DNA损伤修复酶OGG1调节基因转录的作用及机制研究

Reactive oxygen species (ROS) may inflict oxidation modification to various bio-macromolecules including DNA bases. Among 4 DNA bases guanine is the most frequent targetdue to its lowest redox potential, and its oxidized product,7,8-dihydro-8-oxoguanine (8-oxoG), is linked to the pathogenesis of many diseases despite of the lack of direct experimental evidence. It has been reported that depletion of 8-oxoG glycosylase 1 (OGG1), the specific enzyme to repair 8-oxoG lesion in DNA duplex, resulted in the significant decrease of pro-inflammatory geneexpression which are highly ROS signaling-dependent. Our recent publication also documented that OGG1 binds to 8-oxoG within high GC-containing promoters, facilitates the recruitment of site specific transcription factors such as NF-κB, as well as the transcription of pro-inflammatory genes. This unexpected finding urged us to further explore the precise molecular mechanisms how a DNA repair enzyme regulates gene transcription. In the proposed study, we will utilize next generation sequencing skills such as ChIP-Seq, RNA-Seq, as well as various molecular techniques analyzing the interaction between protein-protein and protein-DNA, to define 1) in a whole genome scale, the characters of the binding sites of OGG1 and NF-κB, the transcriptome associated with OGG1 expression as well as the gene ontology annotation and the related pathways revealed by the omics data;2)the precise molecular mechanisms by which OGG1 interacts with NF-κB; 3) the DNA sequence context within the high GC-containing promoters that is required for the OGG1- NF-κB complex-driven transcription. The completion of the proposed study will add insight to the role of DNA repair enzyme OGG1 in gene transcription initiation; unveil a novel mechanism by which NF-κB achieves sophisticated regulation under oxidative stress. More importantly, the structural resolution of the interaction of OGG1- NF-κB complex will benefit the design of inhibitory small compounds that may be utilized in the intervention of chronic inflammatory diseases.

活性氧可引起体内各种生物大分子的氧化，其中DNA碱基是主要作用靶点之一，鸟嘌呤因其氧还电势最低而极易被氧化，形成的损伤产物8-羟鸟嘌呤被认为与多种疾病发生高度相关，但直接实验证据却极度缺乏。我们前期工作发现8-羟鸟嘌呤修复酶OGG1与含有8-羟鸟嘌呤的高GC含量启动子结合后，促进了转录因子NF-kB的招募及活性氧信号依赖的促炎基因转录活化，但DNA损伤修复酶OGG1如何影响基因转录的分子机制尚未深入揭示。本项目研究将利用二代测序技术及蛋白-蛋白、蛋白-核酸作用分析技术深入解析1)全基因组范围内OGG1、NF-kB的染色质免疫沉淀组、OGG1依赖的转录组的关联性及高度代表基因的功能2)OGG1与NF-kB相互作用的分子机制3)OGG1与NF-kB相互作用对启动子区序列环境的要求。项目的完成将揭示DNA修复酶OGG1转录活化的新功能及NF-kB活性调节的新机制，为炎症相关疾病的治疗提供新的靶点

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活性氧（ROS）的侵袭。ROS会导致各种细胞内生物大分子（包括蛋白质、脂类及核酸）的氧化。各种生物大分子中，DNA分子的碱基因拥有较低的氧还电势是ROS的主要作用靶点，而氧化的DNA 碱基长期以来一直被认为是DNA的损伤形式，与炎症、衰老、癌症的发生高度相关。但碱基氧化与机体病理现象间病因学的直接实验证据却极为缺乏。. 组成DNA的4个碱基中鸟嘌呤(G)的氧还电势最低因而最易被氧化，形成的主要氧化产物为8-羟鸟嘌呤(8-oxoG)。OGG1是真核细胞识别并切除8-oxoG的特异性DNA修复酶，通过自然界中极为保守的碱基切除修复(Base Excision Repair, BER)途径对8-oxoG进行修复。本项目研究内容包括：1）全基因组范围内OGG1ChIP-Seq、OGG1依赖的转录组 RNA-Seq、RelA/p65 CHIP-Seq的关联性，及高度代表基因的相关功能；2)OGG1与转录因子NF-ҝB相互作用的分子机制；3）OGG1与转录因子NF-ҝB相互作用对于启动子区DNA序列环境的要求等。研究结果证明启动子区8-oxoG的存在并不影响转录的进行。氧化应激状态下，OGG1半胱氨酸的氧化导致的BER酶促活性暂时抑制。BER功能暂时失活的OGG1可以迅速识别并结合于氧还敏感基因启动子区的8-oxoG，但并不继续行使BER，而是迅速招募位点特异转录因子（如NF-κB），促进转录机器组装，特异性地指导氧化应激条件下相关转录组的活化（如促炎细胞因子、趋化因子TNFα、CXCL2、CCL20、IL-1β等）。OGG1在基因组的富集倾向于启动子等调节区域，相关基因的功能分析显示，OGG1靶基因参与的生物学进程包括氧化应激、细胞氧化还原稳态、负调控细胞死亡、炎症应答等。. 依据本项目的研究结果，我们提出一种新的概念：特定条件下的碱基的氧化，可以视其为一种修饰（如同甲基化）而非损伤，修复酶的识别可以特异性地指导应答氧化应激的转录组快速活化，在基础理论研究方面为基因表达的表观遗传调控机制增添新的内容。另外，针对OGG1活性位点的特异性小分子化合物有望特异性地阻断促炎基因的转录活化，从而治疗过度炎症、慢性炎症等相关疾病，具有重要的转化意义。