【作者】 朱慧芳；

【导师】 邵吉民；

【作者基本信息】 浙江大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 单功能烷化剂N-甲基-N’-硝基-N-亚硝基胍(MNNG)是一种在实验室中普遍使用的模式化学诱变剂和致癌剂,用于研究环境中广泛存在的N-亚硝胺类化学物质的致突变致癌机制。MNNG能与DNA及蛋白质等生物大分子形成加合物,引起突变和其它损伤。本实验室长期致力于MNNG等环境化学污染物引起细胞损伤机制的研究,并对MNNG等诱发非定标性突变以及细胞遗传不稳定的机制提出了以下假设:致突变致癌物经由DNA损伤和非DNA损伤途径诱发一系列信号转导通路的激活,引起相关基因表达发生改变,包括DNA聚合酶表达谱改变,致使DNA复制保真度下降,最终可导致非定标性突变和细胞遗传不稳定的形成。为深入探讨MNNG引起DNA复制保真度下降的机制,我们对具有低复制保真度特点、参与跨DNA损伤合成的Y家族聚合酶Polη、Polι和Polκ的启动子及其对MNNG应答的转录调控机制进行了研究。主要研究方法和结果:a.应用生物信息学软件结合进化足迹法对POLH、POLI和POLK启动子及其转录因子结合位点进行预测,并对它们共同的转录因子和可能的信号通路进行分析,推测E2F和STAT等相关信号通路可能在POLH、POLI和POLK的转录调控中起一定作用。应用报告基因技术分析POLH、POLI和POLK启动子截短体的转录活性,对三个聚合酶的最小启动子区、转录激活区和抑制区进行了定位。b.定量RT-PCR和/或免疫印迹试验检测发现10μM MNNG可以引起POLH、POLI和POLK转录上调。应用突变、报告基因和转录表达分析等技术发现:(1)过表达IRF1上调Polη表达;将POLH启动子区-898位或-590位IRF1结合位点突变后,过表达IRF1不能引起其转录上调,也抑制了MNNG引起的POLH启动子的活性增加,说明IRF1与-898位和-590位结合,参与调控MNNG诱导的POLH的转录上调。(2)MNNG引起POL1的转录激活,Sp1在MNNG诱导POLI的转录调控中起一定作用。(3)MNNG可能通过多个转录因子的协同作用调节POLK的表达,而起主要调控作用的转录因子有待于进一步研究。c.为了研究调控基因表达的“转录因子组”,我们初步建立了DNA pulldown-质谱检测方法,为下一步研究基因的转录调控机制提供有力的工具。主要结论:(1)首次系统研究了Y家族DNA聚合酶POLH和POLI启动子的活性,发现POLH和POLI启动子上的多个反应元件参与基因的转录调控,有的是正向调控,有的则是负向调控;(2)首次证明了IRF1和Sp1分别参与了MNNG诱导的POLH和POLI的转录激活调控;(3)初步建立的DNA pulldown-质谱检测方法可用于鉴定与基因启动子序列结合的多个转录因子。本课题的研究有助于进一步阐明Y家族聚合酶在DNA损伤应答过程中的转录调控机制,为探索化学致突变致癌物的作用机制及干预的新途径提供了新的实验依据。更多还原

【Abstract】 The alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) is widely used in research laboratory as a model carcinogen in studying the mechanisms of N-nitroso alkylating agent-induced mutagenesis and carcinogenesis. It can generate adducts with DNA and proteins, which cause mutation and other damages. Our laboratory has been long studying the mechanisms of cellular damages induced by environmental chemical pollutants such as MNNG. Based on our previous findings, we hypothesize that the carcinogens can trigger a series of cellular signal transduction via DNA damage and non-DNA damage pathways, which alter the expression of related responsive genes, including DNA polymerases with low replication fidelity. The abnormal regulation of the low-fidelity DNA polymerases may ultimately lead to DNA non-targeted mutation and cellular hereditary instability. In this study, we have investigated the transcriptional regulation of the low-fedelity Y-family DNA polymerase Polη, Polι, and Polκ, which carry out translesion DNA synthesis, under the treatment of MNNG, in the hope to further elucidate the molecular mechanisms of chemical mutagenesis and carcinogenesis.Main methods and results:a. Using bioinformatics softwares and phylogenetic footprinting analysis, we predicted the promoters and related transcription factor binding sites of POLH, POLI and POLK. Further analysis to find the potential common transcription factor binding sites in three promoters and related signalling pathways suggests that some transcription factors (e.g. E2F and STAT) may play a role in co-regulation of Y-family DNA polymerase.A series of deletion constructs of the promoters of POLH, POLI, and POLK and reporter gene assays were employed to investigate the transcriptional activities to locate the positive or negative regulation regions in the promoters.b. Quantitative real time RT-PCR and/or western blot analyses revealed that the expression of POLH, POLI, and POLK was up-regulated in human amnion FL cells treated by 10μM MNNG. Further investigations with reporter gene assays, mutation and transcriptional analysis demonstrated: (1) Overexpression of IRF1 increased the expression of Polη. When the IRF1 binding sites located in -898 or -590 relative to the transcription start position of POLH was mutated, overexpression of IRF1 did not enhance the transcriptional activities of POLH promoter. The mutation of IRF1 binding sites in the POLH promoter inhibited the up-regulation of POLH induced by MNNG as well. These results indicate that IRF1 could participate in the up-reguation of POLH in response to MNNG treatment through binding to -898 and -590 sites of the promoter. (2) MNNG transactivated POLI expression, and the transcription factor Sp1 could play an important role in the regulation of POLI expression induced by MNNG (3) Many transcription factors could involve in the transactivation of POLK induced by MNNG, but the key transcription factors need further exploration.c. A DNA pulldown-mass spectrometry assay was established to study many transcription factors that bind to the promoter sequence of one gene.Main conclusions: (1) This is the first systemical study of the transcriptional activity of human POLH and POLI promoter, and the positive and negative regulatory elements located in the promoter regions were analyzed. (2) It is the first finding that IRF1 and Sp1 involve in the transactivation of POLH and POLI induced by MNNG, respectively. (3) The established DNA pulldown-coupling mass spectrometry method could be useful for deepening the understanding of the complicated mechanisms of transcriptional regulation in the future.Taken together, our findings can help to elucidate the molecular mechanisms of the transcriptional regulation of Y-family DNA polymerase in response to DNA damages, and provide new insights into the mechanisms of mutagenesis and carcinogenesis induced by environmental chemical compounds.更多还原