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Bokuan Wu


bo-kuan-wu@uiowa.edu
Mentor: Charles Brenner, Ph.D.
Lab Room: 4-339 BSB
Lab Phone: 319-384-4099

Development of a Cellular Model of Dnmt1 Activation in Carcinogenesis

DNA methyltransferase 1 (Dnmt1) is the major enzyme responsible for repressive, epigenetic modification of DNA in eukaryotes. During carcinogenesis, it becomes dysregulated to cause hypermethylation-mediated silencing of tumor suppressor gene (TSG). Interestingly, several frequent, cancer-specific alterations have been found to significantly increase Dnmt1 activity, like Ras activation, P53 loss and carcinogen treatment. However, the resulting methylomes are not necessarily the same, indicating different mechanisms of Dnmt1 activation will result in different patterns of DNA methylation. Therefore, our purpose is to establish stable clones in immortalized human bronchial epithelial cells (HBEC3) and analyze the methylation consequences of Dnmt1 activation. Moreover, Dnmt1 consists of a series of globular domains N-terminal to the catalytic domain, all of which are implicated in functions essential for maintenance of DNA methylation. However, in our lab, in vitro evidence was recently presented that the replication foci targeting sequence (RFTS) is an inhibitor of DNA-binding and catalytic activity. To test the hypothesis that RFTS is a negative regulator of Dnmt1 activity, cellular expression systems for Dnmt1 mutants were established. Data indicate that the deltaRFTS allele of Dnmt1 is activated for transformation. Expression of Dnmt1-deltaRFTS results in reduced global DNA methylation, and methylation and silencing of TSGs. These data demonstrate that the deltaRFTS form of Dnmt1 is hyperactivated.