Cooperative control of Polycomb Repressive Complexes by long noncoding RNAs, CpG island DNA, and RNA-binding proteins

摘要 这项资助的目标是确定长的非编码RNA(LncRNAs)诱导 多梳抑制复合体(PRC)在基因组特定区域的扩散。这两个人都是 PRC，PRC1和PRC2，产生组蛋白修饰，抑制转录，同时刺激 另一个复合体的活动。原癌基因对大多数主要器官系统的发育及其 调节失调会导致一系列疾病。在已知的表观遗传修饰物中，pRCs是独一无二的 它们在染色质上的扩散是由特定的lncRNA诱导的。然而，尽管进行了数十年的研究，该领域已经 尚未确定lncRNAs如何导致原发癌的传播。突出的模型表明，lncRNA依赖于3- 三维(3D)基因组结构，在不受影响的情况下在染色质中不分青红皂白地传播原癌 由DNA的潜在序列决定。然而，这些模型不能解释中华人民共和国的广泛变异性- 在lncRNA目标域中的依赖修饰，并且不考虑在lncRNA目标域之外， 被称为CpG岛(CGI)的DNA元件将原癌细胞招募到染色质中。此外，目前还不清楚有多少人 InncRNAs导致了原癌的传播。虽然调查人员最初提出数以千计的LncRNA 这一功能后，有效的PRC诱导的LncRNAs的数量保持在近10个。我们的发现已经开始 解决这些未知数。例如，我们发现在小鼠滋养层干细胞中，lncRNA Airn 在表达AIRN之前，使PRC从自主绑定PRC的CGI向外扩散。删除 仅这些CGI中的一个就会导致在Airn域中PRC存放的修改造成数百万数据库的损失。 此外，我们和其他人发现了与pRCs相关的特定RNA结合蛋白，并且至少有两个 这些蛋白质似乎是lncRNAs诱导PRC传播所必需的。因此，与其传播PRC，不如说 不分青红皂白地跨越染色质，我们假设与CGI相关的lncRNAs从 这种联系是由RNA结合蛋白介导的，这些蛋白结合了lncRNA和 原告。此外，我们假设任何染色质结合的RNA都可以诱导原癌的传播，只要它 结合必要的蛋白质。我们现在建议对这些假设进行严格的测试：在目标1下，我们将 确定CGI和CGI结合因子在lncRNAs诱导的原癌扩散中的作用。在目标2下，我们 将确定RNA结合蛋白在由lncRNAs诱导的pRCs传播中的作用。在目标3下，我们将 确定非规范RNA在诱导原癌传播中的作用。如果成功，我们的工作将 展示DNA调控元件、RNA结合蛋白和RNA在PRC功能中的新作用。 我们建立的范例应该应用于核细胞生物学的广泛领域，并提出多种新的途径 以治疗的方式控制基因表达。

ABSTRACT The goal of this grant is to determine the mechanisms through which long noncoding RNAs (lncRNAs) induce the spread of Polycomb Repressive Complexes (PRCs) over specific regions of the genome. Each of the two PRCs, PRC1 and PRC2, deposits histone modifications that repress transcription and simultaneously stimulate the activity of the other complex. PRCs are essential for the development of most major organ systems and their dysregulation causes a wide range of diseases. PRCs are unique amongst known epigenetic modifiers in that their spread over chromatin is induced by specific lncRNAs. However, despite decades of study, the field has not established how lncRNAs induce the spread of PRCs. Prominent models suggest that lncRNAs rely on 3- dimensional (3D) genome structure to spread PRCs indiscriminately across chromatin, without being influenced by the underlying sequence of DNA. Yet, these models cannot account for the extensive variability of PRC- dependent modifications in lncRNA target domains, and do not consider that outside of lncRNA target domains, DNA elements called CpG islands (CGIs) recruit PRCs to chromatin. Moreover, it remains unclear how many lncRNAs induce the spread of PRCs. While investigators originally proposed that thousands of lncRNAs have this function, the number of validated PRC-inducing lncRNAs remains close to ten. Our findings have begun to resolve these unknowns. For example, we discovered that in mouse trophoblast stem cells, the lncRNA Airn causes PRCs to spread outwards from CGIs that bind PRCs autonomously, prior to expression of Airn. Deletion of just one of these CGIs causes a multi-megabase loss of PRC-deposited modifications in the Airn domain. Moreover, we and others found specific RNA-binding proteins that associate with the PRCs, and at least two of these proteins appear to be required for lncRNAs to induce PRC spread. Thus, rather than spreading PRCs indiscriminately across chromatin, we hypothesize that lncRNAs associate with CGIs to spread PRCs from specific points of contact, and, that this association is mediated by RNA-binding proteins that bind lncRNAs and PRCs. Further, we hypothesize that any chromatin-bound RNA can induce the spread of PRCs, so long as it binds the necessary proteins. We now propose rigorous tests of these hypotheses: Under Aim 1, we will determine the role of CGIs and CGI-bound factors in the spread of PRCs induced by lncRNAs. Under Aim 2, we will determine the role of RNA-binding proteins in the spread of PRCs induced by lncRNAs. Under Aim 3, we will determine the roles of non-canonical RNAs in inducing the spread of PRCs. If successful, our work will demonstrate new roles for DNA regulatory elements, RNA-binding proteins, and RNAs in PRC function. Paradigms we establish should apply to broad areas in nuclear cell biology, and suggest multiple new avenues for controlling gene expression therapeutically.