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

X chromosome inactivation (XCI) triggers drastic reorganization of chromatin architecture, which in turn facilitates XCI. However, how the 3D organization is de novo established during XCI in vivo remains poorly understood. Mouse embryos present an ideal model, as they undergo imprinted XCI, X chromosome reactivation (XCR) and subsequent random XCI. Using low-input Hi-C, we revealed a unique chromatin structure of the inactive X chromosome (Xi) during both imprinted and random XCI in early mouse embryos. A bipartite structure featured by two megadomains separated by Xist (X-megadomains), emerges in extraembryonic lineages, derived extraembryonic endoderm (XEN) cells, and also transiently in embryonic lineages. X-megadomains then diminish in adult tissues, where previously reported Dxz4-delineated megadomains (D-megadomains) appear in a strain-specific manner. Mechanistically, X-megadomains coincide with developmentally controlled activities of regulatory regions near Xist (XRRs), and loss of XRR disrupts X-megadomains. CUT&RUN revealed strong enrichment of CTCF and cohesin near Xist, despite global depletion, on Xi in embryos and XEN cells. X-megadomains are also impaired when Xist is silenced, accompanied by global cohesin restoration, or when residual cohesin is degraded, suggesting that X-megadomain formation is promoted by both global cohesin depletion and local cohesin loading. Importantly, the loss of cohesin leads to ectopic activation of regulatory elements and genes near Xist. Hence, these data reveal programming of chromatin architecture during de novo establishment of mammalian XCI in vivo, and suggest that cohesin mediates self-insulation of gene activity of essential escapees amid global silencing. Overall design: Small scale in situ high throughput chromatin conformation capture (sisHi-C) for WT and mutant XEN cells

X染色体失活（X chromosome inactivation, XCI）会引发染色质三维结构的剧烈重塑，进而促进XCI进程。然而，在体内XCI过程中，三维基因组组织如何从头建立，目前仍有待深入阐明。小鼠胚胎是研究该科学问题的理想模型，因其先后经历印记型XCI、X染色体激活（X chromosome reactivation, XCR）以及后续的随机XCI过程。本研究借助低起始量Hi-C测序技术，在小鼠早期胚胎的印记型与随机XCI过程中，均揭示了失活X染色体（inactive X chromosome, Xi）独特的染色质结构特征。
在胚外谱系、诱导生成的胚外内胚层（XEN）细胞中，以及胚胎谱系的瞬时阶段，均出现了由Xist分隔形成的两个巨型结构域构成的二分体结构（X-巨型结构域，X-megadomains）。后续X-巨型结构域在成体组织中逐渐消失，取而代之的是以品系特异性方式出现的、此前已有报道的由Dxz4界定的巨型结构域（D-megadomains）。
从机制层面分析，X-巨型结构域与Xist邻近调控区域（Xist adjacent regulatory regions, XRRs）的发育调控活性高度重合，且XRRs的缺失会破坏X-巨型结构域的形成。CUT&RUN实验结果显示，尽管胚胎与XEN细胞的Xi整体黏连蛋白（cohesin）存在全局耗竭，但Xist邻近区域却显著富集CCCTC结合因子（CTCF）与黏连蛋白。当Xist被沉默时，X-巨型结构域的形成会受到损伤，同时伴随黏连蛋白的全局恢复；当残留黏连蛋白被降解时，同样会出现X-巨型结构域受损的表型，这表明X-巨型结构域的形成既依赖于全局黏连蛋白耗竭，也离不开局部黏连蛋白的装载。
值得关注的是，黏连蛋白的缺失会导致Xist邻近的调控元件与基因发生异位激活。综上，本研究揭示了哺乳动物体内XCI从头建立过程中染色质架构的编程模式，并表明黏连蛋白可介导全局沉默背景下关键逃逸基因的基因活性自我绝缘。
总体实验设计：针对野生型与突变型XEN细胞开展小规模原位高通量染色质构象捕获（sisHi-C）实验。