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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 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 1Mb 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.Weidentify a novel distal regulatory region of Tsix within its TAD, which produces a long intervening RNA, Linx. In addition to uncovering a new principle ofcis-regulatory architecture ofmammalian 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 megabases3,4, and contains several elements known to affect Xist activity, including its repressive antisense transcript Tsix and its regulators Xite, DXPas34 and Tsx5,6. However, additional control elements must exist, as single-copy transgenes encompassing Xist and up to 460 kb of flanking sequences are unable to recapitulate proper Xist regulation7. To characterize the cisregulatory landscape of the Xic in an unbiased approach, we performed 5C2 across a 4.5-Mb region containing Xist. We designed 5C-Forward and 5C-Reverse oligonucleotides following an alternating scheme2, thereby simultaneously interrogating nearly 250,000 possible chromosomal contacts in parallel, with a mean resolution of 10-20 kb (Fig. 1a; see Supplementary Methods). Analysis of undifferentiated mouse embryonic stem cells (ESCs) revealed that long-range (.50 kb) contacts preferentially occur within a series of discrete genomic blocks, each covering 0.2-1Mb (Fig. 1b). These blocks...