Structural organization of the inactive X chromosome

X-chromosome inactivation (XCI) entails a massive structural reorganization of the inactive X (Xi). However the molecular architecture of the Xi is unknown. Here we show that the Xi lacks typical autosomal features such as active/inactive compartments and topologically associating domains (TADs), except around a small number of genes that escape XCI and remain expressed. Escaping genes form TADs and retain DNA accessibility at promoter-proximal and CTCF binding sites, indicating that these loci can avoid Xist-mediated erasure of chromosomal structure. We further show that genesilencing competent Xist RNA is sufficient to induce segregation of the Xi into two ‘mega-domains’ separated by a boundary that includes the DXZ4 macrosatellite. Deletion of this boundary prior to XCI results in fusion of the megadomains and altered patterns of escape that correlate with changes in TAD structure following differentiation and XCI . Our results suggest a critical role for the boundary locus and Xist RNA in shaping the structure of the Xi and modulating escape from XCI. Our findings also point to roles of transcription and CTCF binding in TAD formation in the context of facultative heterochromatin. Allele specific Hi-C, Allele specific ATAC-seq and Allele specific RNA-seq. Mouse 129/cast female. Processed data for two reanalyzed RNA-seq Samples, GSM1305895 and GSM1305900, are included in this study and are linked to the Series record, GSE72697_rpkm_bedGraph_ESC.tar.gz and GSE72697_rpkm_bedGraph_NPC-deltaFT.tar.gz.