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: 7365505
• 负责人: ALEXEI V TULIN
• 金额: $ 31.86万
• 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/7365505
• 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; Development; 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; Localized; Maps; Mass Spectrum Analysis; Measures; Metabolism; Methods; Modeling; Mutate; Mutation; N-terminal; Nicotinamide adenine dinucleotide; 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; Purpose; Recombinants; Regulation; Relative (related person); Resources; Role; Sepharose; Serine; Site; Solutions; Specific qualifier value; Standards of Weights and Measures; Sucrose; System; Tail; Tertiary Protein Structure; Testing; Therapeutic; 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 structure; research study; tandem mass spectrometry

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）：该提案的目的是研究蛋白质的聚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）酶的相对活性，该酶利用NAD来创建PADPR修饰的蛋白质，以及聚（ADP-核糖）糖脂酶（PARG）酶，该酶（PARG）酶，该酶的去除Moieties。我在果蝇中的研究首先揭示了PARP蛋白在建立无声染色质结构域以及染色质松动和转录激活中的至关重要作用。随后，增加活性PARP1蛋白的表达增加与凝结和沉默的染色质结构域的形成有关，而在刺激PARP酶活性，染色质否定质并变为转录活性之后。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理，以及（2）局部PARP激活的机理。在初步研究中，我们通过使用串联亲和力纯化（TAP）策略以及蔗糖梯度纯化和液相色谱串联质谱法（LC-MS/MS），成功地通过使用串联亲和力纯化（TAP）策略来成功鉴定了与染色质相关的PARP1伙伴。使用标准的遗传方法与免疫染色和共聚焦显微镜结合使用标准的遗传方法，在功能上分析了由这种方法引起的候选相互作用者对PARP1的影响。基于这项工作，我们已经确定了变体H2AV是一种促进PARP1靶向染色质的蛋白质。其他组对组蛋白H2AV的最新研究表明，这种蛋白质会触发DNA修复，凋亡和异染色质形成。 Ser137-已显示磷酸化的H2AV与体内局部PARP1激活的焦点共定位。在此提案中，我们将重点关注PARP1靶向染色质的两个方面。首先，我们将表征H2AV控制PARP1蛋白掺入染色质并调节染色质指导的PARP活性的机制。其次，我们将评估PARP1蛋白结构域的功能，定义指定与染色质的PARP1相互作用的功能。我们将以以下特定目的解决这些问题：目标1。表征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。目的3。研究自动化和磷酸化在调节体内染色质的PARP1靶向中的作用。 与公共卫生陈述相关：了解生物体如何利用PARP蛋白来调节染色质和转录的能力将提供对生长，发育和发病机理至关重要的遗传过程的基本见解。