SMCHD1 Merges Chromosome Compartments and Assists Formation of Super-Structures on the Inactive X.

Mammalian chromosomes are partitioned into A/B compartments and topologically associated domains (TADs). The inactive X (Xi) chromosome, however, adopts a distinct conformation without evident compartments or TADs. Here, through exploration of an architectural protein, structural-maintenance-of-chromosomes hinge domain containing 1 (SMCHD1), we probe how the Xi is reconfigured during X chromosome inactivation. A/B compartments are first fused into "S1" and "S2" compartments, coinciding with Xist spreading into gene-rich domains. SMCHD1 then binds S1/S2 compartments and merges them to create a compartment-less architecture. Contrary to current views, TADs remain on the Xi but in an attenuated state. Ablating SMCHD1 results in a persistent S1/S2 organization and strengthening of TADs. Furthermore, loss of SMCHD1 causes regional defects in Xist spreading and erosion of heterochromatic silencing. We present a stepwise model for Xi folding, where SMCHD1 attenuates a hidden layer of Xi architecture to facilitate Xist spreading.

Disease-Associated Short Tandem Repeats Co-localize with Chromatin Domain Boundaries.

More than 25 inherited human disorders are caused by the unstable expansion of repetitive DNA sequences termed short tandem repeats (STRs). A fundamental unresolved question is why some STRs are susceptible to pathologic expansion, whereas thousands of repeat tracts across the human genome are relatively stable. Here, we discover that nearly all disease-associated STRs (daSTRs) are located at boundaries demarcating 3D chromatin domains. We identify a subset of boundaries with markedly higher CpG island density compared to the rest of the genome. daSTRs specifically localize to ultra-high-density CpG island boundaries, suggesting they might be hotspots for epigenetic misregulation or topological disruption linked to STR expansion. Fragile X syndrome patients exhibit severe boundary disruption in a manner that correlates with local loss of CTCF occupancy and the degree of FMR1 silencing. Our data uncover higher-order chromatin architecture as a new dimension in understanding repeat expansion disorders.

The Cohesin Release Factor WAPL Restricts Chromatin Loop Extension.

The spatial organization of chromosomes influences many nuclear processes including gene expression. The cohesin complex shapes the 3D genome by looping together CTCF sites along chromosomes. We show here that chromatin loop size can be increased and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. Cohesin's DNA release factor WAPL restricts this loop extension and also prevents looping between incorrectly oriented CTCF sites. We reveal that the SCC2/SCC4 complex promotes the extension of chromatin loops and the formation of topologically associated domains (TADs). Our data support the model that cohesin structures chromosomes through the processive enlargement of loops and that TADs reflect polyclonal collections of loops in the making. Finally, we find that whereas cohesin promotes chromosomal looping, it rather limits nuclear compartmentalization. We conclude that the balanced activity of SCC2/SCC4 and WAPL enables cohesin to correctly structure chromosomes.

Chromatin gene-gene loops support the cross-regulation of genes with related function.

Chromatin loops between gene pairs have been observed in diverse contexts in both flies and vertebrates. Combining high-resolution Capture-C, DNA fluorescence in situ hybridization, and genetic perturbations, we dissect the functional role of three loops between genes with related function during Drosophila embryogenesis. By mutating the loop anchor (but not the gene) or the gene (but not loop anchor), we disentangle loop formation and gene expression and show that the 3D proximity of paralogous gene loci supports their co-regulation. Breaking the loop leads to either an attenuation or enhancement of expression and perturbs their relative levels of expression and cross-regulation. Although many loops appear constitutive across embryogenesis, their function can change in different developmental contexts. Taken together, our results indicate that chromatin gene-gene loops act as architectural scaffolds that can be used in different ways in different contexts to fine-tune the coordinated expression of genes with related functions and sustain their cross-regulation.

Cohesin mediates DNA loop extrusion and sister chromatid cohesion by distinct mechanisms.

