Repair of Oxidative Genome Damage Associated with Gene Activation

修复与基因激活相关的氧化基因组损伤

• 批准号: 8639248
• 负责人: Sankar Mitra
• 金额: $ 30.31万
• 依托单位: METHODIST HOSPITAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-15 至 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8639248
• 关键词: Alkaline Single-Cell Gel Electrophoresis Assay; Apoptosis; BCL2 gene; Binding; Biological Assay; Cancer Etiology; Cells; Chromatin; Chromatin Structure; Comet Assay; Complex; Cytosol; DNA Modification Process; Dioxygenases; Disease; Down-Regulation; Elements; Enhancers; Enzymes; Epigenetic Process; Estrogen Receptors; Estrogens; Excision; Family; Family member; Gene Activation; Gene Expression Regulation; Gene Targeting; Generations; Genes; Genetic Transcription; Genome; Health; Histone H3; Histones; Human Genome; Hydrogen Peroxide; In Vitro; Inflammation; Kinetics; Ligand Binding; Ligands; Link; Lysine; MCF7 cell; Malignant Neoplasms; Mediating; Methylation; Mitochondria; Monitor; N-terminal; Nuclear; Nucleic Acid Regulatory Sequences; Nucleotide Excision Repair; PARP inhibition; Pathway interactions; Process; Production; Proteins; Reaction; Reactive Oxygen Species; Reagent; Recruitment Activity; Relative (related person); Repair Complex; Repression; Resources; Respiration; Response Elements; Role; Signal Transduction; Single Strand Break Repair; Site; Specificity; System; Testing; Therapeutic; Toxic effect; Transcription Initiation; Transcriptional Activation; Tretinoin; Tumor Necrosis Factor-alpha; XRCC1 gene; arginyllysine; base; chromatin immunoprecipitation; demethylation; experience; gene repression; genome-wide; histone modification; human APEX1 protein; induced pluripotent stem cell; inhibitor/antagonist; mutant; new therapeutic target; oxidation; oxidative damage; prevent; promoter; protein complex; receptor; repaired; sensor

DESCRIPTION (provided by applicant): Reactive oxygen species (ROS), generated in mitochondria and cytosol, globally induce single-strand breaks (SSBs) and oxidized bases in the genome. Previously unrecognized, localized oxidative damage in promoter/enhancer regions, are induced by nuclear ROS produced during oxidative demethylation of methyl Lys/Arg in histones H3/H4 by lysine specific demethylase (LSD1in KMD family), and Jumanji (JMJ) family enzymes, and of methyl CpG sites in promoter sequences by TET dioxygenases during transcriptional activation. Ligands including estrogen (E2), retinoic acid (RA) and TNFα activate hundreds of target genes by binding to cognate receptors, which then form multi-protein complexes to unfold chromatin via complex histone modifications and CpG demethylation as a prerequisite for transcription initiation. We observed transient formation of single-strand breaks (SSBs), localized to the cis elements, presumably caused by ROS products of demethylases followed by repair which is initiated by SSB sensor PARP1, XRCC1 and end processing enzymes, e.g., APE1, PNKP. By monitoring site-specific repair using quantitative PCR, global genome repair by alkaline Comet assay and recruitment of repair proteins at the enhancer site via ChIP analysis, we observed that genome damage and repair rates are fast (completed in 10 min) for E2 activation of the BCL2 gene, moderate (~1 h) for TNFα activation of IL1α promoter and slow for RA activation of the RARβ2 gene (~4 h). Co-IP analysis showed that RA activation increased interaction between LSD1 and APE1and ChIP assay confirmed increased recruitment of PARP1 and other SSBR proteins at the enhancer site. This universal phenomenon of regulatory region specific damage induction and repair during gene activation has implications in damage signaling and toxicity which we will characterize by pursuing the following aims. Aim 1. To test the hypothesis that enhancer/promoter-specific oxidized bases and SSBs in BCL2, IL1β and RARβ2 genes and their repair are universally induced during gene activation. We will determine relative contribution of histone vs. CpG demethylation to the SSB production, elucidate the mechanism of recruitment of repair proteins and characterize the repair sub-pathways and end processing enzymes recruited at the SSB site. Aim 2. To test the hypothesis that repair proteins are recruited at promoter/enhancer sites by demethylases to form repair complexes. We will characterize complexes of demethylases with repair proteins, possibly including those of nucleotide excision repair. Aim 3. To unravel the role of PARP1 in repair of promoter/enhancer-specific SSBs induced during gene activation. We will test if PARP inhibition or depletion abrogates repair and transcriptional activation in all systems, and if SSB repair inhibition induces apoptosis after transcription activation only in replicating cells. Our state-of the-art approaches and sophisticated reagents will establish a new paradigm about gene activation-dependent genome damage and repair with therapeutic potential.

