The bipartite TAD organization of the X-inactivation centre ensures opposing developmental regulation of Tsix and Xist

The mouse X-inactivation center (Xic) locus represents a powerful model for understanding the links between genome architecture and gene regulation, with the non-coding genes Xist and Tsix showing opposite developmental expression patterns while being organized as an overlapping sense/antisense unit. The Xic is organized into two topologically associating domains (TADs) but the role of this architecture in orchestrating cis-regulatory information remains elusive. To explore this, we generated genomic inversions within the Xic that swap the Xist/Tsix transcriptional unit and place their promoters in each other's TAD. We found that this led to a switch in their expression dynamics: Xist became precociously and ectopically up-regulated, both in male and female pluripotent cells, while Tsix expression aberrantly persisted during differentiation. The topological partitioning of the Xic is thus critical to ensure proper developmental timing of X-inactivation. Our study illustrates how genomic architecture can hardwire the potential for mammalian developmental regulation by cis-regulatory landscapes. Overall design: Genomic deletions and inversions were genereated in the Xic of mouse male ES cells (E14Tg2a) and female ES cells (PGK#106, PGK12.1-derived). Consequences of these inversions were analysed using high-throughput Chromosome Conformation Capture (5C and Capture-C), RNAseq and RAP.