Identifying the epigenetic regulation of arsenic exposure - Revision 2

 描述(申请人提供)：染色质上必须适当地发生多种大分子蛋白质相互作用，以驱动染色体生物学的功能方面，如基因转录、DNA复制、重组、修复和姐妹染色单体分离。分析蛋白质如何在体内与染色质相互作用来指导这些活动仍然是一个重大的挑战，因为它们之间的联系具有时间性和动态性。我们最近开发了一种名为染色质亲和纯化与质谱学(CHAP-MS)的技术，该技术提供了对染色体天然1kb片段的浓缩，用于对蛋白质相互作用和相关的组蛋白翻译后修饰(PTM)进行位点特异性鉴定。在这项拨款申请中，我们计划使用这一尖端方法来确定组蛋白PTM和在萌芽酵母的砷反应位置调节转录的蛋白质。我们推测，CHAP-MS将为所有组蛋白修饰和调控酿酒酵母砷基因转录的蛋白质提供全面和公正的鉴定。我们将追求以下目标来验证这一假设：使用CHAP-MS来定义酿酒酵母中组蛋白PTM和调控砷反应位点转录的蛋白质。

 DESCRIPTION (provided by applicant): A multitude of macromolecular protein interactions must properly occur on chromatin to drive functional aspects of chromosome biology like gene transcription, DNA replication, recombination, repair and sister chromatid segregation. Analyzing how proteins interact in vivo with chromatin to direct these activities remains a significant challenge due to the temporal and dynamic nature of their associations. We have recently developed a technique termed Chromatin Affinity Purification with Mass Spectrometry (ChAP-MS) that provides for the enrichment of a native 1 kb section of a chromosome for site-specific identification of protein interactions and associated histone posttranslational modifications (PTMs). In this grant application, we plan to use this cutting-edge approach to define the histone PTMs and proteins regulating transcription at the arsenic response locus in budding yeast. We hypothesize that ChAP-MS will provide for a comprehensive and unbiased identification of all histone modifications and proteins regulating transcription at the arsenic locus in S. cerevisiae. We will pursue the following Aim to test this hypothesis: Use ChAP-MS to define the histone PTMs and proteins regulating transcription at the arsenic response locus in S. cerevisiae.