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

描述(申请人提供)：哺乳动物的剂量补偿会导致雌性X染色体的转录沉默。这个过程被称为“X染色体失活”(XCI)，是表观遗传调控的例证，并已成为理解非编码RNA(NcRNA)和基于染色质的机制进行基因调控的范例。XCI由“X失活中心”(XIC)控制，XIC是一个与X相连的区域，以大量产生ncRNA的基因而闻名。XCI的启动受两个这样的ncRNA基因座的调控，Xist和它的反义伴侣TSix。TSix的表达阻止了沉默的启动，而Xist的表达则引导了整个染色体的失活。虽然我们对XIST如何反对XIST的理解在过去10年中不断提高，但XIST如何诱导XCI在很大程度上仍未解决。在过去的一年里，我的实验室在了解沉默可能如何诱导方面取得了重大进展：(I)我们已经确定第一个Xist RNA相互作用因子是多梳抑制复合体2(PRC2)，它是负责三甲基化H3-赖氨酸27(H3-K27me3)的蛋白质复合体。我们已经证明，PRC2的催化亚基Ezh2是特异的RNA结合亚基。Xist RNA通过RNA 5‘端的一个重复的茎环结构直接结合这个亚基，称为“Repeat A”。(Ii)我们还发现Repeat A是一个独立的转录单位，我们称之为REPA。1.6kb的REPA RNA最初将PRC2招募到X，是诱导全长Xist RNA所必需的，后者又与PRC2结合，并沿整个X传播H3-K27甲基化。(Iii)反义tSix RNA也与PRC2结合。我们认为，REPA和TSix之间对PRC2的竞争决定了XCI是否以及何时启动。(4)奇怪的是，DICER缺陷细胞在Xist定位、PRC2募集和H3-K27甲基化方面存在缺陷，这表明RNAi途径也可能在Polycomb募集和功能中发挥作用。综上所述，这些数据表明，ncRNA位于Polycomb蛋白及其靶基因之间的界面上，并表明ncRNA可能是染色质修饰物--通常缺乏序列特异性DNA结合亚基--可能定向到特定基因座的一般机制。本研究旨在了解多梳复合体通过核糖核酸靶向遗传位点的机制。我们将研究REPA、TSix和PRC2是如何相互作用来触发XCI的启动，解决沉默是如何通过Xist RNA和PRC2沿X传播的，然后确定ncRNA是否在将PRC2靶向其他基因组区域方面发挥作用。 公共卫生相关性：由于人们日益意识到ncRNA和Polycomb蛋白与人类疾病的关系，因此拟议的研究具有重大的公共卫生相关性。例如，许多印迹障碍(如Beckwith-Wiedemann综合征、Prader Willi和Angelman综合征)可以追溯到控制一组相连疾病基因表达的大非编码RNA的异常表达。Polycomb蛋白对人类健康的广泛影响可以在两种情况下得到证明：第一，维持胚胎干细胞的多能性；第二，当Polycomb蛋白表达异常时，在癌症的发展中(例如，结肠癌、膀胱癌、子宫内膜癌和淋巴癌)。由于我们的研究集中在多聚核糖体蛋白和特定遗传靶点之间的ncRNA，我们预计所获得的知识将加强对正常发育过程以及当ncRNA和相互作用的多聚集合体蛋白未被适当调控时疾病过程的理解。

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.