Poly ADP-ribose Polymerase in Chromatin and Transcriptional Regulation

染色质和转录调控中的聚 ADP-核糖聚合酶

• 批准号: 7760967
• 负责人: ALEXEI V TULIN
• 金额: $ 31.87万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-15 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7760967
• 关键词: ADP ribosylation; Address; Affinity; Affinity Chromatography; Apoptosis; Binding; Biological Assay; Bos taurus PARP protein; Cells; Chimeric Proteins; Chromatin; Complex; Confocal Microscopy; Core Protein; Coupled; DNA Repair; Data; Detection; Developmental Gene; Drosophila genus; Enzymes; Fluorescence Recovery After Photobleaching; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Processes; Genetic Transcription; Goals; Growth and Development function; Heat-Shock Response; Heterochromatin; Histone H2A; Histones; In Vitro; Individual; Knowledge; Liquid Chromatography; Maps; Mass Spectrum Analysis; Measures; Metabolism; Methods; Modeling; Mutate; Mutation; N-terminal; Organism; Pathogenesis; Peptides; Pharmaceutical Preparations; Phosphorylation; Point Mutation; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Process; Protein Binding; Protein Isoforms; Proteins; Proteolysis; Public Health; Recombinants; Regulation; Relative (related person); Resources; Role; Sepharose; Serine; Site; Solutions; Specific qualifier value; Sucrose; System; Tail; Tertiary Protein Structure; Testing; Transcriptional Activation; Transcriptional Regulation; Transgenic Organisms; Variant; Work; base; casein kinase II; crosslink; fly; in vitro Assay; in vivo; insight; mutant; novel; poly ADP-ribose glycohydrolase; programs; protein activation; protein complex; protein structure; research study; tandem mass spectrometry; therapeutic development

DESCRIPTION (provided by applicant): The goal of this proposal is to investigate the mechanisms by which poly ADP-ribosylation (pADPr) of proteins regulates gene expression. The level of protein pADPr reflects the relative activities of the poly(ADP-ribose) polymerase (PARP) enzyme, which utilizes NAD to create pADPr-modified proteins, and the poly(ADP-Ribose) glycohydrolase (PARG) enzyme, which removes pADPr moieties. My studies in Drosophila first revealed vital roles for PARP protein in the establishment of silent chromatin domains as well as in the chromatin loosening and transcriptional activation of a subset of inducible chromosomal loci. Subsequently, increased expression of inactive PARP1 protein has been implicated in the formation of condensed and silent chromatin domains, whereas upon the stimulation of PARP enzymatic activity, chromatin decondenses and becomes transcriptionally active. At present, the main gaps in our understanding of the PARP1-dependent transcriptional regulation are (1) the mechanism of PARP protein targeting to specific chromatin domains, and (2) the mechanism of local PARP activation. In preliminary studies, we have successfully identified novel chromatin-associated PARP1 partners by use of a Tandem Affinity Purification (TAP) strategy together with sucrose gradient purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Candidate interactors arising from this approach were functionally analyzed for influence on PARP1, using standard genetic approaches combined with immunostaining and confocal microscopy. Based on this work, we have identified the variant histone H2Av as a protein that promotes targeting of PARP1 to chromatin. Recent studies of histone H2Av by other groups have suggested this protein triggers DNA repair, apoptosis and heterochromatin formation. Ser137- phosphorylated H2Av has been shown to co-localize with foci of local PARP1 activation in vivo. In this proposal, we will focus on two aspects of PARP1 targeting to chromatin. First, we will characterize the mechanism through which H2Av controls PARP1 protein incorporation to chromatin and regulates chromatin-directed PARP activity. Second, we will evaluate functions of the PARP1 protein domains, defining which specify PARP1 interactions with chromatin. We are will address these questions with the following specific aims: Aim 1. To characterize the mechanism by which H2Av targets PARP1 to chromatin. Aim 2. To determine the role of core histones in the PARP1 protein binding to chromatin. Aim 3. To investigate the roles of automodification and phosphorylation in regulating PARP1 targeting to chromatin in vivo. Relevance to Public Health Statement: Understanding how an organism can utilize PARP protein ability to modulate chromatin and transcription will provide fundamental insight into genetic processes that are essential for growth, development, and pathogenesis.

描述（由申请人提供）：本提案的目的是研究蛋白质的多聚 ADP 核糖基化 (pADPr) 调节基因表达的机制。蛋白质 pADPr 的水平反映了聚（ADP-核糖）聚合酶（PARP）和聚（ADP-核糖）糖水解酶（PARG）的相对活性，前者利用 NAD 产生 pADPr 修饰的蛋白质，后者去除 pADPr部分。我对果蝇的研究首先揭示了 PARP 蛋白在沉默染色质结构域的建立以及染色质松弛和诱导染色体位点子集的转录激活中的重要作用。随后，非活性 PARP1 蛋白表达的增加与浓缩和沉默染色质结构域的形成有关，而在 PARP 酶活性的刺激下，染色质解浓缩并变得转录活跃。目前，我们对PARP1依赖性转录调控的理解的主要差距是（1）PARP蛋白靶向特定染色质结构域的机制，以及（2）局部PARP激活的机制。在初步研究中，我们通过使用串联亲和纯化 (TAP) 策略以及蔗糖梯度纯化和液相色谱-串联质谱 (LC-MS/MS)，成功鉴定了新型染色质相关 PARP1 伴侣。使用标准遗传方法结合免疫染色和共聚焦显微镜，对这种方法产生的候选相互作用因子对 PARP1 的影响进行了功能分析。基于这项工作，我们确定了组蛋白 H2Av 变体是一种促进 PARP1 靶向染色质的蛋白质。其他研究小组最近对组蛋白 H2Av 的研究表明，这种蛋白会触发 DNA 修复、细胞凋亡和异染色质形成。 Ser137-磷酸化的 H2Av 已被证明与体内局部 PARP1 激活的焦点共定位。在本提案中，我们将重点关注 PARP1 靶向染色质的两个方面。首先，我们将描述 H2Av 控制 PARP1 蛋白掺入染色质并调节染色质导向的 PARP 活性的机制。其次，我们将评估 PARP1 蛋白结构域的功能，定义其指定 PARP1 与染色质的相互作用。我们将通过以下具体目标来解决这些问题： 目标 1. 描述 H2Av 将 PARP1 靶向染色质的机制。目标 2. 确定核心组蛋白在 PARP1 蛋白与染色质结合中的作用。目标 3. 研究自身修饰和磷酸化在体内调节 PARP1 靶向染色质中的作用。 与公共卫生的相关性声明：了解生物体如何利用 PARP 蛋白能力来调节染色质和转录，将为了解生长、发育和发病机制所必需的遗传过程提供基本见解。