DNA碱基损伤修复酶OGG1调控NF-κB磷酸化修饰的作用及机制研究

8-oxoguanine DNA glycosylase-1 (OGG1) is a DNA damage repair enzyme that specifically recognizes 8-hydroxyguanine (8-oxoG) in eukaryotic cells. However, recent studies have shown that 8-oxoG is not only a lesion to be repaired, but also an epigenetic modification. Under oxidative stress, OGG1 binding to 8-oxoG in the promoter, plays a role in the recruitment of NF-κB and the assembly of transcriptional machinery, to assure a prompt launch of the immediately responsive transcriptome.Studies have shown that the phosphorylation of NF-κB subunit RelA/p65 S276 may selectively promote the expression of genes regulated by ROS signaling, but it is urgent to reveal which genes are regulated and what the molecular mechanism is. Our preliminary results showed that OGG1 promoted phosphorylation of RelA/p65 Ser276. 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 determine ①Ogg1-mediated phosphorylation of RelA/p65 S276 is involved in the selective expression of the gene; ② which protein kinase interaction with OGG1 promotes phosphorylation of RelA/p65 S276; ③ the precise molecular mechanisms by which OGG1 interacts with protein kinase and RelA/p65, and the DNA sequence context within the high GC-containing promoters that is required for the OGG1 and p-RelA/p65 S276 complex-driven transcription. The completion of the proposed study will reveal the transcriptional regulation and molecular mechanism of DNA damage repair enzyme OGG1.

OGG1是真核细胞特异识别8-羟鸟嘌呤的DNA损伤修复酶，然而近期研究结果表明8-羟鸟嘌呤不只是一种需要修复的损伤，而且还具有表观遗传修饰作用。氧化应激条件下OGG1与启动子区内8-羟鸟嘌呤的结合可招募NF-κB促进先天免疫促炎基因的转录。有研究报道提出NF-κB的RelA亚基S276磷酸化可能选择性地促进ROS信号调控的基因表达，但全基因组哪些基因受其调控、分子机制是什么等问题亟待揭示。我们前期实验数据显示OGG1能促进NF-κB的RelA亚基276位丝氨酸的磷酸化。因此本项目将利用二代测序技术、蛋白-蛋白及蛋白-核酸作用分析技术，①明确OGG1介导RelA S276磷酸化修饰参与的选择性表达基因组及其生物学意义；②鉴定与OGG1相互作用蛋白激酶；③解析蛋白质相互作用分子机制以及8-羟鸟嘌呤的核苷酸序列环境特征。项目的完成将更加深入地揭示DNA损伤修复酶OGG1的转录调控作用及分子机制。

活性氧由多种内源性生理过程产生，并因环境因素而加剧。ROS攻击DNA，最常见的氧化产物是8-oxo-7,8-二氢鸟嘌呤(8-oxoG)。8-oxoG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 (BER) 途径对8-oxoG进行修复。越来越多的证据表明，8-oxoG不仅仅是一种氧化损伤，而且作为一种DNA表观遗传修饰，通过与其特异修复蛋白OGG1的相互作用实现对氧化应激基因的转录调控。OGG1与底物结合，招募NF-κB，促进促炎细胞因子/趋化因子的转录。NF-κB活性依赖于翻译后修饰，其中RelA/p65 Ser276磷酸化受ROS密切影响，可选择性上调。但是，RelA/p65 Ser276磷酸化是如何响应ROS信号进而选择性调控NF-κB部分靶点的机制尚不清楚。.基于上述科学问题，我们认为在高度氧化的细胞环境下，OGG1不仅能招募NF-κB到基因的启动子区，是否进一步促进RelA/p65 S276磷酸化修饰进而开启快速应答ROS转录组的基因转录。本项目的研究内容包括：1）OGG1是否介导了RelA/p65 S276磷酸化参与基因的选择性表达；2）OGG1与哪种蛋白激酶相互作用促进RelA/p65 S276的磷酸化；3）解析OGG1-蛋白激酶-RelA/p65的相互作用的分子机制。.在项目中，发现1）OGG1与丝裂原激活蛋白激酶1 (MSK1)之间的相互作用是RelA/p65丝氨酸276磷酸化所必需的。2）ROS清除或OGG1耗尽/抑制阻碍了MSK1与RelA/p65之间的相互作用，从而降低了phospho-Ser276的水平，导致编码ROS响应性细胞因子/趋化因子的基因表达显著降低，但NFKBIs没有。3）阻断OGG1与DNA底物的结合以基因特异性的方式阻止了RelA/p65、Pol II和p-RNAP II在启动子募集。我们的数据强调了OGG1在影响RelA/p65的S276磷酸化以选择性转录调节ROS反应和立即激活的促炎细胞因子/趋化因子方面的重要性。该研究不仅对磷酸化编码的调控机制有了新的认识，而且巩固了好氧细胞中进化而来的启动子定位底物结合OGG1的严密而精细的控制，以满足及时基因转录激活的需要。