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

描述(申请人提供)：核糖体RNA基因(RRNA)的转录和加工与控制细胞生长和增殖的过程密切相关。RRNA的表达受多种信号的调节，如营养物质的可获得性、生长 因素和细胞压力。不足为奇的是，越来越多的证据表明，rRNA合成上调的巨大变化可能是癌细胞最重要的标志之一。转录终止因子(TTF-I)是rRNA转录中的重要因子，也决定了rDNA启动子的整体结构。TTF-I通过招募不同的染色质重构体和修饰物来协调激活和沉默基因的表观遗传状态。在活性rRNA基因中，TTF-I结合到终止子T0位点，并招募Cockayne综合征蛋白B(CSB)，在ATP依赖的反应中改变核小体在启动子中的位置，激活基因的转录。此外，TTF-I可以招募核仁Remodeler复合体(NORC)在转录中起相反的作用，并促进rRNA基因沉默。虽然关于每种机制的一般特征的许多细节是已知的，但缺乏关于TTF-I在原子水平上的rRNA基因的表观遗传调控中的作用的机制观点。在这个新的R21中，我们建议开发一个系统来了解：1)TTF-I是如何与染色质模板结合的，以及在这种初始结合之后发生的结构变化；2)TTF-I是如何招募CSB的，以及当招募到核小体的位置时发生的变化。我们将使用组合的功能分析来描绘TTF-I和CSB的相互作用，以及结合X射线结晶学、单粒子重建电子显微镜(EM)和小角X射线散射(SAXS)的混合结构方法。除了首次获得重塑复合体和导致rRNA基因激活的变化的详细VW外，在这一应用中开发的系统还将引导我们未来通过招募NORC复合体和调节TTF-I细胞丰度的蛋白质复合体来实现基因沉默的研究，例如它与肿瘤抑制基因ARFp19和MDM2的相互作用。这项工作将与蒙特塞拉特·萨姆索博士密切合作，后者将提供单颗粒重建EM方面的专业知识，而迪帕克·巴斯蒂亚博士将提供生化分析方面的技术帮助。

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.