Abstract:
Global DNA hypomethylation promotes genomic instability through the accumulation of DNA damage and resulted in an increasing rate of mutation. To stabilize the genome, cells possess an epigenetic mechanism to reduce DNA tension that causes DNA damages, named Replication-Independent Endogenous DNA Double-Strand Breaks (RIND-EDSBs). Due to their biological roles, this type of physiologic EDSBs act as youth-associated genomic-stabilizing DNA gaps (Youth-DNA-GAPs). They are localized within methylated genome and maintained by non-histone HMGB1 and SIRT1 proteins. Reduction of RIND-EDSBs was found in hypomethylated genome, including cancer and aging. Therefore, this study aimed to explore the role of HMGB1-mediated DNA methylation in genomic instability prevention. First, using IRS-EDSB LMPCR and DI-PLA methods, we demonstrated that HMGB1, specifically Box-A domain produces Youth-DNA-GAPs. Second, the results from PLA assay revealed that HMGB1 forms complex with Youth-DNA-GAPs and SIRT1, and SIRT1 protected Youth-DNA-GAPs from γH2AX. Third, we conducted two novel PCR, DNA immunoprecipitate 8-OHdG followed by IRS-EDSB LMPCR and IRS-SSB PCR combined with EDSB-SSB PCR to study genome distribution pattern of Youth-DNA-GAPs, and found that Youth-DNA-GAPs are located far from DNA damages and can stabilize genome in long distance. Finally, to study the mechanism of HMGB1-mediated DNA methylation, Alu siRNA and AGO4 transfection were performed. We showed that DNA methylation preventing genomic instability is HMGB1 dependent. Therefore, HMGB1 mediates DNA methylation by producing and maintaining methylated RIND-EDSB or Youth-DNA-GAPs in methylated genome to prevent genomic instability.
