Spreading of Xist RNA and Polycomb complexes along the inactive X-chromosome.

项目总结 我们的研究计划是统一的，努力了解X染色体的潜在机制 失活(XCI)及多梳抑制复合体2(PRC2)与Long之间的功能相互作用 非编码RNA(LncRNA)通过X染色体传播沉默过程。XCI为剂量 哺乳动物的代偿过程导致围着床期一条X染色体沉默 胚胎。X-失活中心(XIC)-控制X-连锁区域的起始、扩散和 沉默的维持-含有大量编码功能性LncRNAs的基因，包括 原型，Xist，一个17kb的RNA，包裹在不活跃的X(Xi)上，启动沉默。走向理解 机制，我们在2008年确定多梳抑制复合体2(PRC2)是一个相互作用的蛋白质伙伴 用于Xist RNA。PrC2是负责H3赖氨酸27(H3K27me3)三甲基化的表观遗传复合体。在……下面 在NIH的这个奖项中，我们发现了装载Xist-Polycomb络合物所需的“成核中心” 在传播之前，将YY1定义为系绳，并证明了Xist RNA只是 PRC2的催化亚单位EZH2的数千个RNA伙伴。我们假设监管机构 PrC2和RNA之间的相互作用将成为表观遗传调控中反复出现的主题。两大 该领域尚未解决的问题是如何实现PrC2和RNA之间的相互作用特异性以及如何 它们以特定于基因位点的方式调节基因表达。在过去的资金周期中，我们取得了重大进展 在解决这些问题方面取得了进展。我们已经：(I)定位了Xist RNA和PrC2的XI结合位点， (Ii)在XI上进行Xist-PrC2关系的单分子成像，(Iii)定义相互作用 Xist‘s Repeat A基序与PrC2各亚基的特异性和调控相互作用，(Iv) 确定了一种体内伴侣，有助于确定Xist和PrC2之间的相互作用特异性，以及(V) 揭示Smchd1(一种染色体结构蛋白)在Xist-PRC2区域传播中的作用 复合体。此外，我们还发现了EZH2的一种令人惊讶的非规范活动--AN活动 不依赖于其组蛋白甲基转移酶的活性，并触发正弦重复序列的位点特异性切割 应激反应过程中的RNA(B2)。未来五年，我们将解决关键的后续问题。首先，我们 目的为Polycomb-RNA相互作用定义特定的基序，并了解ATRX如何影响 互动。其次，我们将研究XIST-PRC2在 XI通过Xist的突变和高阶xi结构所需因子的扰动来实现。最后，我们 将研究EZH2如何触发RNA的位点特异性切割，并确定这一活动是否影响XCI。

PROJECT SUMMARY Our research program is unified by an effort to understand the mechanisms underlying X-chromosome inactivation (XCI) and how functional interactions between Polycomb repressive complex 2 (PRC2) and long noncoding RNA (lncRNA) spread the silencing process through the X-chromosome. XCI is the dosage compensation process in mammals that leads to silencing of one X-chromosome in the peri-implantation embryo. The X-inactivation center (Xic) — the X-linked region that controls the initiation, spread, and maintenance of silencing — harbors a large number of genes encoding functional lncRNAs, including the prototype, Xist, a 17-kb RNA that “coats” the inactive X (Xi) to initiate silencing. Towards understanding mechanisms, in 2008 we identified Polycomb repressive complex 2 (PRC2) as an interacting protein partner for Xist RNA. PRC2 is the epigenetic complex responsible for trimethylating H3 lysine27 (H3K27me3). Under this NIH award, we have identified of a “nucleation center” required for loading of Xist-Polycomb complexes onto the Xi prior to spreading, defined YY1 as the tether, and demonstrated that Xist RNA is but one of thousands of RNA partners for EZH2, the catalytic subunit of PRC2. We have hypothesized that regulatory interactions between PRC2 and RNA would become a recurrent theme in epigenetic regulation. Two major unsolved problems in the field are how the interaction specificity between PRC2 and RNA is achieved and how they regulate gene expression in a locus-specific manner. In the past funding cycle, we have made significant progress towards addressing these problems. We have: (i) mapped Xi-binding sites for Xist RNA and PRC2, (ii) performed single-molecule imaging of the Xist-PRC2 relationship on the Xi, (iii) defined the interaction specificity and regulatory interactions between Xist's Repeat A motif and various subunits of PRC2, (iv) identified an in vivo chaperone that helps determine the interaction specificity between Xist and PRC2, and (v) revealed a role for SMCHD1 (a chromosome architectural protein) in the regional spreading of Xist-PRC2 complexes. We have furthermore discovered a surprising non-canonical activity of EZH2 — an activity independent of its histone methyltransferase activity and that triggers a site-specific cleavage of a SINE repeat RNA (B2) during the stress response. In the next five years, we will address key follow-up questions. First, we aim to define specific motifs for the Polycomb-RNA interaction and understand how ATRX influences the interaction. Second, we will investigate underlying mechanisms for the Xist-PRC2 "spreading" function on the Xi through mutagenesis of Xist and perturbation of factors required for higher order Xi structures. Finally, we will examine how EZH2 triggers site-specific cleavage of RNA and determine whether this activity impacts XCI.