Dissecting the mechanism of epigenetic spreading by targeted degradation of architectural proteins

通过靶向降解建筑蛋白来剖析表观遗传扩散的机制 在雌性哺乳动物中，一条X染色体被沉默，以平衡X连锁基因与雄性的剂量， 这一过程称为X染色体失活(XCI)。XCI由一个长的非编码RNA Xist介导，它 在不活跃的X区蔓延，引发抑制因素的招募，如Polycomb抑制 复合体1和2(Prc1/2)、异染色质形成和基因沉默。通过这种方式，XCI充当了 异染色质标记传播的模型，这一过程对于基因沉默是必不可少的 哺乳动物的基因组，但还不是很清楚。XCI还显著改变了三维(3D) X染色体的染色质结构。特别是，染色体构象研究表明，X 染色体和哺乳动物基因组的其余部分由结构蛋白CTCF和 粘附素进入拓扑相关结构域(TADS)，即染色质在其内部的百万碱基大小的区域 相互作用的形成比跨域边界更频繁。哺乳动物的染色体也是有组织的 独立于CTCF和粘附素，在活动和非活动之间实现更长范围的“区隔”相互作用 区域(A/B车厢)。虽然TAD和隔室在分化期间通常保持稳定， 这两种结构在XCI期间都发生了戏剧性的变化。具体地说，TADS和舱室 在XCI期间衰减，而不活动的X被构造成巨型区域。如此戏剧性 染色体构象的重组在任何其他细胞过程中都没有看到，一个关键的问题是 X染色体构象的这些变化是XCI所必需的，还是仅仅是XCI的副产品 让整个染色体沉默。为了解决这个问题，我培育了雌性小鼠胚胎干细胞 (MESCs)，其中结构蛋白CTCF、RAD21和WAPL可以使用Dtag快速降解 XCI期间的降级系统。使用我的CTCF和RAD21 degron细胞系，我首先提出了削弱TADS和 加强XCI早期分舱，研究这些变化对Xist传播和 XCI的后续步骤。相反，我建议用我的WAPL degron细胞系来增强TADS和 削弱早期XCI的隔层，并研究这些相反变化对Xist传播的影响。通过 阐明XIST扩散需要哪些(如果有的话)3D染色质结构，我希望不仅能阐明 关于XCI，但也关于XCI外异染色质扩散的机制-以及3D基因组的作用 基因调控方面的组织也是如此。

Dissecting the mechanism of epigenetic spreading by targeted degradation of architectural proteins In female mammals, one X chromosome is silenced in order to balance dosage of X-linked genes with males, a process known as X chromosome inactivation (XCI). XCI is mediated by a long noncoding RNA Xist, which spreads across the inactive X, triggering recruitment of repressive factors such as Polycomb Repressive Complexes 1 and 2 (PRC1/2), heterochromatin formation and gene silencing. In this way, XCI serves as a model for the spreading of heterochromatin marks, a process which is essential for gene silencing across the mammalian genome, but which is not well understood. XCI also dramatically alters the three-dimensional (3D) chromatin structure of the X chromosome. In particular, chromosome conformation studies revealed that the X chromosome, along with the rest of the mammalian genome is organized by architectural proteins CTCF and cohesin into Topologically Associating Domains (TADs), megabase-sized regions within which chromatin interactions form more frequently than across domain borders. Mammalian chromosomes are also organized independently of CTCF and cohesin into longer range ‘compartmental’ interactions between active and inactive regions (A/B compartments). While TADs and compartments generally remain stable during differentiation, both structures are dramatically altered during XCI. Specifically, TADs and compartments are greatly attenuated during XCI, with the inactive X instead being structured into megadomains. As such dramatic restructuring of chromosome conformation is not seen during any other cellular process, a pivotal question is whether these changes in X chromosome conformation are required for XCI or are merely a byproduct of silencing the entire chromosome. To address this question, I generated female mouse embryonic stem cells (mESCs) in which architectural proteins CTCF, RAD21, and WAPL can be rapidly degraded using the dTAG degron system during XCI. Using my CTCF and RAD21 degron cell lines, I first propose to weaken TADs and strengthen compartments in early XCI and study the impact of these changes on Xist spreading and subsequent steps of XCI. In contrast, I propose to use my WAPL degron cell lines to strengthen TADs and weaken compartments in early XCI and study the impact of these opposing changes on Xist spreading. By elucidating which, if any, 3D chromatin structures are needed for Xist spreading, I expect to shed light not only on XCI, but also on the mechanism of heterochromatin spreading outside of XCI—and the role of 3D genome organization in gene regulation in general as well.