Cohesin connects CTCF-binding sites and other genomic loci in cis to form chromatin loops and replicated DNA molecules in trans to mediate sister chromatid cohesion. Whether cohesin uses distinct or related mechanisms to perform these functions is unknown. Here, we describe a cohesin hinge mutant that can extrude DNA into loops but is unable to mediate cohesion in human cells. Our results suggest that the latter defect arises during cohesion establishment. The observation that cohesin's cohesion and loop extrusion activities can be partially separated indicates that cohesin uses distinct mechanisms to perform these two functions. Unexpectedly, the same hinge mutant can also not be stopped by CTCF boundaries as well as wild-type cohesin. This suggests that cohesion establishment and cohesin's interaction with CTCF boundaries depend on related mechanisms and raises the possibility that both require transient hinge opening to entrap DNA inside the cohesin ring.

CTCF and R-loops are boundaries of cohesin-mediated DNA looping.

Cohesin and CCCTC-binding factor (CTCF) are key regulatory proteins of three-dimensional (3D) genome organization. Cohesin extrudes DNA loops that are anchored by CTCF in a polar orientation. Here, we present direct evidence that CTCF binding polarity controls cohesin-mediated DNA looping. Using single-molecule imaging, we demonstrate that a critical N-terminal motif of CTCF blocks cohesin translocation and DNA looping. The cryo-EM structure of the cohesin-CTCF complex reveals that this CTCF motif ahead of zinc fingers can only reach its binding site on the STAG1 cohesin subunit when the N terminus of CTCF faces cohesin. Remarkably, a C-terminally oriented CTCF accelerates DNA compaction by cohesin. DNA-bound Cas9 and Cas12a ribonucleoproteins are also polar cohesin barriers, indicating that stalling may be intrinsic to cohesin itself. Finally, we show that RNA-DNA hybrids (R-loops) block cohesin-mediated DNA compaction in vitro and are enriched with cohesin subunits in vivo, likely forming TAD boundaries.

Interplay between CTCF boundaries and a super enhancer controls cohesin extrusion trajectories and gene expression.

To understand how chromatin domains coordinate gene expression, we dissected select genetic elements organizing topology and transcription around the Prdm14 super enhancer in mouse embryonic stem cells. Taking advantage of allelic polymorphisms, we developed methods to sensitively analyze changes in chromatin topology, gene expression, and protein recruitment. We show that enhancer insulation does not rely strictly on loop formation between its flanking boundaries, that the enhancer activates the Slco5a1 gene beyond its prominent domain boundary, and that it recruits cohesin for loop extrusion. Upon boundary inversion, we find that oppositely oriented CTCF terminates extrusion trajectories but does not stall cohesin, while deleted or mutated CTCF sites allow cohesin to extend its trajectory. Enhancer-mediated gene activation occurs independent of paused loop extrusion near the gene promoter. We expand upon the loop extrusion model to propose that cohesin loading and extrusion trajectories originating at an enhancer contribute to gene activation.

A Conserved Noncoding Locus Regulates Random Monoallelic Xist Expression across a Topological Boundary.

cis-Regulatory communication is crucial in mammalian development and is thought to be restricted by the spatial partitioning of the genome in topologically associating domains (TADs). Here, we discovered that the Xist locus is regulated by sequences in the neighboring TAD. In particular, the promoter of the noncoding RNA Linx (LinxP) acts as a long-range silencer and influences the choice of X chromosome to be inactivated. This is independent of Linx transcription and independent of any effect on Tsix, the antisense regulator of Xist that shares the same TAD as Linx. Unlike Tsix, LinxP is well conserved across mammals, suggesting an ancestral mechanism for random monoallelic Xist regulation. When introduced in the same TAD as Xist, LinxP switches from a silencer to an enhancer. Our study uncovers an unsuspected regulatory axis for X chromosome inactivation and a class of cis-regulatory effects that may exploit TAD partitioning to modulate developmental decisions.

A central role for canonical PRC1 in shaping the 3D nuclear landscape.

