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

Xist RNA 和 Polycomb 复合物沿着非活性 X 染色体扩散。

• 批准号: 10178063
• 负责人: JEANNIE T LEE
• 金额: $ 69.03万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10178063
• 关键词: 3-Dimensional; ATRX gene; Achievement; Address; Architecture; Award; Base Sequence; Binding; Binding Sites; Biochemical; Biochemistry; Biology; Catalytic Domain; Cellular biology; Chromatin; Chromosomes; Complex; Development; Dosage Compensation (Genetics); EZH2 gene; Epigenetic Process; Funding; Gene Expression; Genes; Genetic; Genomics; Goals; Histones; In Vitro; Link; Literature; Maintenance; Mammals; Maps; Methods; Modeling; Molecular Chaperones; Mutagenesis; Nature; Nucleic Acids; Polycomb; Process; Proteins; Publishing; RNA; RNA-Protein Interaction; Reaction; Recurrence; Regulation; Research; Resolution; Role; Short Interspersed Nucleotide Elements; Site; Specificity; Stress; Structure; Systems Biology; Transcript; United States National Institutes of Health; Untranslated RNA; Work; X Chromosome; X Inactivation; YY1 Transcription Factor; base; biological adaptation to stress; cohesin; deletion analysis; epigenetic regulation; epigenomics; follow-up; frontier; genome-wide; histone methyltransferase; implantation; in vivo; mammalian genome; molecular imaging; natural Blastocyst Implantation; nuclease; programs; prototype; response; single molecule

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.

项目概要 我们的研究计划是通过努力了解 X 染色体的机制来统一的 失活 (XCI) 以及 Polycomb 抑制复合物 2 (PRC2) 和长链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是剂量 哺乳动物的补偿过程导致植入周围的一条 X 染色体沉默 胚胎。 X 失活中心 (Xic) — 控制起始、传播和传播的 X 连锁区域 沉默的维持——拥有大量编码功能性lncRNA的基因，包括 原型 Xist 是一种 17 kb RNA，它“包裹”不活跃的 X (Xi) 以启动沉默。走向理解 机制，2008 年，我们确定了 Polycomb 抑制复合物 2 (PRC2) 作为相互作用的蛋白伙伴 对于 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 在这个 NIH 奖项中，我们确定了加载 Xist-Polycomb 复合物所需的“成核中心” 在传播之前将其连接到 Xi 上，将 YY1 定义为系链，并证明 Xist RNA 只是其中之一 EZH2（PRC2 的催化亚基）有数千个 RNA 伴侣。我们假设监管 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实现 它们以位点特异性的方式调节基因表达。在过去的融资周期中，我们取得了重大进展 解决这些问题的进展。我们已经：（i）绘制了 Xist RNA 和 PRC2 的 Xi 结合位点， (ii) 对 Xi 上的 Xist-PRC2 关系进行单分子成像，(iii) 定义相互作用 Xist 的重复 A 基序和 PRC2 的各个亚基之间的特异性和调控相互作用，(iv) 确定了一个体内伴侣，有助于确定 Xist 和 PRC2 之间的相互作用特异性，以及 (v) 揭示了 SMCHD1（一种染色体结构蛋白）在 Xist-PRC2 区域传播中的作用 复合物。我们还发现了 EZH2 的一项令人惊讶的非规范活动——一种活动 独立于其组蛋白甲基转移酶活性，并触发 SINE 重复序列的位点特异性切割 应激反应期间的RNA (B2)。未来五年，我们将解决关键的后续问题。首先，我们 旨在定义 Polycomb-RNA 相互作用的特定基序并了解 ATRX 如何影响 相互作用。其次，我们将研究 Xist-PRC2 在 Xi 通过 Xist 的诱变和高阶 Xi 结构所需因素的扰动来实现。最后，我们 将检查 EZH2 如何触发 RNA 的位点特异性切割，并确定该活动是否影响 XCI。