Polycomb complexes targeted to specific loci by non-coding RNA co-factors

通过非编码 RNA 辅助因子靶向特定位点的多梳复合物

• 批准号: 8265845
• 负责人: JEANNIE T LEE
• 金额: $ 48.25万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8265845
• 关键词: Address; Affect; Angelman Syndrome; Antisense RNA; Awareness; Beckwith-Wiedemann Syndrome; Binding; Catalytic Domain; Cells; Chromatin; Chromosomes; Colon Carcinoma; Complex; DNA Binding; Data; Defect; Development; Developmental Process; Disease; Disease Clusterings; Dosage Compensation (Genetics); Endometrial Carcinoma; Epigenetic Process; Event; Female; Functional RNA; Gene Expression; Gene Expression Regulation; Gene Silencing; Gene Targeting; Genes; Genetic; Genetic Transcription; Genomics; Hand; Health; Heterochromatin; Human; In Vitro; Investigation; Knowledge; Laboratories; Length; Link; Maintenance; Malignant Neoplasms; Malignant neoplasm of urinary bladder; Mammals; Maps; Mediating; Methylation; Molecular; Names; Pathway interactions; Play; Polycomb; Process; Proteins; RNA; RNA Binding; RNA Interference; RNA-Protein Interaction; Recruitment Activity; Regulation; Research; Research Proposals; Role; Structure; Testing; Untranslated RNA; X Chromosome; X Inactivation; base; embryonic stem cell; human DICER1 protein; human disease; imprint; improved; in vivo; lymphatic cancer; mutant; novel; pluripotency; protein complex; public health relevance; sex; stem

DESCRIPTION (provided by applicant): Dosage compensation in the mammal results in transcriptional silencing of one X-chromosome in the female sex. Known as "X-chromosome inactivation" (XCI), this process exemplifies epigenetic regulation and has served as a paradigm for understanding gene regulation by noncoding RNA (ncRNA) and chromatin-based mechanisms. XCI is controlled by the "X-inactivation center" (Xic), an X- linked region known for an abundance of genes that produce ncRNA. The initiation of XCI is regulated by two such ncRNA loci, Xist and its antisense partner, Tsix. Expression of Tsix blocks initiation of silencing, whereas expression of Xist guides chromosome-wide inactivation. While our understanding of how Tsix opposes Xist has continued to improve over the past 10 years, how Xist induces XCI remains largely unsolved. In the past year, my laboratory has made significant progress towards understanding how silencing might be induced: (i) We have identified the first Xist RNA-interacting factor as Polycomb repressive complex 2 (PRC2), the complex of proteins responsible for trimethylating H3-lysine27 (H3- K27me3). We have shown that Ezh2, the catalytic subunit of PRC2, is the specific RNA-binding subunit. Xist RNA directly binds this subunit via a repeated stem-loop structure at the 5' end of the RNA known as "Repeat A." (ii) We have also discovered that Repeat A is an independent transcription unit, which we named RepA. The 1.6 kb RepA RNA initially recruits PRC2 to the X and is required for induction of full-length Xist RNA, which in turn binds PRC2 also and spreads H3-K27 methylation along the whole X. (iii) The antisense Tsix RNA binds PRC2 as well. We propose that competition between RepA and Tsix for PRC2 determines if and when XCI will initiate. (iv) Curiously, Dicer-deficient cells show defects in Xist localization, PRC2 recruitment, and H3-K27 methylation, suggesting that the RNAi pathway may also play a hand in Polycomb recruitment and function. Taken together, these data implicate ncRNA at the interface between Polycomb proteins and their target genes, and suggest that ncRNA may be a general mechanism by which chromatin modifers - which often lack sequence- specific DNA-binding subunits - may be directed to specific loci. This research proposal is aimed at understanding mechanisms by which Polycomb complexes are targeted to genetic loci by ncRNA. We will examine how RepA, Tsix, and PRC2 interact to trigger the initiation of XCI, address how silencing spreads along the X through Xist RNA and PRC2, and then determine whether ncRNA plays a role in targeting PRC2 to other genomic regions. PUBLIC HEALTH RELEVANCE: The proposed research to study ncRNA and Polycomb proteins is of significant public health relevance due to the growing awareness of their involvement in human disease. For example, a number of imprinting disorders (e.g., Beckwith-Wiedemann Syndrome, Prader Willi and Angelman Syndromes) can be traced to aberrant expression of large noncoding RNAs that control expression of a linked cluster of disease genes. The far-ranging effects of Polycomb proteins for human health can be illustrated in two situations: First, in the maintenance of pluripotency for embryonic stem (ES) cells; and second, in the development of cancer when expression of Polycomb proteins is abnormal (e.g., colon, bladder, endometrial, and lymphatic cancers). Because our research focuses on ncRNA at the interface between Polycomb proteins and specific genetic targets, we expect that the knowledge gained will enhance understanding of normal developmental processes as well as disease processes when ncRNA and interacting Polycomb proteins are not properly regulated.