Polycomb group (PcG) proteins silence gene expression by chemically and physically modifying chromatin. A subset of PcG target loci are compacted and cluster in the nucleus; a conformation that is thought to contribute to gene silencing. However, how these interactions influence gross nuclear organization and their relationship with transcription remains poorly understood. Here we examine the role of Polycomb-repressive complex 1 (PRC1) in shaping 3D genome organization in mouse embryonic stem cells (mESCs). Using a combination of imaging and Hi-C analyses, we show that PRC1-mediated long-range interactions are independent of CTCF and can bridge sites at a megabase scale. Impairment of PRC1 enzymatic activity does not directly disrupt these interactions. We demonstrate that PcG targets coalesce in vivo, and that developmentally induced expression of one of the target loci disrupts this spatial arrangement. Finally, we show that transcriptional activation and the loss of PRC1-mediated interactions are separable events. These findings provide important insights into the function of PRC1, while highlighting the complexity of this regulatory system.

RNA Interactions Are Essential for CTCF-Mediated Genome Organization.

The function of the CCCTC-binding factor (CTCF) in the organization of the genome has become an important area of investigation, but the mechanisms by which CTCF dynamically contributes to genome organization are not clear. We previously discovered that CTCF binds to large numbers of endogenous RNAs, promoting its self-association. In this regard, we now report two independent features that disrupt CTCF association with chromatin: inhibition of transcription and disruption of CTCF-RNA interactions through mutations of 2 of its 11 zinc fingers that are not required for CTCF binding to its cognate DNA site: zinc finger 1 (ZF1) or zinc finger 10 (ZF10). These mutations alter gene expression profiles as CTCF mutants lose their ability to form chromatin loops and thus the ability to insulate chromatin domains and to mediate CTCF long-range genomic interactions. Our results point to the importance of CTCF-mediated RNA interactions as a structural component of genome organization.

Emerging regulatory mechanisms of noncoding RNAs in topologically associating domains.

Topologically associating domains (TADs) are integral to spatial genome organization, instructing gene expression, and cell fate. Recently, several advances have uncovered roles for noncoding RNAs (ncRNAs) in the regulation of the form and function of mammalian TADs. Phase separation has also emerged as a potential arbiter of ncRNAs in the regulation of TADs. In this review we discuss the implications of these novel findings in relation to how ncRNAs might structurally and functionally regulate TADs from two perspectives: moderating loop extrusion through interactions with architectural proteins, and facilitating TAD phase separation. Additionally, we propose future studies and directions to investigate these phenomena.

Stratifying TAD boundaries pinpoints focal genomic regions of regulation, damage, and repair.

Advances in chromatin mapping have exposed the complex chromatin hierarchical organization in mammals, including topologically associating domains (TADs) and their substructures, yet the functional implications of this hierarchy in gene regulation and disease progression are not fully elucidated. Our study delves into the phenomenon of shared TAD boundaries, which are pivotal in maintaining the hierarchical chromatin structure and regulating gene activity. By integrating high-resolution Hi-C data, chromatin accessibility, and DNA double-strand breaks (DSBs) data from various cell lines, we systematically explore the complex regulatory landscape at high-level TAD boundaries. Our findings indicate that these boundaries are not only key architectural elements but also vibrant hubs, enriched with functionally crucial genes and complex transcription factor binding site-clustered regions. Moreover, they exhibit a pronounced enrichment of DSBs, suggesting a nuanced interplay between transcriptional regulation and genomic stability. Our research provides novel insights into the intricate relationship between the 3D genome structure, gene regulation, and DNA repair mechanisms, highlighting the role of shared TAD boundaries in maintaining genomic integrity and resilience against perturbations. The implications of our findings extend to understanding the complexities of genomic diseases and open new avenues for therapeutic interventions targeting the structural and functional integrity of TAD boundaries.

Visualization of Transvection in Living Drosophila Embryos.

How remote enhancers interact with appropriate target genes persists as a central mystery in gene regulation. Here, we exploit the properties of transvection to explore enhancer-promoter communication between homologous chromosomes in living Drosophila embryos. We successfully visualized the activation of an MS2-tagged reporter gene by a defined developmental enhancer located in trans on the other homolog. This trans-homolog activation depends on insulator DNAs, which increase the stability-but not the frequency-of homolog pairing. A pair of heterotypic insulators failed to mediate transvection, raising the possibility that insulator specificity underlies the formation of chromosomal loop domains. Moreover, we found that a shared enhancer co-activates separate PP7 and MS2 reporter genes incis and intrans. Transvecting alleles weakly compete with one another, raising the possibility that they share a common pool of the transcription machinery. We propose that transvecting alleles form a trans-homolog "hub," which serves as a scaffold for the accumulation of transcription complexes.

