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

描述（由申请方提供）：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。被称为“X染色体失活”（XCI），这一过程证实了表观遗传调控，并已作为一个范例，了解基因调控的非编码RNA（ncRNA）和染色质为基础的机制。XCI由“X-失活中心”（Xic）控制，Xic是一个已知具有大量产生ncRNA的基因的X-连锁区域。XCI的起始受两个这样的ncRNA基因座Xist及其反义配偶体Tsix调节。Tsix的表达阻断沉默的起始，而Xist的表达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如何诱导XCI仍然没有得到解决。 在过去的一年里，我的实验室在理解沉默是如何被诱导方面取得了重大进展：（i）我们已经确定了第一个Xist RNA相互作用因子作为Polycomb抑制复合物2（PRC 2），负责三甲基化H3-赖氨酸27（H3-K27 me 3）的蛋白质复合物。我们已经证明，Ezh 2，PRC 2的催化亚基，是特异性的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的RepA RNA最初将PRC 2募集到X上，并且是诱导全长Xist RNA所必需的，全长Xist RNA又结合PRC 2，并且沿着整个X传播H3-K27甲基化。(iii)反义Tsix RNA也结合PRC 2。我们建议RepA和Tsix之间对PRC 2的竞争决定了XCI是否以及何时启动。(iv)奇怪的是，Dicer缺陷细胞在Xist定位、PRC 2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。总之，这些数据暗示在Polycomb蛋白和它们的靶基因之间的界面处的ncRNA，并且表明ncRNA可能是染色质修饰剂（其通常缺乏序列特异性DNA结合亚基）可被引导至特定基因座的一般机制。 这项研究计划旨在了解Polycomb复合物通过ncRNA靶向遗传位点的机制。我们将研究RepA，Tsix和PRC 2如何相互作用以触发XCI的启动，解决沉默如何通过Xist RNA和PRC 2沿着X沿着传播，然后确定ncRNA是否在将PRC 2靶向其他基因组区域中发挥作用。 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，因为人们越来越意识到它们与人类疾病有关。例如，许多印迹障碍（例如，Beckwith-Wiedemann综合征，Prader Willi和Angelman综合征）可以追溯到控制疾病基因连锁簇表达的大型非编码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说明：第一，维持胚胎干（ES）细胞的多能性;第二，当Polycomb蛋白的表达异常时（例如，结肠癌、膀胱癌、子宫内膜癌和淋巴癌）。由于我们的研究重点是多梳蛋白和特定遗传靶点之间界面的ncRNA，我们预计所获得的知识将增强对正常发育过程以及当ncRNA和相互作用的多梳蛋白未得到适当调节时的疾病过程的理解。

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.