Stepwise de novo establishment of inactive X chromosome architecture in early development [XEN cells CUT&RUN]

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. CUT&RUN for CTCF and SMC3 in WT and mutant XEN cells

X染色体失活（X chromosome inactivation, XCI）会引发基因活性与染色体构象的剧烈重编程。然而，哺乳动物体内失活X染色体（inactive X chromosome, Xi）的三维组织结构如何从头建立，目前仍未完全阐明。本研究借助全面的阶段特异性与谱系特异性Hi-C（高通量染色体构象捕获，High-throughput Chromosome Conformation Capture）图谱分析，在小鼠早期胚胎的失活X染色体上鉴定出一种独特的、由Xist分隔的超大结构域（X-megadomains）。X超大结构域在印记型XCI过程中的胚外谱系中出现，也存在于衍生的胚外内胚层（extraembryonic endoderm, XEN）细胞中，并在随机型XCI过程中的胚胎谱系中瞬时出现，早于以Dxz4为边界的超大结构域（Dxz4-delineated megadomains, D-megadomains）在后续阶段以品系特异性方式形成。从机制层面来看，X超大结构域边界的形成，与Xist邻近调控区域（XRR）内发育调控的增强子活性及黏连蛋白（cohesin）结合的时序高度吻合。我们精准定位到一个对X超大结构域边界至关重要的亚区域XRRa。当在XEN细胞中敲除XRRa或降解黏连蛋白时，X超大结构域的结构会受到显著破坏。尤为关键的是，这一结构破坏现象伴随Xist邻近区域调控元件与基因的异位激活，这表明黏连蛋白在调控元件上的装载可促进X超大结构域的形成，并在全局基因沉默过程中限制局部的基因活性。最终，在小鼠植入前胚胎中敲除XRRa会严重损害Xist的激活与XCI的起始进程。因此，本研究的数据不仅揭示了体内从头XCI过程中染色体折叠的阶段性特征，还支持了如下模型：在XCI早期阶段，依赖调控元件的基因激活与黏连蛋白装载，可同时促进必要的转录活性，并在全局沉默过程中促成后续的自我绝缘。对野生型（wild type, WT）与突变型XEN细胞开展CTCF（CCCTC结合因子，CCCTC-binding factor）与SMC3（结构维持蛋白3，Structural Maintenance of Chromosomes 3）的CUT&RUN（染色质靶向切割与释放，Cleavage Under Targets & Release Using Nuclease）实验。