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.

Summary The molecular mechanisms underlying folding of mammalian chromosomes remain poorly understood. The transcription factor CTCF is a candidate regulator of chromosomal structure. Using the auxin-inducible degron system in mouse embryonic stem cells, we show that CTCF is absolutely and dose-dependently required for looping between CTCF target sites and insulation of topologically associating domains (TADs). Restoring CTCF reinstates proper architecture on altered chromosomes, indicating a powerful instructive function for CTCF in chromatin folding. CTCF remains essential for TAD organization in non-dividing cells. Surprisingly, active and inactive genome compartments remain properly segregated upon CTCF depletion, revealing that compartmentalization of mammalian chromosomes emerges independently of proper insulation of TADs. Further, our data support that CTCF mediates transcriptional insulator function through enhancer-blocking but not as a direct barrier to heterochromatin spreading. Beyond defining the functions of CTCF in chromosome folding these results provide new fundamental insights into the rules governing mammalian genome organization.

Summary A new level of chromosome organization, Topologically Associating Domains (TADs), was recently uncovered by chromosome-confirmation-capture (3C) techniques. To explore TAD structure and function, we developed a polymer model that can extract the full repertoire of chromatin conformations within TADs from population-based 3C data. This model predicts actual physical distances and to what extent chromosomal contacts vary between cells. It also identifies interactions within single TADs that stabilize boundaries between TADs and allows us to identify and genetically validate key structural elements within TADs. Combining the model’s predictions with high-resolution DNA FISH and quantitative RNA FISH for TADs within the X-inactivation center (Xic), we dissect the relationship between transcription and spatial proximity to cis-regulatory elements. We demonstrate that contacts between potential regulatory elements occur in the context of fluctuating structures rather than stable loops and propose that such fluctuations may contribute to asymmetric expression in the Xic during X inactivation.