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会导致女性性别中一个X染色体的转录沉默。该过程被称为“ X染色体灭活”（XCI），例如表观遗传调节，并已成为理解非编码RNA（NCRNA）和基于染色质质机制的基因调节的范式。 XCI由“ X灭活中心”（XIC）控制，这是一个X链接的区域，以产生NCRNA的丰富基因而闻名。 XCI的启动受两个这样的NCRNA基因座及其反义伴侣TSIX的调节。 TSIX阻止沉默的启动的表达，而XIST的表达引导均可灭活染色体。尽管我们对TSIX如何反对XIST的理解在过去10年中持续改善，但XIST如何诱导XCI仍未得到解决。 在过去的一年中，我的实验室取得了重大进展，以了解如何诱导沉默：（i）我们将第一个XIST RNA相互作用因子确定为PolyComb抑制性复合物2（PRC2），这是负责三甲基化H3-赖氨酸的蛋白质络合物（H3- k27me3）。我们已经表明，EZH2是PRC2的催化亚基，是特定的RNA结合亚基。 XIST RNA通过重复的茎环结构在RNA的5'末端直接结合该亚基，称为“重复A”。 （ii）我们还发现重复A是一个独立的转录单元，我们将其命名为Repa。 1.6 kb repa RNA最初将PRC2募集到X中，并且是诱导全长Xist RNA所必需的，这又依次结合PRC2并沿整个X散布H3-K27甲基化。我们建议REPA和TSIX之间的PRC2竞争决定了XCI是否以及何时启动。 （iv）奇怪的是，缺陷型细胞在XIST定位，PRC2募集和H3-K27甲基化中显示出缺陷，这表明RNAi途径也可能在Polycomb募集和功能方面发挥作用。综上所述，这些数据暗示了ncRNA在多角膜蛋白及其靶基因之间的界面上，并表明NCRNA可能是一种通用机制，染色质修饰剂通常缺乏序列 - 特异性的DNA结合亚基 - 可以定向到特定基因座。 该研究建议旨在理解NCRNA靶向遗传基因座的polycomb复合物的机制。我们将研究REPA，TSIX和PRC2如何相互作用以触发XCI的启动，解决如何通过XIST RNA和PRC2沿X沿X传播，然后确定NCRNA是否在将PRC2靶向其他基因组区域中起作用。 公共卫生相关性：拟议的研究NCRNA和PolyComb蛋白的研究具有重要的公共卫生相关性，因为它们越来越了解其参与人类疾病的意识。例如，可以追溯到许多印记疾病（例如贝克维斯·威德曼综合症，普拉德·威利和安吉尔曼综合症）可追溯到控制一个连接的疾病基因簇表达的大型非编码RNA的表达。可以在两种情况下说明多角膜蛋白对人体健康的极远程作用：首先，在维持胚胎茎（ES）细胞的多能性方面；其次，当多肉蛋白的表达异常时（例如，结肠，膀胱，子宫内膜和淋巴结癌）时，癌症的发展。由于我们的研究重点是多肉蛋白与特定遗传靶标之间的界面上的NCRNA，因此我们期望获得的知识将增强对正常发育过程的理解，以及当NCRNA和相互作用的多肉蛋白不适当调节时疾病过程。