RNA Regulation of Transcription Factor Activity

项目总结/摘要 灵活和反应灵敏的转录控制是所有生命过程的核心，因此这一过程是 在多个层面上进行监管。在上一次资助期间，我们发现了一种强大的，意想不到的RNA- 来自两大类转录因子（TF）的代表性蛋白质中固有的结合活性： 高迁移率族蛋白（HMGB）家族的多能性因子Sox 2和糖皮质激素核激素 受体（GR）。发现它们的RNA结合活性是由它们的DNA结合结构域介导的， 与结合到它们各自的启动子或增强子序列的DNA竞争。此外，我们发现， 这种活性在体外是结构特异性的而不是序列特异性的。两种TF均强烈不利于结合 ssRNA。相反，多能性因子Sox 2结合dsRNA区域，而GR仅结合RNA 发夹结构 这些观察结果提出了关于TF RNA结合活性在转录调控中的作用的关键问题。 监管，并为这里描述的研究计划提供直接动力。我们的佐证 在小鼠胚胎干细胞中发现Sox 2的RNA结合证实了直接RNA结合的发生 in vivo.下一步是进一步定义和理解体内RNA靶点的特异性， 确定TF RNA相互作用对转录程序的影响。这包括改进我们的 了解是什么驱动了这种RNA结合，并确定这种活性在其他TF中的普遍程度。 我们已经制定了一个综合和战略研究计划，以实现这些目标。目标1利用 我们观察到Sox 2直接与细胞中的RNA相互作用，并在GR中发展了这一关键的研究路线， 开发包括eRNA在内的全面的RNA相互作用组。我们将研究转录因子是否 利用它们的RNA结合活性提供一种替代的染色质缔合策略， 和基因调控。最后，我们利用我们的生物化学见解来设计和验证DNA， RNA结合功能分离突变体，这将使我们能够直接测量RNA丢失的影响， 与转录组结合。在目标2中，我们转向理解RNA缔合的分子性质， Sox 2和GR，首先通过使用eCLIP策略来鉴定共有结合基序， 通过TF/RNA复合物的高分辨率结构测定。在本计划的目标3中，我们将 建立这些观察的一般性，以其他重要的转录因子，无论是内部和外部 HMGB和核激素受体家族，分别以Sox 2和GR为代表。 总之，这项建议描述了一个全面的计划，寻求确定RNA的程度和作用， 通过与经典转录因子的相互作用调节转录程序。的 该计划的影响很大，因为了解全基因组普遍转录的作用， 这种活动如何影响不同的细胞过程仍然是生物学中一个主要的未解之谜。

Project Summary/Abstract Nimble and responsive transcriptional control is central to all processes of life, and hence this process is regulated at a number of levels. During the last grant period, we discovered a robust, and unexpected, RNA- binding activity inherent in representative proteins from two major classes of transcriptional factors (TFs): the pluripotency factor Sox2 of the high mobility group box (HMGB) family and the glucocorticoid nuclear hormone receptor (GR). Their RNA-binding activity was found to be mediated by their DNA-binding domains and directly compete with DNA binding to their respective promoter or enhancer sequences. Furthermore, we found that this activity was structure-specific rather than sequence specific in vitro. Both TFs strongly disfavored binding ssRNA. Instead, the pluripotency factor Sox2 bound dsRNA regions while GR bound exclusively to RNA hairpin structures. These observations raise critical questions regarding the role of TF RNA-binding activity in transcriptional regulation and provide the direct motivation for the research program described here. Our corroborating discovery of RNA association of Sox2 in mouse embryonic stem cells confirms that direct RNA binding occurs in vivo. The next steps are to further define and understand the specificity for the in vivo RNA targets and determine the impact TF RNA interactions has on the transcriptional program. This includes refining our understanding of what drives this RNA binding and determine how pervasive the activity is in other TFs. We have developed an integrated and strategic research program to achieve these goals. Aim 1 capitalizes on our observation that Sox2 directly interacts with RNAs in cells and develops this critical line of inquiry in GR to develop a comprehensive RNA interactome including eRNAs. We will investigate whether transcription factors use their RNA-binding activity to provide an alternate chromatin association strategy that impacts localization and gene regulation. Finally, we take advantage of our biochemical insights to design and validate DNA- and RNA-binding separation-of-function mutants, which will allow us to directly measure the impact of loss of RNA binding on the transcriptome. In Aim 2, we turn to understanding the molecular nature of RNA association to Sox2 and GR, first through the use of eCLIP strategies to identify consensus binding motifs and second through high resolution structure determination of TF/RNA complexes. In Aim 3 of this program, we will establish the generality of these observations to other important transcription factors, both within and beyond the HMGB and nuclear hormone receptor families, as represented by Sox2 and GR, respectively. Together, this proposal describes a comprehensive program seeking to determine the extent and role of RNA binding in modulating the transcriptional program through interaction with classic transcription factors. The impact of this program is high because understanding the roles of genome-wide pervasive transcription and how this activity influences diverse cellular processes remains a major unanswered question in biology.