描述（由申请人提供）：哺乳动物中的剂量补偿导致雌性中一条X染色体的转录沉默。这一过程被称为“X 染色体失活”(XCI)，它体现了表观遗传调控，并成为理解非编码 RNA (ncRNA) 和基于染色质的机制进行基因调控的范例。 XCI 受“X 失活中心”(Xic) 控制，这是一个 X 连锁区域，以大量产生 ncRNA 的基因而闻名。 XCI 的启动由两个这样的 ncRNA 位点 Xist 及其反义伙伴 Tsix 调节。 Tsix 的表达阻止沉默的启动，而 Xist 的表达则引导染色体范围的失活。虽然我们对 Tsix 如何对抗 Xist 的理解在过去 10 年中不断提高，但 Xist 如何引发 XCI 的问题在很大程度上仍然没有得到解决。 在过去的一年中，我的实验室在了解如何诱导沉默方面取得了重大进展：（i）我们已经确定了第一个 Xist RNA 相互作用因子，即 Polycomb 抑制复合物 2 (PRC2)，这是负责三甲基化 H3- 的蛋白质复合物。赖氨酸27 (H3-K27me3)。我们已经证明PRC2的催化亚基Ezh2是特异性RNA结合亚基。 Xist RNA 通过 RNA 5' 端的重复茎环结构（称为“重复 A”）直接结合该亚基。 (ii)我们还发现Repeat A是一个独立的转录单位，我们将其命名为RepA。 1.6 kb RepA RNA 最初将 PRC2 招募到 X，并且是诱导全长 Xist RNA 所必需的，全长 Xist RNA 反过来也结合 PRC2 并沿着整个 X 传播 H3-K27 甲基化。 (iii) 反义 Tsix RNA 结合 PRC2出色地。我们建议 RepA 和 Tsix 之间对 PRC2 的竞争决定 XCI 是否以及何时启动。 (iv) 奇怪的是，Dicer 缺陷细胞在 Xist 定位、PRC2 募集和 H3-K27 甲基化方面表现出缺陷，这表明 RNAi 途径也可能在 Polycomb 募集和功能中发挥作用。总而言之，这些数据表明 ncRNA 存在于 Polycomb 蛋白与其靶基因之间的界面处，并表明 ncRNA 可能是染色质修饰剂（通常缺乏序列特异性 DNA 结合亚基）可定向至特定位点的通用机制。 本研究计划旨在了解 Polycomb 复合物通过 ncRNA 靶向遗传位点的机制。我们将研究 RepA、Tsix 和 PRC2 如何相互作用以触发 XCI 的启动，解决沉默如何通过 Xist RNA 和 PRC2 沿 X 传播，然后确定 ncRNA 是否在将 PRC2 靶向其他基因组区域中发挥作用。 公共卫生相关性：由于人们越来越认识到 ncRNA 和 Polycomb 蛋白与人类疾病的关系，因此拟议的研究 ncRNA 和 Polycomb 蛋白具有重要的公共卫生相关性。例如，许多印记疾病（例如贝克威斯-维德曼综合症、普瑞德威利综合症和天使曼综合症）可以追溯到控制疾病基因连锁簇表达的大非编码RNA的异常表达。 Polycomb 蛋白对人类健康的广泛影响可以通过两种情况来说明：第一，维持胚胎干 (ES) 细胞的多能性；其次，在癌症的发展过程中，当 Polycomb 蛋白表达异常时（例如结肠癌、膀胱癌、子宫内膜癌和淋巴癌）。因为我们的研究重点是 Polycomb 蛋白和特定遗传靶标之间界面的 ncRNA，所以我们期望所获得的知识将增强对正常发育过程以及当 ncRNA 和相互作用的 Polycomb 蛋白未得到适当调节时的疾病过程的理解。