Mechanism of chromatin remodeling and gene silencing by the lncRNAs Xist and Tsix

lncRNA Xist 和 Tsix 的染色质重塑和基因沉默机制

• 批准号: 8988214
• 负责人: Kathrin Plath
• 金额: $ 39.38万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8988214
• 关键词: Architecture; Cell physiology; ChIP-seq; Chromatin; Chromosome Structures; Chromosomes; Complex; Data; Development; Disease; Distal; Elements; Ensure; Exhibits; Female; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genetic Transcription; Genome; Genomics; Goals; Link; Malignant Neoplasms; Mammalian Cell; Mediating; Mediator of activation protein; Molecular; Molecular Conformation; Nuclear; Pattern; Polycomb; Positioning Attribute; Process; Proteins; RNA; RNA Polymerase II; RNA Sequences; Regulation; Role; Site; Testing; Untranslated RNA; Work; X Chromosome; X Inactivation; base; cell type; chromatin remodeling; insight; novel; public health relevance; research study; scaffold; temporal measurement; transcriptome sequencing

 DESCRIPTION (provided by applicant): The goal of this proposal is to reveal mechanisms by which the long non-coding (lnc) RNAs Xist and Tsix find their genomic target sites and control gene expression at these sites during the process of X chromosome inactivation (XCI). XCI is a remarkable paradigm of lncRNA-mediated chromatin regulation, controlled by the oppositely transcribed RNAs Xist and Tsix, that transcriptionally silence one of the two X chromosomes in female mammalian cells. While Xist induces silencing of the X chromosome, Tsix negatively regulates Xist early in development to ensure that Xist only becomes active on one X chromosome. We already know several functions of Xist, including spreading in cis from its site of transcription to cover the entire X chromosome, excluding RNA Polymerase II, triggering the accumulation of repressive chromatin marks, and inducing a chromosome-wide compaction, demonstrating the remarkable versatility of this lncRNA. The mechanisms by which Xist carries out these functions remain poorly understood. In particular, with few exceptions, we still do not know domains of Xist or interacting proteins that mediate these various roles. However, our preliminary data yield new insight into the roles of Xist and Tsix. Specifically, we have been able to define three domains of Xist as mediators of distinct functions, consistent with the idea that distinct RNA domains carry out different functions. We hypothesize that these domains are required for the efficient association of Xist with the X, ensuring its function in cis; the targetng of Xist to active genes, which is a prerequisite for their subsequent silencing; and for changes in chromosome conformation, respectively. To further define the mechanisms by which these domains act, we have also identified a critical protein interaction partner of one of these domains. Moreover, our recent work on Xist spreading suggests an intriguing link between Xist and genome organization, that led to the hypothesis that Xist utilizes long-range chromatin interactions to spread across the X chromosome, and, upon tethering to distal sites acts as a nuclear organization factor that creates a new 3D topology of the chromosome to induce gene silencing. Finally, by comparing the RNA localization of Tsix and Xist on the X chromosome, we found that Tsix can spread along the X, but in contrast to Xist, only spreads locally around its site of transcription. We speculate that specific RNA sequences in Xist and Tsix direct these distinct localization patterns. Based on our findings, we are well positioned to unveil regulatory mechanisms by which Xist and Tsix control XCI, with these Aims: 1) To understand how Xist RNA mediates its various functions through specific RNA domains. 2) To investigate the link between 3D chromosome structure and Xist. 3) To characterize the function and localization of Tsix. Each aim will be supported by outstanding collaborators. Together, our proposed studies will provide novel insights into XCI, and should significantly advance our understanding of the role of nuclear architecture on nuclear function. Exploring the differences between Xist and Tsix will also yield a better understanding of how lncRNAs locate to their genomic targets.

 描述（由申请人提供）：本提案的目的是揭示长非编码（lnc）RNA Xist 和 Tsix 在 X 染色体失活（XCI）过程中找到其基因组靶位点并控制这些位点基因表达的机制。 XCI 是 lncRNA 介导的染色质调节的一个显着范例，由相反转录的 RNA Xist 和 Tsix 控制，它们在转录上沉默雌性哺乳动物细胞中两条 X 染色体之一。 Xist 会诱导 X 染色体沉默，而 Tsix 在发育早期对 Xist 进行负调控，以确保 Xist 仅在一条 X 染色体上变得活跃。我们已经知道 Xist 的几个功能，包括从其转录位点顺式扩展以覆盖整个 X 染色体，排除 RNA 聚合酶 II，触发抑制性染色质标记的积累，并诱导全染色体压缩，证明了这种 lncRNA 的非凡多功能性。 Xist 执行这些功能的机制仍然知之甚少。特别是，除了少数例外，我们仍然不知道 Xist 的结构域或介导这些不同作用的相互作用蛋白。然而，我们的初步数据对 Xist 和 Tsix 的角色产生了新的见解。具体来说，我们已经能够 将 Xist 的三个结构域定义为不同功能的介体，这与不同的 RNA 结构域执行不同功能的观点一致。我们假设这些结构域是 Xist 与 X 有效关联所必需的，确保其顺式功能； Xist 靶向活性基因，这是随后沉默的先决条件；以及对于变化 分别是染色体构象。为了进一步定义这些结构域的作用机制，我们还确定了这些结构域之一的关键蛋白质相互作用伙伴。此外，我们最近关于 Xist 传播的工作表明 Xist 和基因组组织之间存在有趣的联系，这导致了这样的假设：Xist 利用长程染色质相互作用在 X 染色体上传播，并且在束缚到远端位点时充当核组织因子，创建染色体的新 3D 拓扑以诱导基因沉默。最后，通过比较Tsix和Xist在X染色体上的RNA定位，我们发现Tsix可以沿着X传播，但与Xist相反，仅在其转录位点周围局部传播。我们推测 Xist 和 Tsix 中的特定 RNA 序列指导着这些不同的定位模式。根据我们的发现，我们有能力揭示 Xist 和 Tsix 控制 XCI 的调控机制，目标如下：1) 了解 Xist RNA 如何通过特定 RNA 结构域介导其各种功能。 2) 研究3D染色体结构与Xist之间的联系。 3) 表征Tsix的功能和定位。每个目标都将得到杰出合作者的支持。总之，我们提出的研究将为 XCI 提供新颖的见解，并应显着增进我们对核结构对核功能的作用的理解。探索 Xist 和 Tsix 之间的差异也将有助于更好地了解 lncRNA 如何定位其基因组靶标。