Polycomb-Dependent Chromatin Looping Contributes to Gene Silencing during Drosophila Development.

Interphase chromatin is organized into topologically associating domains (TADs). Within TADs, chromatin looping interactions are formed between DNA regulatory elements, but their functional importance for the establishment of the 3D genome organization and gene regulation during development is unclear. Using high-resolution Hi-C experiments, we analyze higher order 3D chromatin organization during Drosophila embryogenesis and identify active and repressive chromatin loops that are established with different kinetics and depend on distinct factors: Zelda-dependent active loops are formed before the midblastula transition between transcribed genes over long distances. Repressive loops within polycomb domains are formed after the midblastula transition between polycomb response elements by the action of GAGA factor and polycomb proteins. Perturbation of PRE function by CRISPR/Cas9 genome engineering affects polycomb domain formation and destabilizes polycomb-mediated silencing. Preventing loop formation without removal of polycomb components also decreases silencing efficiency, suggesting that chromatin architecture can play instructive roles in gene regulation during development. VIDEO ABSTRACT.

Lamins Organize the Global Three-Dimensional Genome from the Nuclear Periphery.

Lamins are structural components of the nuclear lamina (NL) that regulate genome organization and gene expression, but the mechanism remains unclear. Using Hi-C, we show that lamins maintain proper interactions among the topologically associated chromatin domains (TADs) but not their overall architecture. Combining Hi-C with fluorescence in situ hybridization (FISH) and analyses of lamina-associated domains (LADs), we reveal that lamin loss causes expansion or detachment of specific LADs in mouse ESCs. The detached LADs disrupt 3D interactions of both LADs and interior chromatin. 4C and epigenome analyses further demonstrate that lamins maintain the active and repressive chromatin domains among different TADs. By combining these studies with transcriptome analyses, we found a significant correlation between transcription changes and the interaction changes of active and inactive chromatin domains These findings provide a foundation to further study how the nuclear periphery impacts genome organization and transcription in development and NL-associated diseases.

Unpacking chromatin remodelling in germ cells: implications for development and evolution.

Germ cells reflect the evolutionary history and future potential of a species. Understanding how the genome is organised in gametocytes is fundamental to understanding fertility and its impact on genetic diversity and evolution of species. Here, we explore principles of chromatin remodelling during the formation of germ cells and how these are affected by genome reshuffling.

Inversion of a topological domain leads to restricted changes in its gene expression and affects interdomain communication.

The interplay between the topological organization of the genome and the regulation of gene expression remains unclear. Depletion of molecular factors (e.g. CTCF) underlying topologically associating domains (TADs) leads to modest alterations in gene expression, whereas genomic rearrangements involving TAD boundaries disrupt normal gene expression and can lead to pathological phenotypes. Here, we targeted the TAD neighboring that of the noncoding transcript Xist, which controls X-chromosome inactivation. Inverting 245 kb within the TAD led to expected rearrangement of CTCF-based contacts but revealed heterogeneity in the 'contact' potential of different CTCF sites. Expression of most genes therein remained unaffected in mouse embryonic stem cells and during differentiation. Interestingly, expression of Xist was ectopically upregulated. The same inversion in mouse embryos led to biased Xist expression. Smaller inversions and deletions of CTCF clusters led to similar results: rearrangement of contacts and limited changes in local gene expression, but significant changes in Xist expression in embryos. Our study suggests that the wiring of regulatory interactions within a TAD can influence the expression of genes in neighboring TADs, highlighting the existence of mechanisms of inter-TAD communication.

Developmental and evolutionary comparative analysis of a regulatory landscape in mouse and chicken.

