In eukaryotes transcriptional regulation often involves multiple long-range elements and is influenced by the genomic environment1. 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 non-protein-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.
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We thank T. Pollex and T. Forné for experimental help; the imaging facility PICTIBiSA@BDD for technical assistance, D. Gentien and C. Hego for microarray hybridizations. We thank K. Bernhard, F. Stewart and A. Smith for protocols and material for 2i culture and EpiSC differentiation. We are grateful to members of the E.H. laboratory for critical input. This work was funded by grants from the Ministère de la Recherche et de l’Enseignement Supérieur and the ARC (to E.P.N.); a HFSP Long term fellowship (LT000597/2010-L) (to E.G.S.). EU EpiGeneSys FP7 Network of Excellence no. 257082, the Fondation pour la Recherche Medicale, ANR, ERC Advanced Investigator award no. 250367 and EU FP7 SYBOSS grant no. 242129 (to E.H.). N.B. was supported by BMBF (FORSYS) and EMBO (fellowship ASTF 307-2011). J.D., B.R.L. and N.L.v.B. were supported by NIH (R01 HG003143) and a W. M. Keck Foundation Distinguished Young Scholar Award.
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Current Genetics (2019)