Investigation of the mechanism by which the human papillomavirus type-16 E6 oncoprotein induces telomerase in epithelial cells

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Investigation of the mechanism by which the human papillomavirus type-16 E6 oncoprotein induces telomerase in epithelial cells

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Title: Investigation of the mechanism by which the human papillomavirus type-16 E6 oncoprotein induces telomerase in epithelial cells
Author: Gewin, Lindy Carol, 1974-
Abstract: The human papillomavirus (HPV) type-16 E6 oncoprotein activates telomerase in human epithelial cells by inducing expression of hTERT, the catalytic and rate-limiting subunit of telomerase. Another well established function of E6 is to bind the cellular E6-associated protein (E6-AP) to form an E3 ubiquitin ligase that targets p53 for ubiquitination and degradation via the proteasome. Telomerase activation, rather than p53 degradation, is essential for the immortalizing activity of E6 in epithelial cells. In our investigation into the mechanism by which E6 induces hTERT expression we found that the E boxes (c-Myc/Max binding sites) were required for promoter activation. c-Myc protein levels, however, did not change upon E6 expression and c-Myc protein was localized to the endogenous hTERT promoter regardless of promoter activity. Therefore, though c-Myc may be necessary for hTERT induction it is not sufficient. Activation of hTERT was associated with increased levels of acetylated histories H3 and H4 at the hTERT promoter upon E6 expression indicating that chromatin modifications are involved. Furthermore, analysis of several E6 mutant proteins revealed a strong correlation between the ability of E6 to bind E6-AP and its ability to induce hTERT. Using shRNAs to reduce E6-AP expression, we demonstrated that E6-AP is required for E6-mediated telomerase induction. In a search for new targets of E6/E6-AP involved in telomerase activation, we identified NFX1-91, a transcriptional repressor of the hTERT promoter in reporter assays. The stability of NFX1-91 is reduced in cells expressing E6 in a proteasome-dependent manner. Evidence that NFX1-91 repressed the endogenous hTERT promoter was found by decreasing expression of NFX1-91 with shRNAs resulting in derepression of the hTERT promoter and elevated levels of telomerase activity in primary human keratinocytes. We propose that the induction of telomerase by HPV-16 E6 requires binding to cellular E6-AP and involves targeting of NFX1-91, a newly identified repressor of telomerase, for ubiquitination and degradation.
Description: Thesis (Ph. D.)--University of Washington, 2004
URI: http://hdl.handle.net/1773/5006

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