Structural and Functional Studies of lncRNAs in Gene Activation

项目摘要 在过去的二十年里，测序技术的突破表明，哺乳动物的基因组 编码数千个长的非编码RNA（lncRNA）。已知的人类lncRNA的数量 失调疾病的发病率正在迅速上升。新出现的证据表明lncRNA是至关重要的 基本细胞过程和疾病进展的调节因子。尽管它们作为潜在的 尽管lncRNA是生物标志物和治疗靶点，但lncRNA的分子机制仍然知之甚少。的 本研究的主要目的是更详细地阐明lncRNA激活 转录。许多lncRNA与表观遗传机制相关;这些包括组蛋白修饰 复合物，如polycomb抑制复合物2，和核小体重塑复合物，如 SWI/SNF（SWITCH/蔗糖不可发酵）复合物。当RNA-PRC 2相互作用不活跃时， 研究中，lncRNA在核小体重塑中的作用仍然未被表征。在此，我们寻求 了解lncRNA在基于SWI/SNF的基因激活中的功能。SWI/SNF复合体是已知的肿瘤 抑制子，其亚基在各种癌症中突变。最近，lncRNA已被证明可以相互作用， SWI/SNF复合物在哺乳动物和植物中的作用。在本提案中，我们将利用lncTCF 7来研究 lncRNA在基于SWI/SNF的基因激活中的作用。先前的研究表明，lncTCF 7激活Wnt 通过募集SWI/SNF复合物进行信号传导。然而，这些相互作用的生物化学和结构基础 是不成立的。在初步研究中，我们鉴定了SND 1（葡萄球菌核酸酶和Tudor结构域 含有1）作为lncTCF 7的顶级相互作用配偶体。SND 1是一种转录辅激活因子，已知它 加入SWI/SNF家族。在这里，我们提出了一种新的机制，表明lncTCF 7招募了 SWI/SNF复合物通过其与SND 1的相互作用间接地与SND 1结合。此外，我们的RNA化学探测研究发现， 哺乳动物基因组中保守的lncTCF 7的结构域和区域。我们假设这些 保守区域和结构元件在招募蛋白质伴侣和激活蛋白质中起关键作用。 在特定位点的转录。我们将通过以下目标来测试这一假设：在目标1中，我们将定义 lncTCF 7结构域的功能作用。目标2将确定lncTCF 7-SND 1相互作用在招募中的作用 SWI/SNF复合物和激活转录。在目标3中，我们将确定lncTCF 7的3D结构和IncTCF 7的分子结构。 lncTCF 7-SND 1复合物。总之，这些研究将提供lncTCF 7的生物化学和结构知识， 分子功能，并扩大我们对lncRNA如何调节基因转录的理解，并有助于 疾病状态。

Project Summary Over the last two decades, breakthroughs in sequencing technologies have revealed that mammalian genomes encode thousands of long non-coding RNAs (lncRNAs). The numbers of human lncRNAs that are known to be dysregulated in diseases are rising at a rapid pace. Emerging evidence suggests that lncRNAs are critical regulators of fundamental cellular processes and disease progression. Despite their significance as potential biomarkers and therapeutic targets, the molecular mechanisms of lncRNAs remain poorly understood. The primary goal of this research is to elucidate in greater detail the mechanisms by which lncRNAs activate transcription. Many lncRNAs are associated with epigenetic machinery; these include histone-modifying complexes, such as the polycomb repressive complex 2, and nucleosome remodeling complexes, such as the SWI/SNF (SWItch/Sucrose Non-Fermentable) complex. While the RNA-PRC2 interactions are under active investigation, the role of lncRNAs in nucleosome remodeling remains uncharacterized. Here, we seek to understand the function of lncRNAs in SWI/SNF-based gene activation. The SWI/SNF complex is a known tumor suppressor, and its subunits are mutated in various cancers. Recently, lncRNAs have been shown to interact with the SWI/SNF complex in mammals and plants. In this proposal, we will utilize lncTCF7 to investigate the role of lncRNAs in SWI/SNF-based gene activation. Previous studies have shown that lncTCF7 activates Wnt signaling by recruiting the SWI/SNF complex. However, the biochemical and structural basis of these interactions is not established. In preliminary studies, we identified SND1 (Staphylococcal Nuclease and Tudor Domain Containing 1) as the top interaction partner of lncTCF7. SND1 is a transcriptional coactivator, and it is known to associate with the SWI/SNF family. Here, we propose a novel mechanism suggesting that lncTCF7 recruits the SWI/SNF complex indirectly via its interaction with SND1. Further, our RNA chemical probing studies identified structural domains and regions of lncTCF7 conserved across mammalian genomes. We hypothesize that these conserved regions and structured elements play critical roles in recruiting protein partners and activating transcription at specific loci. We will test this hypothesis through the following aims: In Aim 1, we will define the functional role of lncTCF7’s domains. Aim 2 will determine the role of lncTCF7-SND1 interaction in recruiting the SWI/SNF complex and activating transcription. In Aim 3, we will determine the 3D structure of lncTCF7 and the lncTCF7-SND1 complex. Together, these studies will provide biochemical and structural knowledge of lncTCF7’s molecular function and expand our understanding of how lncRNAs regulate gene transcription and contribute to disease states.