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

 描述(申请人提供)：这项建议的目标是揭示在X染色体失活(XCI)过程中，长非编码(LNC)RNA Xist和TSix找到它们的基因组目标位置并控制这些位置上的基因表达的机制。XCI是LncRNA介导的染色质调节的一个显着范例，由相反转录的RNAXist和TSix控制，在转录上沉默雌性哺乳动物细胞中两条X染色体中的一条。虽然Xist诱导X染色体沉默，但tSix在发育早期负向调节Xist，以确保Xist只在一条X染色体上活跃。我们已经知道Xist的几个功能，包括从它的转录位点顺式扩散到覆盖整个X染色体，不包括RNA聚合酶II，触发抑制染色质标记的积累，并诱导整个染色体的紧凑，证明了这种lncRNA的非凡的多功能性。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的结构域或相互作用的蛋白质介导这些不同的作用。然而，我们的初步数据为Xist和TSix的作用提供了新的见解。具体地说，我们已经能够 将XIST的三个结构域定义为具有不同功能的介体，这与不同的RNA结构域执行不同功能的想法一致。我们假设，这些结构域对于Xist与X的有效结合是必需的，确保它在顺式基因中的功能；Xist针对活性基因的靶向，这是它们随后沉默的先决条件；以及 染色体构象。为了进一步确定这些结构域的作用机制，我们还确定了其中一个结构域的关键蛋白质相互作用伙伴。此外，我们最近在XIST传播方面的工作表明了XIST和基因组组织之间有趣的联系，这导致了XIST利用远程染色质相互作用在X染色体上传播的假设，并且，一旦拴在远端位置，XIST就作为一个核组织因子，创建了一个新的染色体3D拓扑结构来诱导基因沉默。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方向扩散，但与Xist相反，TSix只在其转录位点周围局部扩散。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。基于我们的发现，我们很好地揭示了Xist和TSix控制XCI的调控机制，目的如下：1)了解Xist RNA如何通过特定的RNA结构域介导其各种功能。2)探讨3D染色体结构与Xist的关系。3)研究TSix的功能和定位。每个目标都将得到杰出的合作者的支持。总之，我们建议的研究将为XCI提供新的见解，并应显著促进我们对核结构对核功能的作用的理解。探索Xist和TSix之间的差异也将更好地理解lncRNAs如何定位到它们的基因组目标。

 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.