Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Dr. Joseph Torchia

Abstract

The ZNF217 transcription factor is an oncogene found within the 20q13 amplicon and is amplified and overexpressed in many cancers including breast and ovarian. Overexpression of ZNF217 leads to increased cell proliferation, survival, and causes resistance to TGFβ's anti-proliferative effects.

ZNF217 is a core constituent of a transcriptional complex that includes CoREST, HDAC1/2, LSD1, and the CtBP1/2. In this study, I have combined genome-wide biochemical approaches to identify genes directly regulated by ZNF217. I have identified the tumor suppressor and cell cycle inhibitor, p15ink4b, as a direct target of the ZNF217 complex and demonstrated that ZNF217 represses the p15ink4b gene by promoting a repressive chromatin environment and facilitating promoter DNA hypermethylation that involves a novel interaction with DNMT3A.

Furthermore, treatment of cells with TGFβ triggers DNA demethylation of the p15ink4b promoter and the release of ZNF217/CoREST/DNMT3A complex. Subsequently, a novel activation complex is recruited that consists of SMAD2/3, CBP, and the DNA glycosylase TDG which precedes increases in p15ink4b protein expression. Knockdown of TDG, or its functional homolog MBD4, prevents TGF-β-dependent demethylation of the p15ink4b promoter suggesting that the demethylation occurs through an active mechanism and is required for TGFβ dependant activation of gene expression. DNA immunoprecipitation experiments indicate that 5mC undergoes conversion to 5hmC in response to TGFβ treatment. AID/APOBEC2 deaminases are also required for the DNA demethylation by TGFβ supporting a mechanism whereby 5mC is hydroxylated to 5hmC and then deaminated to 5hmU which is reverted to the unmethylated cytosine by the BER enzymes.

Overexpression of ZNF217 inhibits promoter demethylation and expression of the p15ink4b gene in response to TGFβ by preventing recruitment of SMAD2/3/TDG complex. These findings suggest that the coregulator balance at promoters of genes is an important determinant of gene regulation and oncogenic amplifications such as ZNF217 can upset this balance causing deregulation of many genes. Taken together, these results establish the ZNF217 complex as a negative regulator of the p15ink4b gene and may constitute an important link between amplification of ZNF217, increased cell proliferation and loss of TGFβ responsiveness in cancer.

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