DNA糖基化酶OGG1促进NF-κB结合其DNA靶序列的机制研究

发布时间：2018-03-26 07:22

本文选题：8-羟基鸟嘌呤DNA糖基化酶1(OGG1)　切入点：8-羟基鸟嘌呤(8-oxoG)　出处：《东北师范大学》2017年博士论文

【摘要】：有氧呼吸的有机体时刻都在面临自身代谢产生的,或外界环境因子造成的活性氧(Reactive Oxygen Species,ROS)的侵袭。细胞内ROS水平超过清除酶系统处理能力的氧化状态被称为氧化应激(oxidative stress)。氧化应激会导致各种细胞内生物大分子(包括蛋白质、脂质及核酸)的氧化。ROS在DNA上造成的氧化性碱基的主要类型是8-羟基鸟嘌呤(8-oxoG)。8-羟基鸟嘌呤DNA糖基化酶1(OGG1)是特异性修复8-oxoG的修复蛋白,通过OGG1起始的碱基切除修复途径(OGG1-BER)将8-oxoG识别出来并更正为正确的碱基。为了研究8-oxoG在DNA中积累的效应和OGG1在病理生理过程中的作用,研究人员培育出Ogg1敲除(Ogg1-/-)的小鼠。出乎意料的是,基因组上异常过量的8-oxoG并不影响胚胎发育或小鼠的寿命;而且,Ogg1-/-的小鼠对炎症没有明显的反应,表现为细胞内促炎因子的表达降低和炎症细胞浸润不足。由于OGG1的功能受到影响时,促炎基因的表达降低,因此我们假设:氧化应激时,OGG1结合到到转录因子识别位点内或邻近部位的8-oxoG时,能够调节转录因子的结合和基因的表达。基因启动子区域的损伤碱基,尤其通过氧化反应产生的损伤碱基,能够改变DNA-蛋白质之间的相互作用,从而调节基因表达的效应。我们认为这种损伤本身,以及DNA损伤修复的过程,能够影响基因表达的模式;研究这一过程所涉及的分子机制有助于我们清楚理解DNA损伤修复和转录调节之间的关系。在本研究中,我们用细胞因子(TNFα)处理细胞以模拟氧化应激的状态,结合siRNA介导的基因沉默、染色质免疫沉淀(ChIP)和ChIP-偶联的测序(ChIP-seq)、双报告实验、蛋白质免疫印迹、RT-qPCR和凝胶迁移实验(EMSA)等实验技术探讨基因的启动子区8-oxoG损伤碱基的存在对于转录的影响,以及OGG1修复蛋白参与转录调节的作用机制。本研究的结果表明:(1)氧化应激时,OGG1修复蛋白能够迅速识别染色质中启动子区域的氧化损伤底物,随后招募转录因子募集到结合的保守区域,这不仅有效促进了转录因子的大量结合,同时在时间上大大缩短了转录因子识别染色质中的保守序列所需要的时间,为氧化应激状态下特异性基因的及时表达提供良好的先决条件;(2)OGG1修复蛋白还能够影响NF-κB调节的基因启动子活性,从而直接调节基因的转录水平;(3)活性氧不仅氧化攻击DNA,产生氧化性损伤碱基,蛋白质也会被氧化修饰,OGG1蛋白的半胱氨酸被氧化导致碱基切除活性受到抑制,基因组的完整性为转录的发生提供可能;(4)损伤碱基的存在和DNA修复酶能够特异性地调节基因表达,在一定时间和空间内,DNA损伤和转录调控具有相互促进的作用;(5)通过以上分子机制的作用,OGG1修复蛋白在NF-κB起始的转录调控过程中产生的分子效应是促进基因的表达。我们的研究揭示出DNA损伤修复酶OGG1转录活化的新机制,即OGG1识别启动子区氧化损伤的产物8-oxoG时能够促进并加速NF-κB与DNA的结合,从而有助于基因的表达;机体的组织、细胞在遭受病原物、环境因子的侵袭时ROS迅速升高,先天性免疫反应所需要的促炎细胞因子、趋化因子的表达高度依赖ROS信号,而这类基因的启动子都是高GC含量的,并含有多个NF-κB结合位点,有机体的氧化损伤应答是一种非常重要的先天免疫防御机制,我们的研究表明传统观念上的DNA损伤在特定的时间和空间内具有重要的转录应答功能;氧化应激条件下,OGG1识别损伤的机制可能成为限定NF-κB转录调节的特异性前提条件。
[Abstract]:Aerobic organisms are always in the face of their metabolism, active oxygen caused or external environmental factors (Reactive Oxygen, Species, ROS) of the invasion. The oxidation state of the intracellular level of ROS than the scavenging enzyme processing ability of the system is called oxidative stress (oxidative stress). Oxidative stress can lead to a variety of cell biological macromolecules in (including protein, lipid and nucleic acid) main types of oxidative base oxidation caused by.ROS on DNA is 8- 8-hydroxyguanine (8-oxoG).8- oxoguanine DNA glycosylase 1 (OGG1) repair protein specific 8-oxoG repair, OGG1 started by the base excision repair pathway (OGG1-BER) 8-oxoG identified and corrected for the correct base. In order to study 8-oxoG effect and OGG1 accumulation in DNA in the pathophysiologic process, the researchers created Ogg1 knockout (Ogg1-/) mice unexpectedly. Is that the genome 8-oxoG abnormal excess does not affect embryonic development or the lifespan of mice; moreover, Ogg1-/- mice showed no response to inflammation, intracellular expression of pro-inflammatory cytokines and reduce the infiltration of inflammatory cells. Due to lack of the function of OGG1 is affected, decrease the expression of proinflammatory genes, so we assume that oxidative stress, OGG1 binding to transcription factor recognition sites within or adjacent parts of the 8-oxoG, capable of binding and gene expression of transcription factor gene promoter. The damage to the base region, especially the base damage by oxidation reaction to produce, can change the interaction