Stepwise de novo establishment of inactive X chromosome architecture in early development [XEN cells RNA-seq]

X chromosome inactivation (XCI) triggers a drastic reprogramming of gene activities and chromosome architecture. However, how the 3D organization of the inactive X chromosome (Xi) is de novo established in vivo in mammals remains poorly understood. By comprehensive stage- and lineage- specific Hi-C mapping, we identified a unique Xist-separated megadomain structure (X-megadomains) on the Xi in mouse early embryos. X-megadomains emerge in extraembryonic lineages during imprinted XCI, in derived extraembryonic endoderm (XEN) cells, and transiently in the embryonic lineages during random XCI, before Dxz4-delineated megadomains (D-megadomains) occur at later stages in a strain-specific manner. Mechanistically, the emergence of X-megadomain boundary coincides with developmentally regulated enhancer activities and cohesin binding in a regulatory region near Xist (XRR). We pinpointed a subregion XRRa that is critical for the X-megadomain boundary. X-megadomains are impaired when XRRa is removed or cohesin is degraded in XEN cells. Importantly, this is accompanied by ectopic activation of regulatory elements and genes near Xist, suggesting that cohesin loading at regulatory elements promotes X-megadomains and confines local gene activities. Finally, the knockout of XRRa in mouse preimplantation embryos severely impairs the activation of Xist and the initiation of XCI. Hence, our data not only reveal stepwise chromosome folding during de novo XCI in vivo, but also support a model that regulatory element-dependent gene activation and cohesin loading simultaneously promote essential transcription activities and subsequent self-insulation amid global silencing during the early stage of XCI. Overall design: RNA-seq data for WT and mutant XEN cells Please note that, in the *allelic_FPKM.txt.gz files, the "_Xa" and "_Xi" columns contain the FPKM values of genes in the active X chromosome and in the inactive X chromosome, respectively.