Transcriptional Regulation Of Y-family DNA Polymerase In Response To The Carcinogen N-methyl-N'-nitro-N-nitrosoguanidine

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.