This study investigates the spatial organization of the X-inactivation centre (Xic) in mice, revealing that it is partitioned into topologically associating domains (TADs). Using 5C and super-resolution microscopy, the researchers analyzed a 4.5 Mb region containing Xist and found that TADs are present both before and after cell differentiation, on the active and inactive X chromosomes. These TADs align with, but do not rely on, epigenetic features such as H3K27me3 or H3K9me2 blocks and lamina-associated domains. TADs also align with coordinately regulated gene clusters, and disrupting a TAD boundary leads to 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. A novel distal regulatory region of Tsix within its TAD produces a long intervening RNA, Linx. The study also shows that TADs are not determined by domain-wide histone modifications, but rather by spatial partitioning of chromosomes into TADs. The Xic was originally defined by deletions and translocations, and contains several elements known to affect Xist activity, including its repressive antisense transcript Tsix and its regulators. 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 regulation. The study also shows that TAD organization is conserved during differentiation and XCI, and that the inactive X has a more random chromosomal organization than its active homologue. The study further reveals that TADs may be used to coordinate gene expression patterns during development, and that TAD boundaries can have a critical role in high-order chromatin folding and proper long-range transcriptional control. The study also identifies a new principle of cis-regulatory architecture of mammalian chromosomes, and sets the stage for the full genetic dissection of the X-inactivation centre. The findings suggest that TADs may underlie regulatory domains previously proposed on the basis of functional and synteny conservation studies. The study also highlights the role of Linx in the long-range transcriptional regulation of Tsix, either through its chromosomal association with Xite and/or via the RNA it produces. The study provides new insights into the cis-regulatory architecture of chromosomes that orchestrates transcriptional dynamics during development, and paves the way to dissecting the constellation of control elements of Xist and its regulators within the Xic.This study investigates the spatial organization of the X-inactivation centre (Xic) in mice, revealing that it is partitioned into topologically associating domains (TADs). Using 5C and super-resolution microscopy, the researchers analyzed a 4.5 Mb region containing Xist and found that TADs are present both before and after cell differentiation, on the active and inactive X chromosomes. These TADs align with, but do not rely on, epigenetic features such as H3K27me3 or H3K9me2 blocks and lamina-associated domains. TADs also align with coordinately regulated gene clusters, and disrupting a TAD boundary leads to 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. A novel distal regulatory region of Tsix within its TAD produces a long intervening RNA, Linx. The study also shows that TADs are not determined by domain-wide histone modifications, but rather by spatial partitioning of chromosomes into TADs. The Xic was originally defined by deletions and translocations, and contains several elements known to affect Xist activity, including its repressive antisense transcript Tsix and its regulators. 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 regulation. The study also shows that TAD organization is conserved during differentiation and XCI, and that the inactive X has a more random chromosomal organization than its active homologue. The study further reveals that TADs may be used to coordinate gene expression patterns during development, and that TAD boundaries can have a critical role in high-order chromatin folding and proper long-range transcriptional control. The study also identifies a new principle of cis-regulatory architecture of mammalian chromosomes, and sets the stage for the full genetic dissection of the X-inactivation centre. The findings suggest that TADs may underlie regulatory domains previously proposed on the basis of functional and synteny conservation studies. The study also highlights the role of Linx in the long-range transcriptional regulation of Tsix, either through its chromosomal association with Xite and/or via the RNA it produces. The study provides new insights into the cis-regulatory architecture of chromosomes that orchestrates transcriptional dynamics during development, and paves the way to dissecting the constellation of control elements of Xist and its regulators within the Xic.