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: 10001559
• 负责人: JEANNIE T LEE
• 金额: $ 70.44万
• 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/10001559
• 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; 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.

项目摘要 我们的研究计划是统一的努力，以了解X染色体的机制 多梳抑制复合物2（PRC 2）和长链DNA之间的功能性相互作用 非编码RNA（lncRNA）通过X染色体传播沉默过程。XCI是剂量 哺乳动物中的补偿过程，导致着床期一条X染色体沉默 胚胎X-失活中心（Xic）-控制启动、扩散和转移的X连锁区域。 沉默的维持-含有大量编码功能性lncRNA的基因，包括 原型，Xist，一个17 kb的RNA，“外套”的失活X（Xi）启动沉默。走向理解 2008年，我们确定了Polycomb抑制复合物2（PRC 2）作为相互作用的蛋白质伴侣， 对于Xist RNA。PRC 2是负责三甲基化H3赖氨酸27（H3 K27 me 3）的表观遗传复合物。下 这个NIH奖，我们已经确定了一个“成核中心”的加载Xist-Polycomb复合物所需的 在传播之前，将YY 1定义为系链，并证明Xist RNA只是 数以千计的RNA伴侣EZH 2，PRC 2的催化亚基。我们假设监管机构 PRC 2和RNA之间的相互作用将成为表观遗传调控中的一个反复出现的主题。两大 该领域未解决的问题是如何实现PRC 2和RNA之间的相互作用特异性以及如何实现PRC 2和RNA之间的相互作用特异性。 它们以基因座特异性方式调节基因表达。在过去的融资周期中， 在解决这些问题方面取得进展。我们已经：（i）绘制了Xist RNA和PRC 2的Xi结合位点， (ii)在Xi上进行Xist-PRC 2关系的单分子成像，（iii）定义相互作用 Xist的重复序列A基序和PRC 2的各种亚基之间的特异性和调节相互作用，（iv） 鉴定了有助于确定Xist和PRC 2之间的相互作用特异性的体内伴侣，和（v） 揭示了SMCHD 1（一种染色体结构蛋白）在Xist-PRC 2区域传播中的作用 配合物我们还发现了EZH 2的一种令人惊讶的非典型活性， 不依赖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 RNA（B2）在应激反应中的作用。在今后五年中，我们将处理关键的后续问题。一是 目的是定义Polycomb-RNA相互作用的特定基序，并了解ATRX如何影响 互动其次，我们将研究Xist-PRC 2“传播”功能的潜在机制， Xi通过诱变Xist和扰动高阶Xi结构所需的因素。最后我们 将研究EZH 2如何触发RNA的位点特异性切割，并确定这种活性是否影响XCI。