Repair of Oxidative Genome Damage Associated with Gene Activation

描述（由申请人提供）：线粒体和胞质溶胶中产生的活性氧（ROS）可全面诱导基因组中的单链断裂（SSB）和氧化碱基。在转录激活过程中，组蛋白H3/H4中的甲基Lys/Arg通过赖氨酸特异性去甲基化酶（KMD家族中的LSD 1）和Jumanji（JMJ）家族酶氧化去甲基化，启动子序列中的甲基CpG位点通过泰特双加氧酶氧化去甲基化，从而产生细胞核ROS，这是以前未认识到的启动子/增强子区域的局部氧化损伤。包括雌激素（E2）、视黄酸（RA）和TNF？在内的配体通过与同源受体结合激活数百个靶基因，然后通过复杂的组蛋白修饰和CpG去甲基化形成多蛋白复合物以展开染色质，这是转录起始的先决条件。我们观察到定位于顺式元件的单链断裂（SSB）的瞬时形成，推测是由脱甲基酶的ROS产物引起的，随后是由SSB传感器PARP 1、XRCC 1和末端加工酶（例如，APE 1、PNKP。通过使用定量PCR监测位点特异性修复，通过碱性彗星试验监测全局基因组修复，以及通过ChIP分析在增强子位点募集修复蛋白，我们观察到基因组损伤和修复速率很快对于BCL 2基因的E2激活，IL 1启动子的TNF激活中度（~1 h），RAR 2基因的RA激活缓慢（~4 h）。Co-IP分析表明RA激活增加了LSD 1和APE 1之间的相互作用，ChIP分析证实了增强子位点上PARP 1和其他SSBR蛋白的募集增加。在基因活化过程中，这种调控区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有影响，我们将通过追求以下目标来表征。目标1。验证BCL 2、IL 1 <$和RAR <$2基因中增强子/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诱导的假设。我们将确定组蛋白与CpG去甲基化对SSB产生的相对贡献，阐明修复蛋白的募集机制，并表征在SSB位点募集的修复子途径和末端加工酶。目标二。为了检验修复蛋白在启动子/增强子位点被脱甲基酶募集以形成修复复合物的假设。我们将表征脱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PARP 1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抑制或耗竭是否在所有系统中废除修复和转录激活，以及SSB修复抑制是否仅在复制细胞中在转录激活后诱导凋亡。我们的国家 现有技术的方法和复杂的试剂将建立关于具有治疗潜力的基因激活依赖性基因组损伤和修复的新范例。

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 TNF¿ 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 TNF¿ activation of IL1¿ promoter and slow for RA activation of the RAR¿2 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, IL1¿ and RAR¿2 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.