Role of Transcriptional Factor YY1 on HPV16 Gene Expression and the Modulator Role of SAP30 on YY1

Autor: Nu En Huang, 黃女恩
Rok vydání: 2003
Druh dokumentu: 學位論文 ; thesis
Popis: 91
Incidence of human papillomavirus (HPV) infection is generally considered as frequent throughout the world. Both benign and malignant tissues develop as the result of either low or high-risk types of HPV infection. More than 11% of the global cancer cases in female are due to HPV infections. HPV genotype 16 alone constitutes approximately 50% of it and is regarded as the most prevalent viral type associated with cervical cancers. The E6 and E7 oncogenes of high-risk HPVs have been shown to inactivate the p53 and pRb cellular tumor suppressor proteins, respectively, which are thought to be important for immortalization and carcinogenesis processes. The transcription of HPV16 E6 and E7 oncogenes is controlled by P97 promoter and modulated by regulatory elements within the long control region (LCR). Most of the cis-responsive elements that influence viral transcription and replication are located in the LCR. The progression from a mild dysplasia lesion to a cervical carcinoma is usually accompanied by deregulation of the intracellular control mechanisms of HPV transcription in the basal cells, resulting in a continuously up-regulated E6/E7 expression. It is therefore of particular interest for us to study the molecular mechanisms that result in the enhancement of E6/E7 expression during HPV induced tumorigenesis. In the first part of this thesis, using a serum free cell line SFR, we have observed an elevation of HPV16 E7 associated with the lag phase of cell growth. Elevated HPV16 E7 level could be suppressed by replenishing with fresh medium 12 hours before cell harvest. More cells were pushed from G0/G1 to S phase as the result of changing medium. An obvious relation between cell growth status and HPV16 E7 level was demonstrated. Having proved that HPV16 gene transcription was enhanced in the lag phase of cell growth, through a series of experiments, we observed the quantitative difference of cellular factors binding to LCR-A (nt 7152-7454) and LCR-C (nt 7855-97) regions with obvious association with cell growth status. Two specific binding sites, A1 site (nt 7434-7448) and C1 site (nt 59-88), were identified within LCR-A and LCR-C, respectively, and the binding factors were identified to contain YY1. The transcriptional regulation functions of these two YY1 binding sites on HPV16 LCR were tested using CAT assay. YY1 binding to A1 site exhibited an activation effect on HPV16 gene expression, while C1 site showed repression effect. Functional analysis of the A1 and C1 YY1 binding sites in different cervical carcinoma cell lines presented similar results. YY1 phosphorylation status did show differences on cellular factor binding to HPV16 A1 and C1 sites. However, no significant difference on either protein level or phosphorylation status of YY1 was observed in relation to different cell growth phases. In the second part of this thesis, we switch to study the modulator function of YY1. YY1 is a highly conserved, multifunctional transcription factor. The diverse activities of YY1 are regulated and sometimes modified by interaction with various other proteins. Using yeast two-hybrid screening system, SAP30 was identified as a protein that associates with YY1 and enhance YY1-mediated repression in a dose-dependent manner. SAP30 is a 30 kDa nuclear protein and a component of the human histone deacetylase complex. In this study, the interaction of SAP30 and YY1 was confirmed both in vitro and in vivo. The interaction domains between YY1 and SAP30 were mapped to the C-terminal segment of YY1 (295-414) and 91 amino acids at the C-terminal region of SAP30. The observation that YY1, SAP30 and HDAC1 form a complex in vivo provides evidence that YY1 also recruits HDAC1 indirectly via its binding to SAP30. These results describe a novel mechanism for YY1-mediated repression.
Databáze: Networked Digital Library of Theses & Dissertations
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