The Characterization Of The Functions Of UHRFs In DNA Methylation Regulation And ABH2in RDNA Transcription

Alkylation lesions in DNA and RNA are lethal and mutagenic resulting from endogenous and environmental agents. If they could’t be repaired timely, they would get the integrity of the cell genome destroyed, cause irreversible changes of genetic information, disrupt replication and transcription, trigger S-phase arrest, sister chromatid exchange, DNA breaks and accumulation of p53and initiate apoptosis. They can also lead to the related disease, such as cancers. Alkylating agents are also used as cytostatic drugs in cancer therapy, but there are a certain number of problems, such as the drug resistance. AlkB-like proteins belong to the superfamily of iron(II) and2-oxoglutarate dependent dioxygenases and can directly repair these lesions. Furthermore, modulation of the functions of these enzymes will be helpful for the usefulness of alkylating therapeutic drugs.ABH2is the primary enzyme of guarding mammalian genomes against the alkylation damage. Though it is regarded as a housekeeping enzyme, it is shown that the expression level is high in some tumors but lower in others. It is also found that there are ABH2mutations in tumors. In this study, we found ABH2mainly located in the nucleolus. It contained overlapping nuclear localization sequence(NLS) and nucleolus localization sequence(NoLS) near its N-terminus. By identification of the ABH2associated proteins, we found ABH2interacted with the DNA repair protein Ku70and the nucleoli proteins NCL and NPM1. The interactons were verified further by Co-IP and GST pull-down assay.The results of ChIP and immunol fluorescence staining showed that ABH2bound to the promoter region of rDNA and associated with mitotic chromosomes during the M phase of cell cycle. Using luciferase reporter assay and RNA interference, we confirmed that ABH2stimulated rDNA transcription and this activation correlated to its enzymatic activity. The down-regulation of ABH2led to activation of the DNA-Damage Response in cells, including the rDNA region. In addition, ABH2could response quickly to the DNA damage caused by the alkylating agents and resulted in translocation of the protein from nucleolus to nucleoplasmic.Taken together our results, let us to propse the following hypothesis:ABH2associate with DNA repair protein Ku70and the nucleoli proteins NPM1and NCL, locates directly at the promoter region of rDNA to protect the highly activated rDNA and promote its transcription.Epigenetics is a hot area of research right now, which studies the inheritable phenomenon other than the alteration of the DNA sequence. It mainly consists of histone modifications and DNA methylation. Besides, the epigenetic information needs several protein as "readers" or "effectors". UHRF1is just one of such important readers.Previous studies show that UHRF1was targeted to replication fork through the binding with the hemi-methylated DNA via the SRA domain, and recruited the DNMT1to accomplish the methylation of both strands. In addition, UHRF1could also interact with the methylated H3K9(H3K9me) through its TD domain, but the detailed biological function of this interaction remains unclear. This study indicated that UHRF1could also bind at replication fork through its binding activities with H3K9me, recruit DNMT1and methylate the newly synthesized DNA strands. Further studies on the SRA domain of UHRF1, we found that the the level of DNA methylation was rescued in NP95-/-cells by over-expressing exogenous DNMT1-PCNA fusion protein. This result indicated that the flipout of unmethylated cytosine via SRA was not essential for the DNA methylation maintenance.UHRF2and UHRF1both belong to the UHRF family proteins. They have high similarities each other in both sequence and structure, but are not functioanally redundant in DNA mathylation maintenance. Here, our study suggest that UHRF2negatively regulates DNA methylation by degrading DNMT3a through its E3ligase activity.