Summary The mouse X-inactivation center (Xic) orchestrates initiation of X inactivation by controlling the expression of the non-coding Xist transcript. The full extent of Xist’s regulatory landscape remains to be defined however. Here we use Chromosome Conformation Capture Carbon-Copy and super-resolution microscopy to analyse the spatial organisation of a 4.5Mb region including Xist. We uncover a series of discrete 200kb-1Mb topologically associating domains (TADs), present both before and after cell differentiation and on the active and inactive X. These domains align with several domain-wide epigenomic features as well as co-regulated gene clusters. Disruption of a TAD boundary causes ectopic chromosomal contacts and long-range transcriptional mis-regulation. Xist/Tsix illustrates the spatial segregation of oppositely regulated chromosomal neighborhoods, with their promoters lying in two adjacent TADs, each containing their known positive regulators. This led to the identification of a distal regulatory region of Tsix producing a novel long intervening RNA, Linx, within its TAD. In addition to uncovering a new principle of the cis-regulatory architecture of mammalian chromosomes, our study sets the stage for the full genetic dissection of the Xic.

Cellular senescence is an extremely stable form of cell cycle arrest that limits the proliferation of damaged cells and may act as a natural barrier to cancer progression. In this study, we describe a distinct heterochromatic structure that accumulates in senescent human fibroblasts, which we designated senescence-associated heterochromatic foci (SAHF). SAHF formation coincides with the recruitment of heterochromatin proteins and the retinoblastoma (Rb) tumor suppressor to E2F-responsive promoters and is associated with the stable repression of E2F target genes. Notably, both SAHF formation and the silencing of E2F target genes depend on the integrity of the Rb pathway and do not occur in reversibly arrested cells. These results provide a molecular explanation for the stability of the senescent state, as well as new insights into the action of Rb as a tumor suppressor.