安诺Hi-C携手法国居里研究所刊发Nature文章揭示X染色体沉默之谜

X染色体失活是指雌性哺乳类细胞中两条X染色体中一条失去活性的现象，失活的X染色体在三维结构上到底发生了怎样的变化，导致大部分基因发生沉默？近日，安诺优达携法国居里研究所破译了失活X染色体的三维结构，为研究基因组三维结构和基因表达之间关系提供了强大独特的模型系统 。

　　

X染色体失活是指雌性哺乳类细胞中两条X染色体中一条失去活性的现象，如女性细胞中两条X染色体中一条的基因发生了沉默，女性胚胎中多出一条功能性X染色体可以导致早期发育阶段死亡。长达2m的染色体是经过复杂折叠、形成严密的三维结构，“安置”在直径7um左右的细胞核中；失活的X染色体在三维结构上到底发生了怎样的变化，导致大部分基因发生沉默？近日，安诺优达携法国居里研究所破译了失活X染色体的三维结构，为研究基因组三维结构和基因表达之间关系提供了强大独特的模型系统 。

为了研究失活X染色体的结构，选取来源于高度多态性F1代小鼠胚胎干细胞的神经前体细胞做等位基因特异的Hi-C分析。首先，在胚胎干细胞中做Hi-C分析，ES细胞中还没有发生X染色体沉默；发现活跃的X染色体含有明显的活跃、失活区室化（compartment A/B），也没有拓扑结构域结构（TAD）（图1 ）。

图1 胚胎干细胞染色质三维结构特征

索取安诺优达单细胞基因组和转录组平行测序详细技术资料

但是，在失活的X染色体中没有A/B compartment（图1），而是由含有DXZ4微卫星的200kb铰链区域分割成两个大的相互作用区域；此外，在失活的X染色体中也没有拓扑结构域（TADs）（图2）。

图2 失活的X染色体结构特征

在未分化的雄性小鼠ES细胞中诱导Xist表达，Hi-C分析发现诱导Xist48小时导致X染色体结构发生明显变化（图 3a-b）；但是在不含A重复区域的Xist突变样品中，基因不发生沉默，染色体三维结构没有变化。野生型Xist诱导不会导致TAD结构的显著改变，但是会导致染色体相互作用频率的增加（图3d），Mb级别的结构域分割边界用RNA/DNA FISH验证是正确的（图3c）。

图3 Xist在失活X染色体特殊的三维结构重塑中的作用

这项研究揭示了X染色体沉默的机制，为研究基因组空间组织和基因表达之间关系提供了强大独特的模型系统，同时有助于开发新的方法，对抗女性X染色体相关的疾病，促进相关疾病的研究与治疗。

原文摘要：

Structural organization of the inactive X chromosome in the mouse

X-chromosome inactivation (XCI) involves major reorganization of the X chromosome as it becomes silent and heterochromatic. During female mammalian development, XCI is triggered by upregulation of the non-coding Xist RNA from one of the two X chromosomes. Xist coats the chromosome in cis and induces silencing of almost all genes via its A-repeat region1, 2, although some genes (constitutive escapees) avoid silencing in most cell types, and others (facultative escapees) escape XCI only in specific contexts3. A role for Xist in organizing the inactive X (Xi) chromosome has been proposed4, 5, 6. Recent chromosome conformation capture approaches have revealed global loss of local structure on the Xi chromosome and formation of large mega-domains, separated by a region containing the DXZ4 macrosatellite7, 8, 9, 10. However, the molecular architecture of the Xi chromosome, in both the silent and expressed regions, remains unclear. Here we investigate the structure, chromatin accessibility and expression status of the mouse Xi chromosome in highly polymorphic clonal neural progenitors (NPCs) and embryonic stem cells. We demonstrate a crucial role for Xist and the DXZ4-containing boundary in shaping Xi chromosome structure using allele-specific genome-wide chromosome conformation capture (Hi-C) analysis, an assay for transposase-accessible chromatin with high throughput sequencing (ATAC–seq) and RNA sequencing. Deletion of the boundary disrupts mega-domain formation, and induction of Xist RNA initiates formation of the boundary and the loss of DNA accessibility. We also show that in NPCs, the Xi chromosome lacks active/inactive compartments and topologically associating domains (TADs), except around genes that escape XCI. Escapee gene clusters display TAD-like structures and retain DNA accessibility at promoter-proximal and CTCF-binding sites. Furthermore, altered patterns of facultative escape genes in different neural progenitor clones are associated with the presence of different TAD-like structures after XCI. These findings suggest a key role for transcription and CTCF in the formation of TADs in the context of the Xi chromosome in neural progenitors.