| Summary: | 博士 === 國立陽明大學 === 生化暨分子生物研究所 === 104 === Aberrant DNA methylation is a leading cause of cancer formation. DNA methyltransferases contribute to oncogenesis partly through methylation and inactivation of tumor suppressor genes. Whether DNA demethylation counteracts this oncogenic effect has been a burning issue in cancer epigenetics. In 2009/2010, the ten-eleven translocation gene family protein 1-3 (TET1-3) were identified as DNA demethylation enzymes to convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) through iterative oxidation reactions. Subsequently, 5fC and 5caC can be excised by thymine DNA glycosylase (TDG) and replaced with intact cytosines through base excision repair mechanism. We discovered the first mechanism by which TET1 suppresses tumor invasion/metastasis. We found that TET1 inhibited DNA methylation of tissue inhibitors of metalloproteinases (TIMP2 and TIMP3) to potentiate TIMP2 and TIMP3 expressions, further leading to invasion suppression (Best 5 Article of Cell Reports 2012). How TETs are regulated and maintained in normal cells is poorly understood. In Chapter II, we showed that only TET1 was positively regulated by p53, an important tumor suppressor. Using luciferase reporter and promoter deletion assay, we identified -500 bp to the transcription start site (TSS) as the minimal TET1 promoter for TET1 expression in normal cells. The DNA affinity protein assay (DAPA) revealed that p53 could be pulled down by this minimal TET1 promoter region. Chromatin immunoprecipitation (ChIP) further demonstrated that p53 bound to the minimal TET1 promoter in cells. TET1 expression decreased when p53 was knocked down. Consistently, ectopically expressed p53 activated TET1 expression and promoter activity. Furthermore, the expression and function, ex, 5hmC level, of TET1 were activated when normal cells were treated with DNA damage inducers to activate p53. Consistently, p53-null cells could not activate TET1 upon DNA damage, indicating that both TET1 expression and 5hmC increase in a p53-dependent manner. In summary, our findings indicate that p53 positively regulates and maintains TET1 expression and function. In Chapter III of the study, we analyzed how TET1 expression is downregulated in breast cancer tissues. We used different histone modification antibodies in ChIP assay to illustrate that H3K9me2/3, hallmarks for transcriptional repression, were enriched in TET1 promoter in breast cancer tissues. By analyzing The Cancer Genome Atlas (TCGA) database, we showed that KMT1D/GLP was up-regulated in the majority of breast cancer tissues and inversely correlated with TET1 expression. In addition, depletion of KMT1D/GLP increased the expressions of TET1 and TET2 but not TET3. ChIP assays further showed that the endogenous KMT1D/GLP bound to TET1 promoter region. These results indicate that KMT1D/GLP may contribute to transcriptional silencing of TET1 expression in cancer cells via histone H3K9 methylation. Taken together, in this thesis work, we uncovered the mechanistic role of TET1 in cancer suppression and illustrated the regulation of TET1 in normal or breast cancer tissues. These results not only greatly advance our knowledge of DNA demethylation enzymes under physiological condition and in cancer but also provide an opportunity to develop a potential anti-cancer strategy by targeting TET1 expression. We believe that the study will exert a great impact on the cancer epigenetic field.
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