Molecular Switches: Regulatory RNAs Targeting Nuclear Hormone Receptor Promoters

分子开关：针对核激素受体启动子的调节 RNA

• 批准号: 8294678
• 负责人: BETHANY ANN JANOWSKI
• 金额: $ 24.62万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8294678
• 关键词: Affect; Animal Model; Antisense RNA; Binding; Binding Sites; Biochemical; Bioinformatics; Biological; Biological Assay; Biological Models; Breast Cancer Cell; Cancer cell line; Cells; Cleaved cell; Code; Cultured Cells; DNA; Data; Disease; Epithelial Cells; Functional RNA; Gene Activation; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Genome; Goals; Health; Human; Immunoprecipitation; Investigation; Libraries; Link; MCF7 cell; Mammalian Cell; Mammary gland; Mediating; Messenger RNA; Methods; MicroRNAs; Modeling; Molecular; Nuclear Hormone Receptors; Nucleic Acids; Nucleotides; Overlapping Genes; Pathway interactions; Process; Promoter Regions; Protein Binding; Proteins; Publishing; RNA; RNA Binding; RNA Polymerase II; RNA Stability; Recruitment Activity; Regulation; Repression; Research; Research Personnel; Role; Screening procedure; Site; Small RNA; T47D; Testing; Therapeutic; Transcript; Translating; Variant; Work; antigene; base; chromatin immunoprecipitation; design; gene discovery; heterochromatin-specific nonhistone chromosomal protein HP-1; mRNA Stability; novel; novel strategies; promoter; protein complex; research study; restoration

DESCRIPTION (provided by applicant): Recent results from transcriptome analysis demonstrate that most of the genome is transcribed and that RNA transcripts are generated from both DNA strands. The biological significance of these non-coding RNA transcripts in mammalian cells remains unclear. We propose that non-coding RNA transcripts interact with small non-coding antigene RNAs and proteins at gene promoters to regulate expression. Association between non-coding RNAs at promoters to control gene expression may represent a novel regulatory mechanism. Objectives: Our primary goal (Aim I) is to further delineate the mechanism by which antigene RNAs activate or repress PR gene expression by identifying the proteins involved, and determining how they interact with promoter DNA and RNA transcripts. Cell-based assays will be performed to identify the proteins involved. To understand protein-nucleic acid interactions we will use methods designed to isolate specific proteins bound to promoter DNA within the PR gene or RNA transcripts that are generated from the PR gene. These data will allow us to build a mechanistic model for RNA-mediated control of gene expression at gene promoters. Our second goal is to test whether activating agRNAs increase gene expression by inducing gene transcription, enhancing processive elongation and/or stabilizing RNA transcripts. Testing for agRNA-mediated effects on these mechanisms will aid in understanding key aspects of the agRNA mechanism of action. Our third second goal (Aim III) is to investigate the existence of endogenous antigene RNAs that regulate the PR gene. To identify potential antigene RNAs we will use bioinformatics approaches that screen microRNA libraries for sequences complementary to sites within the PR gene promoter. We will use biochemical and cell-based assays to test involvement of RNA "hits" that are identified in our screen. Significance: Association between antisense RNA transcripts and small non-coding RNAs at promoters to control gene expression may represent a novel regulatory mechanism. Understanding its mechanism will extend our understanding of why most of the genome is transcribed but not translated into protein, and how gene expression is regulated by RNA at promoters. This proposal will impact research by demonstrating that RNA can be used to target gene promoters and activate gene expression. The proposed experiments are designed to provide data that will be generally useful for investigators seeking to manipulate other genes in cultured cells and in animal models. PUBLIC HEALTH RELEVANCE: Understanding how non-coding RNAs selectively activate or repress gene expression of gene promoters may reveal a general mechanism of gene regulation that has not been previously appreciated. Many disease states are due to a decrease in the expression of a critical protein. Exploring the capability of RNA to specifically increase expression of a target gene will encourage new approaches in the treatment of disease and in developing therapeutic leads.

描述（由申请人提供）：最近转录组分析的结果表明，大部分基因组被转录，RNA转录物由两条DNA链产生。这些非编码RNA转录物在哺乳动物细胞中的生物学意义尚不清楚。我们提出非编码RNA转录物与小的非编码抗原RNA和基因启动子上的蛋白质相互作用来调节表达。启动子上非编码rna相互关联控制基因表达可能是一种新的调控机制。目的：我们的主要目标（Aim I）是通过鉴定相关蛋白，并确定它们如何与启动子DNA和RNA转录物相互作用，进一步描述抗原RNA激活或抑制PR基因表达的机制。将进行以细胞为基础的测定来鉴定所涉及的蛋白质。为了了解蛋白质与核酸的相互作用，我们将使用设计的方法分离PR基因内与启动子DNA结合的特定蛋白质或PR基因产生的RNA转录本。这些数据将使我们能够建立一个rna介导的基因启动子基因表达控制的机制模型。我们的第二个目标是测试激活agnas是否通过诱导基因转录、增强过程延伸和/或稳定RNA转录来增加基因表达。测试agRNA介导的对这些机制的影响将有助于理解agRNA作用机制的关键方面。我们的第三个目标（Aim III）是研究调节PR基因的内源性抗原rna的存在。为了鉴定潜在的抗原rna，我们将使用生物信息学方法筛选与PR基因启动子内位点互补的microRNA文库。我们将使用生化和基于细胞的分析来测试在我们的筛选中确定的RNA“命中”的参与。意义：反义RNA转录物与启动子上的小非编码RNA联合控制基因表达可能是一种新的调控机制。了解其机制将扩展我们对为什么大多数基因组被转录而不是翻译成蛋白质的理解，以及基因表达是如何由启动子处的RNA调节的。这一提议将通过证明RNA可以用于靶向基因启动子和激活基因表达来影响研究。拟议的实验旨在为研究人员在培养细胞和动物模型中寻求操纵其他基因提供通常有用的数据。公共卫生相关性：了解非编码rna如何选择性地激活或抑制基因启动子的基因表达，可能揭示以前未被认识到的基因调控的一般机制。许多疾病状态是由于一种关键蛋白表达的减少。探索RNA特异性增加靶基因表达的能力将鼓励疾病治疗的新方法和发展治疗先导。