描述（由申请人提供）：线粒体和细胞质中产生的活性氧（ROS）在整体上诱导基因组中的单链断裂（SSB）和氧化碱基。启动子/增强子区域先前未被识别的局部氧化损伤是由赖氨酸特异性去甲基化酶（KMD 家族中的 LSD1）和 Jumanji (JMJ) 家族酶对组蛋白 H3/H4 中的甲基 Lys/Arg 进行氧化去甲基化过程中产生的核 ROS 以及转录过程中 TET 双加氧酶对启动子序列中的甲基 CpG 位点进行氧化去甲基化过程中产生的细胞核 ROS 诱导的。 激活。包括雌激素 (E2)、视黄酸 (RA) 和 TNFα 在内的配体通过与同源受体结合激活数百个靶基因，然后形成多蛋白复合物，通过复杂的组蛋白修饰和 CpG 去甲基化展开染色质，作为转录起始的先决条件。我们观察到单链断裂 (SSB) 的瞬时形成，定位于顺式元件，可能是由去甲基酶的 ROS 产物引起的，随后由 SSB 传感器 PARP1、XRCC1 和末端加工酶（例如 APE1、PNKP）启动修复。通过使用定量 PCR 监测位点特异性修复、通过碱性彗星测定进行全局基因组修复以及通过 ChIP 分析在增强子位点招募修复蛋白，我们观察到，对于 BCL2 基因的 E2 激活，基因组损伤和修复速度很快（10 分钟内完成）；对于 IL1α 启动子的 TNFα 激活，基因组损伤和修复速度中等（~1 小时）；对于 RARβ2 基因的 RA 激活，基因组损伤和修复速度较慢（~4 h）。 Co-IP 分析表明 RA 激活增加了 LSD1 和 APE1 之间的相互作用，ChIP 测定证实增强子位点处 PARP1 和其他 SSBR 蛋白的募集增加。基因激活过程中调节区域特异性损伤诱导和修复的普遍现象对损伤信号传导和毒性具有影响，我们将通过追求以下目标来表征。目标 1. 检验以下假设：BCL2、IL1β 和 RARβ2 基因中的增强子/启动子特异性氧化碱基和 SSB 及其修复在基因激活过程中普遍被诱导。我们将确定组蛋白与 CpG 去甲基化对 SSB 产生的相对贡献，阐明修复蛋白招募的机制，并表征修复子途径和在 SSB 位点招募的末端加工酶。目标 2. 检验修复蛋白通过去甲基酶在启动子/增强子位点募集以形成修复复合物的假设。我们将表征去甲基酶与修复蛋白的复合物，可能包括核苷酸切除修复的复合物。目标 3. 阐明 PARP1 在修复基因激活过程中诱导的启动子/增强子特异性 SSB 中的作用。我们将测试 PARP 抑制或耗竭是否会消除所有系统中的修复和转录激活，以及 SSB 修复抑制是否仅在复制细胞中在转录激活后诱导细胞凋亡。我们的现状 最先进的方法和复杂的试剂将建立一个关于基因激活依赖性基因组损伤和具有治疗潜力的修复的新范例。