Date of Award

Fall 12-22-2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Pharmacology

First Advisor

Debabrata Chakravarti

Abstract

The THAP (Thanatos associated protein) domain is an evolutionarily conserved zinc-finger motif highly similar to the sequence specific DNA binding domain of Drosophila P element transposase. Emerging data suggest THAP proteins may function in DNA and chromatin dependent processes, including transcription. However, the transcriptional regulatory function, mechanisms of action, and role of most THAP proteins in normal and aberrant cellular processes remain largely unknown.

In this thesis, we demonstrate that several human THAP proteins contain transcriptional repressor activity and specifically identify THAP10 and THAP11 as differentially expressed in human DNA damage and colon cancer progression, respectively. THAP10 and THAP11 repressed basal and VP16 activator driven transcription when tethered to promoters as heterologous Gal4-DNA binding domain fusion proteins and physically associated with histone deacetylases in vitro and in vivo. THAP11 was found to be differentially expressed in the SW480/SW620 cell culture model of human colon cancer progression and immunohistochemical analysis of tissue microarrays similarly revealed increased THAP11 expression concomitant with disease progression. The increase in THAP11 expression in colon cancer tumors and cell lines suggests that THAP11 dependent transcriptional repression may contribute to disease progression. Consistent with this hypothesis we find that knockdown of THAP11 in metastatic SW620 colon cancer cells results in a modest but significant decrease in cell proliferation. Gene expression profiling in THAP11 depleted SW620 cells identified 80 differentially expressed genes, 70% of which were de-repressed by THAP11 knockdown. Directly repressed THAP11 gene targets were found to contain chromatin bound THAP11 near their transcription start sites. THAP11 mediated repression requires the multi-functional transcriptional regulator HCF-1 (Host cell factor-1). THAP11 physically associates with and recruits HCF-1 to repressed promoters and knockdown of HCF-1 is sufficient to de-repress THAP11 target genes.

Collectively, this data provides the first characterization of a directly regulated, THAP11 dependent gene expression program in human cells and suggests THAP11 may be an important transcriptional regulator in human colon cancer. These results, in conjunction with previous findings from our laboratory and others, suggest THAP proteins likely function as biologically relevant transcriptional regulators.

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