Structural and Functional Studies of lncRNAs in Gene Activation

lncRNA 在基因激活中的结构和功能研究

• 批准号: 10637407
• 负责人: Srinivas Somarowthu
• 金额: $ 38.83万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10637407
• 关键词: 3-Dimensional; Adopted; Affinity; Binding Sites; Biochemical; Biological Assay; Biological Models; CRISPR/Cas technology; Cell Proliferation; Cell physiology; Cells; Chemicals; Chromatin; Complex; Cryoelectron Microscopy; DNA; Development; Disease; Disease Progression; Down-Regulation; Elements; Epigenetic Process; Family; Gene Activation; Gene Expression Regulation; Gene Silencing; Genes; Genetic Transcription; Goals; Histones; Human; Immunoprecipitation; Investigation; Knowledge; Literature; Malignant Neoplasms; Mammals; Maps; Micrococcal Nuclease; Molecular; Mutate; Nucleosomes; Nucleotides; Plants; Play; Polycomb; Process; Promoter Regions; Proteins; RNA; Reporting; Research; Resolution; Roentgen Rays; Role; Site; Spectrometry; Structure; Structure-Activity Relationship; Sucrose; Technology; Testing; Transcript; Transcription Coactivator; Tumor Promotion; Tumor Suppressor Proteins; Untranslated RNA; Visualization; WNT Signaling Pathway; cell motility; chromatin remodeling; crosslink; deletion analysis; experimental study; genetic regulatory protein; insight; knock-down; mammalian genome; model building; novel; particle; potential biomarker; promoter; recruit; scaffold; therapeutic target; three dimensional structure; transcription factor; tumor progression

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.

项目摘要 在过去的二十年中，测序技术的突破表明哺乳动物基因组 编码数千个长的非编码RNA（LNCRNA）。已知的人类lncRNA数量 疾病的失调正在快速增长。新兴的证据表明lncrnas至关重要 基本细胞过程和疾病进展的调节剂。尽管它们具有潜力 生物标志物和治疗靶标，LNCRNA的分子机制仍然很少了解。这 这项研究的主要目标是更详细地阐明LNCRNA激活的机制 转录。许多LNCRNA与表观遗传机械有关。这些包括修改Hisstone 复合物，例如Polycomb反射复合物2和核小体重塑络合物，例如 SWI/SNF（开关/蔗糖不可发酵）复合物。虽然RNA-PRC2相互作用处于活动状态 研究，LNCRNA在核小体重塑中的作用仍然没有表征。在这里，我们试图 了解LNCRNA在基于SWI/SNF的基因激活中的功能。 SWI/SNF复合物是已知的肿瘤 抑制剂及其亚基在各种癌症中被突变。最近，LNCRNA被证明是相互作用的 与哺乳动物和植物中的SWI/SNF复合物。在此提案中，我们将利用LNCTCF7调查 LNCRNA在基于SWI/SNF的基因激活中的作用。先前的研究表明，LNCTCF7激活Wnt 通过募集SWI/SNF复合物来传导。但是，这些相互作用的生化和结构基础 没有建立。在初步研究中，我们鉴定 包含1）作为LNCTCF7的顶级交互合作伙伴。 SND1是转录共激活因子，已知它 与SWI/SNF家族交往。在这里，我们提出了一种新型机制，表明LNCTCF7记录了 SWI/SNF复合物通过与SND1的相互作用间接地。此外，我们的RNA化学探测研究确定了 跨哺乳动物基因组保守的LNCTCF7的结构域和区域。我们假设这些 保守的区域和结构化元素在招募蛋白质伙伴和激活中起关键作用 特定基因座的转录。我们将通过以下目的检验该假设：在AIM 1中，我们将定义 LNCTCF7领域的功能作用。 AIM 2将确定LNCTCF7-SND1相互作用在招募中的作用 SWI/SNF复合物和激活转录。在AIM 3中，我们将确定LNCTCF7的3D结构和 LNCTCF7-SND1复合物。这些研究将共同​​提供LNCTCF7的生化和结构知识 分子功能并扩展我们对LNCRNA如何调节基因转录的理解并有助于 疾病状态。