of DNA methyltransferase activity dramatically remodels genome compartmentalization as a consequence of the loss of H3K9me3 and HP1 binding. Interestingly, H3K9me3-HP1α/β is replaced by the neutral inactive state and retains late replication timing. Furthermore, we show that H3K9me3-HP1α/β heterochromatin is permissive to loop extrusion by cohesin but refractory to CTCF, explaining a paucity of visible loop extrusion-associated patterns in Hi-C. Accordingly, CTCF loop extrusion barriers are reactivated upon loss of H3K9me3-HP1α/β, not as a result of canonical demethylation of the CTCF binding motif but due to an intrinsic resistance of H3K9me3-HP1α/β heterochromatin to CTCF binding. Together, our work reveals a dynamic structural and organizational diversity of the inactive portion of the genome and establishes new connections between the regulation of chromatin state and chromosome organization, including an interplay between DNA methylation, compartmentalization and loop extrusion.HighlightsThree inactive chromatin states are distinguishable by long-range contact frequencies in HCT116, respectively associated with H3K9me3, H3K27me3 and a H3K9me2 state with neutral contact preferences.H3K9me3-HP1α/β heterochromatin has a high degree of homotypic affinity and is permissive to loop extrusion but depleted in extrusion barriers.Disrupting DNA methylation causes widespread loss of H3K9me3-HP1α/β and dramatic remodeling of genome compartmentalization.H3K9me3-HP1α/β is replaced by the neutral inactive state, which gains CTCF loop extrusion barriers and associated contact frequency patterns.DNA methylation suppresses CTCF binding via two distinct mechanisms.

DNA loop extrusion by condensins and decatenation by DNA topoisomerase II (topo II) are thought to drive mitotic chromosome compaction and individualization. Here, we reveal that the linker histone H1.8 antagonizes condensins and topo II to shape mitotic chromosome organization. In vitro chromatin reconstitution experiments demonstrate that H1.8 inhibits binding of condensins and topo II to nucleosome arrays. Accordingly, H1.8 depletion in Xenopus egg extracts increased condensins and topo II levels on mitotic chromatin. Chromosome morphology and Hi-C analyses suggest that H1.8 depletion makes chromosomes thinner and longer through shortening the average loop size and reducing the DNA amount in each layer of mitotic loops. Furthermore, excess loading of condensins and topo II to chromosomes by H1.8 depletion causes hyper-chromosome individualization and dispersion. We propose that condensins and topo II are essential for chromosome individualization, but their functions are tuned by the linker histone to keep chromosomes together until anaphase.

The intricate folding of chromatin enables living organisms to store genomic material in an extremely small volume while facilitating proper cell function. Hi‐C is a chromosome conformation capture (3C)‐based technology to detect pair‐wise chromatin interactions genome‐wide, and has become a benchmark tool to study genome organization. In Hi‐C, chromatin conformation is first captured by chemical cross‐linking of cells. Cells are then lysed and subjected to restriction enzyme digestion, before the ends of the resulting fragments are marked with biotin. Fragments within close 3D proximity are ligated, and the biotin label is used to selectively enrich for ligated junctions. Finally, isolated ligation products are prepared for high‐throughput sequencing, which enables the mapping of pair‐wise chromatin interactions genome‐wide. Over the past decade, “next‐generation” sequencing has become cheaper and easier to perform, enabling more interactions to be sampled to obtain higher resolution in chromatin interaction maps. Here, we provide an in‐depth guide to performing an up‐to‐date Hi‐C procedure on mammalian cell lines. These protocols include recent improvements that increase the resolution potential of the assay, namely by enhancing cross‐linking and using a restriction enzyme cocktail. These improvements result in a versatile Hi‐C procedure that enables the detection of genome folding features at a wide range of distances. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Fixation of nuclear conformation Basic Protocol 2: Chromosome conformation capture Basic Protocol 3: Hi‐C sequencing library preparation