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Title: Cell type specific regulation of papillomavirus transcription
Author: Vance, Keith
ISNI:       0000 0001 3542 5656
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2000
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All papillomaviruses have a non-coding region of approximately 500-1000 bp called the upstream regulatory region (URR) or long control region (LCR). The LCR is the transcriptional control unit of the virus. Mucosal epitheliotropic papillomaviruses, for example, BPV-4 and HPV-16 and -18, have a similar LCR organisation: a promoter region, an epithelial specific enhancer, and a highly conserved distribution of DNA binding sites for the viral E2 protein. When compared with the HPV-16 LCR, the BPV-4 LCR has a higher transcriptional response to activation by E2 in its target cell type. E2 up-regulates transcription from the BPV-4 LCR preferentially in primary bovine palate keratinocytes (PalK) when compared with fibroblasts (PalF) (Morgan et al., 1998). The BPV-4 LCR and primary bovine cell system presents a model to study the mechanisms of E2 mediated transcriptional regulation of mucosal epitheliotropic papillomaviruses and the cell type specificity of this regulation. Insertion of multiple E2 sites upstream from the LCR promoter demonstrates that the enhanced epithelial response to E2 is not due to the epithelial specific enhancer in the BPV-4 LCR, but is a property of the BPV-4 promoter region which responds better to several transcriptional activators in PalK cells. This is a promoter specific effect as the tk promoter shows no such epithelial preference to upstream activators. E2 responsive chimaeric promoter constructs suggest that the BPV-4 promoter region upstream of the TATA box determines the cell type selective response of the BPV-4 promoter. Deletion analysis identifies two novel repressor elements, PRE-1 and PRE- 2, that are at least in part responsible for mediating the enhanced epithelial response of this promoter. These elements function in cis to repress the basal activity of the SV40 promoter. PRE-2 binds a specific protein complex in both Pa1K and Pa1F cells. The active DNA binding protein in this complex is a major species of approximately 50 kDa. PRE-2 mutants that do not compete for binding in band shift assays do not repress transcription when multimerised upstream of the SV40 promoter. Also, nonbinding mutations introduced into the BPV-4 promoter show that the PRE-2 binding factor represses the transcriptional response of the BPV-4 promoter to E2. The PRE- 2 binding protein seems to represent a novel transcriptional repressor and regulator of papillomavirus transcription. The E2 amino terminal transactivation domain is essential for regulating transcription and replication of the viral genome. Far western blot analysis shows that the HPV-16 E2 amino terminus is able to interact with different cellular factors in keratinocytes and fibroblasts. The isolation of cDNAs encoding proteins interacting with this region of E2 in HeLa cells (an immortalised keratinocyte cell line) is described. One of the cDNAs encodes the full length L31 ribosomal protein. L31 specifically interacts with the HPV-16 E2 transactivation domain in vitro. Also, an in vitro interaction is demonstrated between E2 and a truncated version of a protein previously shown to bind the cytoplasmic domain of integrin beta4. These E2 interacting factors may represent potential regulators of transcription, replication or cellular growth control.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Cancer