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The three-dimensional organization of a genome plays a critical role in regulating gene expression, yet little is known about the machinery and mechanisms that determine higher-order chromosome structure. Here we perform genome-wide chromosome conformation capture analysis, fluorescent in situ hybridization (FISH), and RNA-seq to obtain comprehensive three-dimensional (3D) maps of the Caenorhabditis elegans genome and to dissect X chromosome dosage compensation, which balances gene expression between XX hermaphrodites and XO males. The dosage compensation complex (DCC), a condensin complex, binds to both hermaphrodite X chromosomes via sequence-specific recruitment elements on X (rex sites) to reduce chromosome-wide gene expression by half. Most DCC condensin subunits also act in other condensin complexes to control the compaction and resolution of all mitotic and meiotic chromosomes. By comparing chromosome structure in wild-type and DCC-defective embryos, we show that the DCC remodels hermaphrodite X chromosomes into a sex-specific spatial conformation distinct from autosomes. Dosage-compensated X chromosomes consist of self-interacting domains (∼1 Mb) resembling mammalian topologically associating domains (TADs). TADs on X chromosomes have stronger boundaries and more regular spacing than on autosomes. Many TAD boundaries on X chromosomes coincide with the highest-affinity rex sites and become diminished or lost in DCC-defective mutants, thereby converting the topology of X to a conformation resembling autosomes. rex sites engage in DCC-dependent long-range interactions, with the most frequent interactions occurring between rex sites at DCC-dependent TAD boundaries. These results imply that the DCC reshapes the topology of X chromosomes by forming new TAD boundaries and reinforcing weak boundaries through interactions between its highest-affinity binding sites. As this model predicts, deletion of an endogenous rex site at a DCC-dependent TAD boundary using CRISPR/Cas9 greatly diminished the boundary. Thus, the DCC imposes a distinct higher-order structure onto X chromosomes while regulating gene expression chromosome-wide.

The nucleus of mammalian cells displays a distinct spatial segregation of active euchromatic and inactive heterochromatic regions of the genome.sup.1,2. In conventional nuclei, microscopy shows that euchromatin is localized in the nuclear interior and heterochromatin at the nuclear periphery.sup.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.sup.3, and depletion between them. Many mechanisms for the formation of compartments have been proposed, such as attraction of heterochromatin to the nuclear lamina.sup.2,4, preferential attraction of similar chromatin to each other.sup.1,4-12, higher levels of chromatin mobility in active chromatin.sup.13-15 and transcription-related clustering of euchromatin.sup.16,17. However, these hypotheses have remained inconclusive, owing to the difficulty of disentangling intra-chromatin and chromatin-lamina interactions in conventional nuclei.sup.18. The marked reorganization of interphase chromosomes in the inverted nuclei of rods in nocturnal mammals.sup.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., Author(s): Martin Falk [sup.1] , Yana Feodorova [sup.2] [sup.3] , Natalia Naumova [sup.4] [sup.5] , Maxim Imakaev [sup.1] , Bryan R. Lajoie [sup.4] [sup.6] , Heinrich Leonhardt [sup.3] , Boris [...]

X-chromosome inactivation (XCI) involves major reorganization of the X chromosome as it becomes silent and heterochromatic. During female mammalian development, XCI is triggered by upregulation of the non-coding Xist RNA from one of the two X chromosomes. Xist coats the chromosome in cis and induces silencing of almost all genes via its A-repeat region (1,2), although some genes (constitutive escapees) avoid silencing in most cell types, and others (facultative escapees) escape XCI only in specific contexts (3). A role for Xist in organizing the inactive X (Xi) chromosome has been proposed (4-6). Recent chromosome conformation capture approaches have revealed global loss of local structure on the Xi chromosome and formation of large mega-domains, separated by a region containing the DXZ4 macrosatellite (7-10). However, the molecular architecture of the Xi chromosome, in both the silent and expressed regions, remains unclear. Here we investigate the structure, chromatin accessibility and expression status of the mouse Xi chromosome in highly polymorphic clonal neural progenitors (NPCs) and embryonic stem cells. We demonstrate a crucial role for Xist and the DXZ4-containing boundary in shaping Xi chromosome structure using allele-specific genome-wide chromosome conformation capture (Hi-C) analysis, an assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) and RNA sequencing. Deletion of the boundary disrupts mega-domain formation, and induction of Xist RNA initiates formation of the boundary and the loss of DNA accessibility. We also show that in NPCs, the Xi chromosome lacks active/inactive compartments and topologically associating domains (TADs), except around genes that escape XCI. Escapee gene clusters display TAD-like structures and retain DNA accessibility at promoter-proximal and CTCF-binding sites. Furthermore, altered patterns of facultative escape genes in different neural progenitor clones are associated with the presence of different TAD-like structures after XCI. These findings suggest a key role for transcription and CTCF in the formation of TADs in the context of the Xi chromosome in neural progenitors., To investigate the structure of the Xi chromosome, we performed allele-specific Hi-C in a clonal neural progenitor cell (NPC) line that was derived from highly polymorphic F1 mouse embryonic stem [...]

