Stepwise de novo establishment of inactive X chromosome architecture in early development [Embryo genomic DNA]. Stepwise de novo establishment of inactive X chromosome architecture in early development [Embryo genomic DNA]

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: Genomic DNA libraries for various genotypes of morula embryos.

X染色体失活（X chromosome inactivation, XCI）可引发基因活性与染色体三维构象的剧烈重编程。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，目前仍未得到充分解析。本研究通过覆盖发育阶段与细胞谱系特异性的全面Hi-C作图分析，在小鼠早期胚胎的失活X染色体上鉴定出一种独特的、由X失活特异性转录本（Xist）分隔的超大结构域（X-megadomains）。X超大结构域（X-megadomains）首先在印记XCI过程中的胚外谱系中出现，可稳定存在于胚外内胚层（extraembryonic endoderm, XEN）细胞中；在随机XCI过程的胚胎谱系中则会暂时性出现，且早于后续以菌株特异性方式形成的、由Dxz4界定的超大结构域（Dxz4-delineated megadomains, D-megadomains）。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（Xist adjacent regulatory region, XRR）内发育调控的增强子活性及黏连蛋白（cohesin）结合的时序高度契合。本研究精准定位到其中一个亚区域XRRa，其对X超大结构域的边界形成至关重要。当在XEN细胞中敲除XRRa或降解黏连蛋白时，X超大结构域的形成会受到显著损伤。重要的是，这一现象同时伴随Xist邻近调控元件与基因的异位激活，表明黏连蛋白在调控元件处的装载可促进X超大结构域的形成，并在XCI早期的全局基因沉默背景下，限制局部基因的活性范围。进一步实验发现，在小鼠植入前胚胎中敲除XRRa会严重损伤Xist的激活与XCI的起始。综上，本研究不仅揭示了体内从头XCI过程中染色体折叠的逐步动态变化，同时支持了如下模型：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，并在全局沉默的背景下介导后续的自我绝缘。总体实验设计：针对不同基因型的桑椹胚胚胎构建基因组DNA文库。