Characterization of TEF-1 and its role in cellular transformation by SV40 large tumour antigen
This thesis is an investigation into the role of the transcription enhancing factor, TEF-1, in cellular transformation by simian virus 40 (SV40) large tumour antigen (TAg). Alternative splicing of TEF-1 transcripts was identified in fibroblast cells, resulting in at least eight TEF-1 mRNA species. Transformation of fibroblast cells by TAg induces alternative splicing of TEF-1 transcripts producing a more abundant transcript lacking an exon encoding 4 amino acids, VTSM. Splicing variants that lacked this exon were detected in mouse pancreatic tumours and in human pancreatic tumour cell lines in contrast to the single 'normal' isoform that was detected from mouse pancreatic tissue. The DNA-binding consensus sequence of TEF-1 is 5'-RCATDCY-3 ' and this sequence matches the high-affinity p53 DNA-binding half site, 5'-RRRCATGYYY-3'. TEF-1 consensus binding sites occur in the p53 responsive elements of p53 target genes. TEF-1 binds to the p53 responsive element of the p21waf1/cip1 promoter. In MCF-7 human mammary epithelial cells, TEF-1 represses transcription from the p2lwaf1/cip1 promoter from at least two different regions, a distal site that requires TEF-1 binding sites and a proximal site requiring two adjacent E-boxes. Isoforms of TEF-1 that are present in transformed cells are better repressors of the p21waf1/cip1 promoter than those found in un-transformed cells. Alternative splicing of TEF-1 transcripts in rat embryo fibroblasts (REF52) occurs in response to mitogenic signalling as demonstrated by TAg transformation and serum starvation experiments. The different isoforms of TEF-1 exhibit various nuclear localization patterns and undergo nucleo-cytoplasmic shuttling in what may be cell cycle-dependent cellular localization of the protein. In TAg transformed cells however, TEF-1 localization is completely nuclear and TEF-1 is able to repress the p21waf1/cip1 promoter more efficiently than in the parental un-transformed cell line. These observations suggest a role for TEF-1 in the regulation of cell cycle genes and also provide a novel mechanism of transformation by TAg involving alternative splicing and disruption of nucleo-cytoplasmic shuttling of TEF-1.