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)：在线粒体和细胞质中产生的活性氧物种(ROS)，在全球范围内诱导基因组中的单链断裂(SSB)和氧化碱基。以前未知的启动子/增强子区域的局部氧化损伤，是由赖氨酸特异性去甲基酶(LSD1 in KMD家族)和Jumanji(JMJ)家族酶氧化去甲基化组蛋白H3/H4中的甲基Lys/Arg产生的核ROS，以及在转录激活过程中由Tet双加氧酶引起的启动子序列中甲基CpG位点的核ROS。雌激素(E_2)、维甲酸(RA)和肿瘤坏死因子α等配体通过与同源受体结合，激活成百上千个靶基因，再通过复合组蛋白修饰和CpG去甲基化形成多蛋白复合体，形成染色质，从而启动转录。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，然后由SSB传感器PARP1、XRCC1和末端加工酶(如APE1、PNKP)启动的修复。通过定量聚合酶链式反应检测定点特异性修复、碱性彗星实验检测全球基因组修复和芯片分析发现bcl2基因E2激活的基因组损伤和修复速率较快(10min内完成)，IL1α启动子的中等损伤修复速率(~1h)，RA激活的RARα2基因损伤修复速率较慢(~4h)。Co-IP分析表明RA激活增加了LSD1和APE1之间的相互作用，CHIP实验证实了PARP1和其他SSBR蛋白在增强子位置的募集增加。这种基因激活过程中调控区域特异性损伤诱导和修复的普遍现象与损伤信号和毒性有关，我们将通过追求以下目标来表征这一现象。目的1.验证bcl2、IL1β和RARβ2基因的增强子/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存在的假说。我们将确定组蛋白和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，并表征在SSB位点招募的修复亚途径和末端加工酶。目的2.验证修复蛋白被去甲基酶招募到启动子/增强子位置形成修复复合体的假说。我们将描述脱甲基酶与修复蛋白的复合体，可能包括核苷酸切除修复的复合体。目的3.揭示PARP1在基因激活过程中诱导的启动子/增强子特异性SSB修复中的作用。我们将测试PARP抑制或耗尽是否会破坏所有系统的修复和转录激活，以及SSB修复抑制是否只在复制细胞转录激活后诱导细胞凋亡。我们的国家 先进的方法和复杂的试剂将建立一种新的范式，关于依赖基因激活的基因组损伤和修复，具有治疗潜力。