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Deletion of DXZ4 on the human inactive X chromosome eliminates superdomains and impairs gene silencing

ABSTRACT: During interphase, the inactive X chromosome (Xi) adopts an unusual 3D configuration known as the Barr body and is largely transcriptionally silent. Despite the importance of X inactivation, little is known about the 3D configuration of Xi and its relationship to gene silencing. We recently showed that in humans, Xi exhibits two distinctive structural features. First, Xi is partitioned into two huge intervals, called superdomains, such that pairs of loci in each superdomain show an enhanced contact frequency with one another. The boundary between the two superdomains lies near DXZ4, a macrosatellite repeat spanning ~300kb, whose Xi allele extensively binds the protein CTCF. Second, Xi exhibits extremely large loops, up to 77Mb long, called superloops. DXZ4 lies at the anchor of several superloops. Here, we use 3D mapping to study the structure of Xi, focusing on the role of DXZ4. We show that superloops and superdomains are conserved across mammals. We develop a novel variant of our in situ Hi-C protocol, dubbed COLA (COncatemer Ligation Assay) to probe the higher order structures formed by the superloops. In COLA, in situ proximity ligation of multiple extremely short fragments produced by the enzyme CviJI is used to efficiently map simultaneous proximity among three or more loci. Using data from Hi-C and COLA, we demonstrate that DXZ4 and other superloop anchors tend to co-locate simultaneously within the same cells, a result that is confirmed by 3D-FISH. Finally, we examine the effects of deleting DXZ4 from Xi in human cells. Using in situ Hi-C, microscopy, and RNA-FISH, we show that superdomains and superloops disappear; that Xi frequently dissociates into multiple separate structures; and that transcriptional silencing on Xi is compromised. Deletion of DXZ4 from the active X chromosome (Xa) has no such effect. Thus, DXZ4 is essential for proper folding and silencing of Xi.

ORGANISM(S): Mus musculus Homo sapiens Macaca mulatta

PROVIDER: GSE71831 | GEO | 2016/07/19

SECONDARY ACCESSION(S): PRJNA292502

REPOSITORIES: GEO

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Project description:During interphase, the inactive X chromosome (Xi) adopts an unusual 3D configuration known as the Barr body and is largely transcriptionally silent. Despite the importance of X inactivation, little is known about the 3D configuration of Xi and its relationship to gene silencing. We recently showed that in humans, Xi exhibits two distinctive structural features. First, Xi is partitioned into two huge intervals, called superdomains, such that pairs of loci in each superdomain show an enhanced contact frequency with one another. The boundary between the two superdomains lies near DXZ4, a macrosatellite repeat spanning ~300kb, whose Xi allele extensively binds the protein CTCF. Second, Xi exhibits extremely large loops, up to 77Mb long, called superloops. DXZ4 lies at the anchor of several superloops. Here, we use 3D mapping to study the structure of Xi, focusing on the role of DXZ4. We show that superloops and superdomains are conserved across mammals. We develop a novel variant of our in situ Hi-C protocol, dubbed COLA (COncatemer Ligation Assay) to probe the higher order structures formed by the superloops. In COLA, in situ proximity ligation of multiple extremely short fragments produced by the enzyme CviJI is used to efficiently map simultaneous proximity among three or more loci. Using data from Hi-C and COLA, we demonstrate that DXZ4 and other superloop anchors tend to co-locate simultaneously within the same cells, a result that is confirmed by 3D-FISH. Finally, we examine the effects of deleting DXZ4 from Xi in human cells. Using in situ Hi-C, microscopy, and RNA-FISH, we show that superdomains and superloops disappear; that Xi frequently dissociates into multiple separate structures; and that transcriptional silencing on Xi is compromised. Deletion of DXZ4 from the active X chromosome (Xa) has no such effect. Thus, DXZ4 is essential for proper folding and silencing of Xi. Hi-C protocol was used on wildtype and DXZ4-deleted cells to examine the structure of Xi. A novel variant of our in situ Hi-C protocol, dubbed COLA (COncatemer Ligation Assay), was developed to probe the higher order structures formed by the superloops. This series also includes RNA-seq data on Retinal Pigmented Epithelial Cells (hTERT-RPE1). At the time of submission, processed data were available only for the RNA-seq samples. Submitter states that processed data files for HiC samples will be added to this series in the future.

