Giorgetti Luca, Lajoie Bryan R, Carter Ava C, Attia Mikael, Zhan Ye, Xu Jin, Chen Chong Jian, Kaplan Noam, Chang Howard Y, Heard Edith, Dekker Job
Nature. 2016 Jul 28;535(7613):575-9. doi: 10.1038/nature18589. Epub 2016 Jul 18.
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 region, although some genes (constitutive escapees) avoid silencing in most cell types, and others (facultative escapees) escape XCI only in specific contexts. A role for Xist in organizing the inactive X (Xi) chromosome has been proposed. 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 macrosatellite. 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 indifferent 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.
X染色体失活（XCI）涉及X染色体在变得沉默和异染色质化时的重大重组。在雌性哺乳动物发育过程中，XCI由两条X染色体之一上的非编码Xist RNA上调触发。Xist以顺式方式覆盖染色体，并通过其A重复区域诱导几乎所有基因沉默，尽管一些基因（组成型逃逸基因）在大多数细胞类型中避免沉默，而其他基因（兼性逃逸基因）仅在特定情况下逃避XCI。有人提出Xist在组织失活X（Xi）染色体中起作用。最近的染色体构象捕获方法揭示了Xi染色体上局部结构的整体丧失以及由包含DXZ4大卫星的区域分隔的大型超级结构域的形成。然而，Xi染色体在沉默和表达区域的分子结构仍不清楚。在这里，我们研究了高度多态性的克隆神经祖细胞（NPC）和胚胎干细胞中小鼠Xi染色体的结构、染色质可及性和表达状态。我们使用等位基因特异性全基因组染色体构象捕获（Hi-C）分析、转座酶可及染色质高通量测序分析（ATAC-seq）和RNA测序，证明了Xist和含DXZ4的边界在塑造Xi染色体结构中的关键作用。边界的缺失破坏了超级结构域的形成，Xist RNA的诱导启动了边界的形成和DNA可及性的丧失。我们还表明，在NPC中，Xi染色体缺乏活跃/不活跃的区室和拓扑相关结构域（TAD），除了逃避XCI的基因周围。逃逸基因簇显示出类似TAD的结构，并在启动子近端和CTCF结合位点保留DNA可及性。此外，不同神经祖细胞克隆中兼性逃逸基因的改变模式与XCI后不同的类似TAD结构的存在有关。这些发现表明转录和CTCF在神经祖细胞Xi染色体背景下TAD形成中起关键作用。