Modifications in gene regulation are driving forces in the evolution of organisms. Part of these changes involve cis-regulatory elements (CREs), which contact their target genes through higher-order chromatin structures. However, how such architectures and variations in CREs contribute to transcriptional evolvability remains elusive. We use Hoxd genes as a paradigm for the emergence of regulatory innovations, as many relevant enhancers are located in a regulatory landscape highly conserved in amniotes. Here, we analysed their regulation in murine vibrissae and chicken feather primordia, two skin appendages expressing different Hoxd gene subsets, and compared the regulation of these genes in these appendages with that in the elongation of the posterior trunk. In the two former structures, distinct subsets of Hoxd genes are contacted by different lineage-specific enhancers, probably as a result of using an ancestral chromatin topology as an evolutionary playground, whereas the gene regulation that occurs in the mouse and chicken embryonic trunk partially relies on conserved CREs. A high proportion of these non-coding sequences active in the trunk have functionally diverged between species, suggesting that transcriptional robustness is maintained, despite considerable divergence in enhancer sequences.

LPAD: using network construction and label propagation to detect topologically associating domains from Hi-C data.

With the development of chromosome conformation capture technique, the study of spatial conformation of a genome based on Hi-C technique has made a quantum leap. Previous studies reveal that genomes are folded into hierarchy of three-dimensional (3D) structures associated with topologically associating domains (TADs), and detecting TAD boundaries is of great significance in the chromosome-level analysis of 3D genome architecture. In this paper, we propose a novel TAD identification method, LPAD, which first extracts node correlations from global interactions of chromosomes based on the random walk with restart and then builds an undirected graph from Hi-C contact matrix. Next, LPAD designs a label propagation-based approach to discover communities and generates TADs. Experimental results verify the effectiveness and quality of TAD detections compared with existing methods. Furthermore, experimental evaluation of chromatin immunoprecipitation sequencing data shows that LPAD performs high enrichment of histone modifications remarkably nearby the TAD boundaries, and these results demonstrate LPAD's advantages on TAD identification accuracy.

From DNA human sequence to the chromatin higher order organisation and its biological meaning: Using biomolecular interaction networks to understand the influence of structural variation on spatial genome organisation and its functional effect.

The three-dimensional structure of the human genome has been proven to have a significant functional impact on gene expression. The high-order spatial chromatin is organised first by looping mediated by multiple protein factors, and then it is further formed into larger structures of topologically associated domains (TADs) or chromatin contact domains (CCDs), followed by A/B compartments and finally the chromosomal territories (CTs). The genetic variation observed in human population influences the multi-scale structures, posing a question regarding the functional impact of structural variants reflected by the variability of the genes expression patterns. The current methods of evaluating the functional effect include eQTLs analysis which uses statistical testing of influence of variants on spatially close genes. Rarely, non-coding DNA sequence changes are evaluated by their impact on the biomolecular interaction network (BIN) reflecting the cellular interactome that can be analysed by the classical graph-theoretic algorithms. Therefore, in the second part of the review, we introduce the concept of BIN, i.e. a meta-network model of the complete molecular interactome developed by integrating various biological networks. The BIN meta-network model includes DNA-protein binding by the plethora of protein factors as well as chromatin interactions, therefore allowing connection of genomics with the downstream biomolecular processes present in a cell. As an illustration, we scrutinise the chromatin interactions mediated by the CTCF protein detected in a ChIA-PET experiment in the human lymphoblastoid cell line GM12878. In the corresponding BIN meta-network the DNA spatial proximity is represented as a graph model, combined with the Proteins-Interaction Network (PIN) of human proteome using the Gene Association Network (GAN). Furthermore, we enriched the BIN with the signalling and metabolic pathways and Gene Ontology (GO) terms to assert its functional context. Finally, we mapped the Single Nucleotide Polymorphisms (SNPs) from the GWAS studies and identified the chromatin mutational hot-spots associated with a significant enrichment of SNPs related to autoimmune diseases. Afterwards, we mapped Structural Variants (SVs) from healthy individuals of 1000 Genomes Project and identified an interesting example of the missing protein complex associated with protein Q6GYQ0 due to a deletion on chromosome 14. Such an analysis using the meta-network BIN model is therefore helpful in evaluating the influence of genetic variation on spatial organisation of the genome and its functional effect in a cell.