Xist RNA and Regulation of Chromatin Structure

描述（申请人摘要）：这项工作的长期目标是 了解含有RNA的复合物如何调节染色质结构和基因 表情X染色体失活是一个特别引人注目的例子， 由RNA调节的染色质结构的变化。Xist基因调节 X染色体失活Xist编码一种非编码RNA， 从雌性体细胞的Xi中提取。在X-灭活Xist启动期间 RNA以顺式方式扩散，包裹在染色体上，染色体将成为Xi，而 转录沉默的传播与转录沉默的顺式传播密切相关。 Xist RNA。我的遗传发育和细胞学分析显示 RNA在X失活开始时稳定。这些结果导致 一种发育调节的Xist RNA -蛋白质相互作用的假说， 促进Xist RNA在Xi上的稳定和扩散。作为一个独立 研究者I已经证明Xist RNA是RNA蛋白质一部分 复合物和反义亲和层析可用于纯化 这个复杂。采用强有力和互补的方法相结合， 包括生化纯化、胚胎干细胞的遗传操作 细胞和分子细胞遗传学，我们提出鉴定蛋白质， 与Xist RNA相互作用。Xist用于调节X-失活的机制是 对Xist相互作用蛋白的了解很少， 这是分析Xist RNA功能分子基础的第一步。 RNA分子很可能更普遍地用于染色质 真核生物中基因表达的重塑和调节，因为RNA是至关重要的 为核心的功能组织，是一个组成部分， 剂量补偿装置在苍蝇和哺乳动物。我们理解 RNA-蛋白质复合物影响染色质结构的机制是 通过鉴定额外的Xist样调节RNA， 分子。我们已经开发了一种分离染色质相关RNA的方法， 用这种方法分离出一种在小鼠着丝粒上积累的RNA。我们 我们希望能够描述这种新的RNA。RNA的特征 以及Xist的蛋白质成分和其他影响 染色质结构将提供深入了解核RNA的作用， 调节基因表达。

DESCRIPTION (APPLICANT'S ABSTRACT): The long-term goal of this work is to understand how RNA containing complexes modulate chromatin structure and gene expression. X chromosome inactivation is a particularly dramatic example of a change in chromatin structure that is regulated by RNA. The Xist gene regulates X-inactivation. Xist encodes a non-coding RNA that is transcribed exclusively from the Xi in female somatic cells. During initiation of X-inactivation Xist RNA spreads in cis to coat the chromosome that will become the Xi, and the spread of transcriptional silencing correlates tightly with the cis-spread of Xist RNA. My genetic, developmental and cytological analysis showed that Xist RNA is stabilized at the initiation of X-inactivation. These results lead to the hypothesis that a developmentally regulated Xist RNA - protein interaction facilitates stabilization and spread of Xist RNA on the Xi. As an independent investigator I have demonstrated that Xist RNA is part of an RNA-protein complex and that antisense affinity chromatography can be employed to purify this complex. Using a combination of powerful and complementary approaches, including biochemical purification, genetic manipulation of embryonic stem cells, and molecular cytogenetics, we propose to identify proteins that interact with Xist RNA. The mechanisms Xist uses to regulate X-inactivation are poorly understood and the identification of Xist-interacting proteins is the first step in analysis of the molecular basis of Xist RNA function. It is highly likely that RNA molecules are more generally used for chromatin remodeling and regulation of gene expression in eukaryotes since RNA is crucial for the functional organization of the nucleus and is an integral part of the dosage compensation apparatus in flies and mammals. Our understanding of the mechanisms by which RNA-protein complexes affect chromatin structure would be greatly facilitated by identification of additional Xist-like regulatory RNA molecules. We have developed a method to isolate chromatin associated RNAs and used this method to isolate an RNA that accumulates on mouse centromeres. We propose to characterize this novel RNA. The characterization of both the RNA and the protein components of Xist and other RNA-protein complexes that affect chromatin structure will provide insight into the role nuclear RNA plays in regulating gene expression.