The study investigates the spatial organization of the regulatory landscape of the X-inactivation center (Xic) in mice, focusing on the 4.5-Mb region containing the non-protein-coding Xist transcript. Using chromosome conformation capture carbon-copy (5C) and super-resolution microscopy, the researchers discovered a series of discrete topologically associating domains (TADs) ranging from 200 kb to 1 Mb. These TADs are present before and after cell differentiation and on both active and inactive X chromosomes. The TADs align with epigenomic features such as H3K27me3 and H3K9me2 blocks but do not rely on them. They also align with coordinately regulated gene clusters. Disruption of TAD boundaries leads to ectopic chromosomal contacts and long-range transcriptional misregulation. The study highlights how TADs enable the spatial segregation of oppositely regulated chromosomal neighborhoods, with the Xist and Tsx promoters lying in adjacent TADs. Additionally, a novel distal regulatory region of Tsx within its TAD produces a long intervening RNA, Linx, which may play a role in long-range transcriptional regulation. The findings provide new insights into the cis-regulatory architecture of mammalian chromosomes and set the stage for further dissection of the X-inactivation center.The study investigates the spatial organization of the regulatory landscape of the X-inactivation center (Xic) in mice, focusing on the 4.5-Mb region containing the non-protein-coding Xist transcript. Using chromosome conformation capture carbon-copy (5C) and super-resolution microscopy, the researchers discovered a series of discrete topologically associating domains (TADs) ranging from 200 kb to 1 Mb. These TADs are present before and after cell differentiation and on both active and inactive X chromosomes. The TADs align with epigenomic features such as H3K27me3 and H3K9me2 blocks but do not rely on them. They also align with coordinately regulated gene clusters. Disruption of TAD boundaries leads to ectopic chromosomal contacts and long-range transcriptional misregulation. The study highlights how TADs enable the spatial segregation of oppositely regulated chromosomal neighborhoods, with the Xist and Tsx promoters lying in adjacent TADs. Additionally, a novel distal regulatory region of Tsx within its TAD produces a long intervening RNA, Linx, which may play a role in long-range transcriptional regulation. The findings provide new insights into the cis-regulatory architecture of mammalian chromosomes and set the stage for further dissection of the X-inactivation center.