The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall, the project provides new insights into the organization and regulation of our genes and genome, and is an expansive resource of functional annotations for biomedical research., Author(s): The ENCODE Project Consortium; Overall coordination (data analysis coordination); Ian Dunham [2]; Anshul Kundaje [3, 82]; Data production leads (data production); Shelley F. Aldred [4]; Patrick J. Collins [4]; [...]

The vast non-coding portion of the human genome is full of functional elements and disease-causing regulatory variants. The principles defining the relationships between these elements and distal target genes remain [...]

In eukaryotes transcriptional regulation often involves multiple long-range elements and is influenced by the genomic environment (1). A prime example of this concerns the mouse X-inactivation centre (Xic), which orchestrates the initiation of X-chromosome inactivation (XCI) by controlling the expression of the nonprotein-coding Xist transcript. The extent of Xic sequences required for the proper regulation of Xist remains unknown. Here we use chromosome conformation capture carbon-copy (5C) (2) and super-resolution microscopy to analyse the spatial organization of a 4.5-megabases (Mb) region including Xist.We discover a series of discrete 200-kilobase to 1 Mb topologically associating domains (TADs), present both before and after cell differentiation and on the active and inactive X. TADs align with, but do not rely on, several domain-wide features of the epigenome, such as H3K27me3 or H3K9me2 blocks and lamina-associated domains. TADs also align with coordinately regulated gene clusters. Disruption of a TAD boundary causes ectopic chromosomal contacts and long-range transcriptional misregulation. The Xist/Tsix sense/antisense unit illustrates how TADs enable the spatial segregation of oppositely regulated chromosomal neighbourhoods, with the respective promoters of Xist and Tsix lying in adjacent TADs, each containing their known positive regulators. We identify a novel distal regulatory region of Tsix within its TAD, which produces a long intervening RNA, Linx. In addition to uncovering a new principle of cis-regulatory architecture of mammalian chromosomes, our study sets the stage for the full genetic dissection of the X-inactivation centre., The X-inactivation centre was originally defined by deletions and translocations as a region spanning several megabases (3,4), and contains several elements known to affect Xist activity, including its repressive antisense [...]

The genome is extensively transcribed into long intergenic non-coding RNAs (lincRNAs), many of which are implicated in gene silencing (1,2). Potential roles of lincRNAs in gene activation are much less understood (3-5). Development and homeostasis require coordinate regulation of neighbouring genes through a process termed locus control (6). Some locus control elements and enhancers transcribe lincRNAs (7-10), hinting at possible roles in long-range control. In vertebrates, 39 Hox genes, encoding homeodomain transcription factors critical for positional identity, are clustered in four chromosomal loci; the Hox genes are expressed in nested anterior-posterior and proximal-distal patterns colinear with their genomic position from 3' to 5'of the cluster (11). Here we identify HOTTIP, alincRNA transcribed from the 5' tip of the HOXA locus that coordinates the activation of several 5' HOXA genes in vivo. Chromosomal looping brings HOTTIP into close proximity to its target genes. HOTTIP RNA binds the adaptor protein WDR5 directly and targets WDR5/MLL complexes across HOXA, driving histone H3 lysine 4 trimethylation and gene transcription. Induced proximity is necessary and sufficient for HOTTIP RNA activation of its target genes. Thus, by serving as key intermediates that transmit information from higher order chromosomal looping into chromatin modifications, lincRNAs may organize chromatin domains to coordinate long-range gene activation., We examined chromosome structure and histone modifications in human primary fibroblasts derived from several anatomic sites (12), and found distinctive differences in the HOXA locus. High throughput chromosome conformation capture [...]

An Amendment to this paper has been published and can be accessed via a link at the top of the paper., Author(s): Martin Falk [sup.1] , Yana Feodorova [sup.2] [sup.3] , Natalia Naumova [sup.4] [sup.5] , Maxim Imakaev [sup.1] , Bryan R. Lajoie [sup.4] [sup.6] , Heinrich Leonhardt [sup.3] , Boris [...]

The genome is extensively transcribed into long intergenic noncoding RNAs (lincRNAs), many of which are implicated in gene silencing. Potential roles of lincRNAs in gene activation are much less understood. Development and homeostasis require coordinate regulation of neighbouring genes through a process termed locus control. Some locus control elements and enhancers transcribe lincRNAs, hinting at possible roles in long-range control. In vertebrates, 39 Hox genes, encoding homeodomain transcription factors critical for positional identity, are clustered in four chromosomal loci; the Hox genes are expressed in nested anterior-posterior and proximal-distal patterns colinear with their genomic position from 3′ to 5′of the cluster. Here we identify HOTTIP, a lincRNA transcribed from the 5′ tip of the HOXA locus that coordinates the activation of several 5′ HOXA genes in vivo. Chromosomal looping brings HOTTIP into close proximity to its target genes. HOTTIP RNA binds the adaptor protein WDR5 directly and targets WDR5/MLL complexes across HOXA, driving histone H3 lysine 4 trimethylation and gene transcription. Induced proximity is necessary and sufficient for HOTTIP RNA activation of its target genes. Thus, by serving as key intermediates that transmit information from higher order chromosomal looping into chromatin modifications, lincRNAs may organize chromatin domains to coordinate long-range gene activation. [ABSTRACT FROM AUTHOR]