between protein DNA- to regulate gene expression, effect. We believe that this injury, and the repair of DNA damage, can influence patterns of gene expression; molecular mechanism of the process involved in the study will help us A clear understanding of the relationship between DNA repair and transcription regulation. In this study, we used cytokine (TNF alpha) treated cells to simulate the oxidative stress state, combined with the siRNA mediated gene silencing and chromatin immunoprecipitation (ChIP) sequencing and coupled ChIP- (ChIP-seq), two experimental report, immune protein Western blot, RT-qPCR and gel shift assay (EMSA) and other experimental techniques to investigate the 8-oxoG gene promoter region of the base damage of the presence of the transcriptional effects, and OGG1 repair proteins involved in the mechanism of transcriptional regulation. The results of this study show that: (1) oxidative stress, OGG1 repair proteins can quickly identify oxidative damage start sub substrate the chromatin, then recruit transcription factor to raise the conservative region combined, which not only effectively promote a large number of transcription factor binding, and in time is greatly shortened to identify transcription factor staining In the conservative sequence of the time required to provide a good prerequisite for the timely expression of specific genes under oxidative stress; (2) OGG1 repair proteins can also affect NF- kappa B regulated gene promoter activity, which directly regulates gene transcription levels; (3) ROS not only oxidative attack DNA, oxidative base damage, protein will be oxidized, cysteine OGG1 protein is due to oxidation of base excision activity was inhibited, integrity of the genome may provide for transcription; (4) there is damage to the base and DNA repair enzyme can specifically regulate gene expression, in a certain period of time and space that is the interaction between the DNA damage and transcriptional regulation; (5) the molecular mechanism and molecular effects of OGG1 repair proteins in transcriptional regulation of NF- kappa B starting in promoting gene expression. The study reveals a new mechanism of DNA damage repair enzymes OGG1 transcription activation, with OGG1 oxidative damage recognition starting sub area can promote and accelerate the production of 8-oxoG NF- kappa B and DNA, which helps to show gene; tissue, cells subjected to pathogen, environmental factors of the invasion of ROS the rapid increase of proinflammatory cytokines required for innate immune response, chemokine expression is highly dependent on the ROS signal, and this kind of gene promoter is high GC content, and contains a number of NF- B binding site, oxidative damage to organism response is an innate immune defense mechanism is very important. Our study shows that, with the important function of transcriptional response in the specific time and space on the traditional concept of DNA damage; oxidative stress conditions, the mechanism of OGG1 damage recognition may become the premise of define specific regulation of NF- kappa B transcription .

【学位授予单位】：东北师范大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q78

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