Project description:A subset of genomic regions, including one of the female X chromosomes and all imprinted genes, are expressed exclusively from a single allele in somatic cells of mammals. To evaluate structural changes associated with allelic silencing, we used mouse F1 hybrid systems in which X inactivation is skewed and alleles can be distinguished based on single nucleotide polymorphisms to analyze chromatin contacts by a new Hi-C assay that uses DNase I for chromatin fragmentation. Both DNase Hi-C and in situ DNase Hi-C reveal a radically different conformation between the two female mouse X chromosomes. Two superdomains of frequent intrachromosomal contacts separated by a hinge region are specifically observed on the inactive X chromosome both in vivo (brain) and in vitro (Patski cells). A bipartite 3D organization of the inactive X has been also reported in human lymphoblastoid cells, suggesting that this conserved structure probably plays an important role in maintenance of gene silencing on the inactive X. Interestingly, we found that the genomic content of the superdomains differs between species, but that part of the hinge region is conserved and located near the Dxz4/DXZ4 locus that binds CTCF on the inactive X. In mouse, the hinge region also contains a minisatellite Ds-TR adjacent to a promoter with strong CTCF binding. Both Dxz4 and Ds-TR bind nucleophosmin and are enriched in nucleolus-associated chromatin, suggesting anchoring to the nucleolus. Genes that escape X inactivation do not cluster but are preferentially located near the periphery of the 3D structure. Regions enriched in CTCF or RNA polymerase are also preferentially located on the periphery of the inactive X while LINE1 elements are preferentially on the interior. Genes subject to silencing exhibit fewer detectable intrachromosomal contacts than escape genes. This transcription-coupled pattern is also evident for imprinted genes, in which more chromatin contacts are detected on the expressed allele, suggesting greater constraint on the organization of expressed genomic regions.

Project description:One of the two X chromosomes in female somatic cells is transcriptionally silenced across cell generations, a classic paradigm of epigenetic regulation. Although most genes are stably silenced, certain X-linked genes escape X-chromosome inactivation (XCI), providing a fundamental dosage difference between females and males. A role for chromosome conformation has been proposed in XCI as the process is accompanied by a massive structural reorganisation. However a detailed molecular understanding of the three-dimensional architecture of the Xi has been lacking. Here we reveal an unusual three-dimensional configuration of the Xi, that provides novel insights into the relationship between gene expression and TAD organisation. Using allele-specific Hi-C, RNA-Seq and ATAC-seq, as well as DNA FISH, we show that the Xi is spatially segregated into two ‘mega-domains’ separated by a 200kb boundary including the DXZ4 macrosatellite, and is globally devoid of typical autosomal structural features such as active/inactive compartments and topologically associating domains (TADs). However, we find that a few regions along the Xi display TAD signatures corresponding to regions containing escape genes, and display DNA accessibility at promoter-proximal TF binding sites. Strikingly, when the DXZ4-containing boundary region is deleted prior to XCI, most facultative escapees are no longer expressed from the Xi, and the escape associated chromatin domains are no longer observed. Deletion of the DXZ4-containing boundary region from the Xi also results in fusion of the two mega-domains. We show that Xist A-repeat containing (gene silencing competent) RNA is sufficient to induce this spatial segregation of the X chromosome. Combined, our results point to a critical role for the DXZ4-boundary region, together with Xist RNA, in shaping the higher order structure of the Xi and in allowing facultative escape from XCI during differentiation. They also point to direct and uncover a close relationships between transcription and TAD formation organisation in the context of the Xi.

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Deletion of DXZ4 on the human inactive X chromosome eliminates superdomains and impairs gene silencing

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