Structural Studies of epigenetic regulation of rRNA gene by TTF-I

TTF-I对rRNA基因表观遗传调控的结构研究

• 批准号: 8691757
• 负责人: Carlos R Escalante
• 金额: $ 16.09万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8691757
• 关键词: Affect; Architecture; Binding; Biochemical; Biogenesis; Biological Assay; Cell Cycle Progression; Cell Nucleolus; Cell Proliferation; Cells; Cellular Stress; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Cockayne Syndrome; Collaborations; Complex; DNA Binding Domain; Development; Down-Regulation; Electron Microscopy; Epigenetic Process; Event; Foundations; Future; Gene Activation; Gene Expression; Gene Silencing; Genes; Genetic Transcription; Goals; Growth Factor; Hybrids; Individual; Lead; Link; MDM2 gene; Maps; Modeling; Molecular; Molecular Profiling; Montserrat; Nucleosomes; Nutrient; Oncogene Proteins; Play; Positioning Attribute; Process; Proteins; Reaction; Recombinant DNA; Recruitment Activity; Regulation; Resolution; Ribosomes; Role; Signal Transduction; Site; Solid; Structure; System; Time; Transcription Process; Transcriptional Activation; Tumor Suppressor Proteins; Up-Regulation; Work; X-Ray Crystallography; cancer cell; cancer therapy; cell growth; chromatin remodeling; design; gene synthesis; novel therapeutics; particle; prevent; promoter; protein B; protein complex; protein protein interaction; public health relevance; rRNA Genes; reconstruction; research study; termination factor; tool; transcription termination; transcription termination factor I; tumor molecular fingerprint

DESCRIPTION (provided by applicant): Transcription and processing of ribosomal RNA genes (rRNA) are intimately linked to processes that control cell growth and proliferaton. Expression of rRNA is regulated by multiple signals such as nutrient availability, growth factors and cellular stress. Not surprising, increasing evidence suggest that large changes in the up-regulation of rRNA synthesis may be one of the most important signatures of cancer cells. Transcription Terminator Factor (TTF-I) is an essential factor in rRNA transcription that also determines the overall architecture of the rDNA promoter. TTF-I orchestrates the epigenetic state of both active and silenced genes through the recruitment of different set of chromatin remodelers and modifiers. In active rRNA genes, TTF-I binds to the terminator T0 site and recruits Cockayne Sindrome Protein B (CSB) that in an ATP dependent reaction modifies the position of nucleosomes in the promoter and activates transcription of the gene. In addition, TTF-I can recruit the Nucleolus Remodeler complex (NoRC) to have the opposite effect in transcription and promotes rRNA gene silencing. Although many details about the general features of each mechanism are known, a mechanistic view of the role of TTF-I in the epigenetic regulation of rRNA genes at the atomic level is lacking. In this new R21 we proposed to develop a system to understand: 1) How TTF-I binds chromatin templates and the structural changes occurring after this initial binding and 2) How TTF-I recruits CSB and the changes that occur upon recruitment in the position of the nucleosomes. We will use a combination of functiona assays to map the interactions of TTF-I and CSB and a hybrid structural approach combining X-ray crystallography, single-particle reconstruction electron microscopy (EM) and small-angle x-ray scattering (SAXS). In addition to obtaining for the first time a detailed viw of the remodeling complexes and the changes leading to the activation of rRNA genes, the system developed in this application will lead us into future studies of gene silencing by th recruitment of the NoRC complex and the protein complexes that regulate the cellular abundance of TTF-I such as its interaction with tumor suppressors ARFp19 and MDM2. This work will be in close collaboration with Dr. Montserrat Samso that will provide expertise in single-particle reconstruction EM and Dr. Deepak Bastia that will provide technical help in the biochemical assays.

描述（由申请人提供）：核糖体RNA基因（RRNA）的转录和处理与控制细胞生长和增殖的过程密切相关。 rRNA的表达受养分可用性等多种信号的调节 因素和细胞应激。毫不奇怪，越来越多的证据表明，rRNA合成上调的大量变化可能是癌细胞最重要的特征之一。 转录终结因子（TTF-I）是RRNA转录的重要因素，它也决定了RDNA启动子的整体结构。 TTF-I通过募集不同的染色质重塑剂和修饰剂的募集来策划活性基因和沉默基因的表观遗传状态。 在活性rRNA基因中，TTF-I与终结剂T0位点结合，并募集Cockayne sindrome蛋白B（CSB），在ATP依赖反应中，核小体在启动子中的位置修饰并激活基因的转录。 此外，TTF-I可以募集核仁重塑仪（NORC）在转录中具有相反的作用，并促进rRNA基因沉默。尽管已知有关每种机制的一般特征的许多细节，但缺乏TTF-I在原子水平上rRNA基因表观遗传调节中的作用的机械观点。 在新的R21中，我们建议开发一个系统来理解：1）TTF-I如何结合染色质模板以及这种初始结合后发生的结构变化； 2）TTF-I如何募集CSB以及在核体位置募集时发生的变化。 我们将使用功能A分析的组合来绘制TTF-I和CSB的相互作用以及结合X射线晶体学，单粒子重建电子显微镜（EM）和小角X射线散射（SAXS）的杂种结构方法。 除了首次获得重塑络合物的详细viw以及导致rRNA基因激活的变化外，在本应用中开发的系统还将导致我们通过募集NORC复合物和蛋白质复合物和蛋白质复合物的未来对基因沉默的研究，这些蛋白质复合物调节了TTF-I与TTF-I的细胞丰富性，例如与TTF-I的细胞丰富性，例如与TTTF-I相互作用的undor pessress 29和Arfpp的相互作用。 这项工作将与Montserrat Samso博士密切合作，该博士将提供单粒子重建EM和Deepak Bastia博士的专业知识，这些知识将在生化测定中提供技术帮助。