Bandara, Lasantha R.; (1994) Characterisation of a cellular transcription factor which co-ordinates cell cycle events with transcription. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

Transcription in eukaryotes is mediated by one of three RNA polymerases. RNA polymerase II transcribes all protein coding genes and requires a number of accessory proteins termed transcription factors for the efficient initiation and stimulation of transcription. The cellular transcription factor DRTF1 was originally defined in embryonal carcinoma (EC) stem cells using the adenovirus E2a promoter and is also known to regulate transcription from a number of cellular genes. DRTFl binds to an E2F motif and resolves as several DNA binding complexes referred to as DRTF1a,b and c, of which the b/c form is able to activate transcription. In this study I have shown that DRTF1/E2F complexes vary in different cell types and that the a complex can be disrupted by viral oncoproteins, such as adenovirus E1a and SV40 large T antigen. This requires regions within these proteins which are also necessary for the efficient transformation and immortalisation of tissue culture cells suggesting DRTF1/E2F may mediate these processes. These viral proteins sequester a number of cellular polypeptides including the tumour suppressor retinoblastoma gene product (pRb), pRb-related p107, cyclins and cyclin-dependent kinases, which regulate cell cycle progression at a number of different levels. These proteins were also shown to be components of the a complex. The pRb gene is frequently mutated in human tumours, an event which is thought to inactivate the negative growth regulatory effects of pRb. Two mutations identified in small cell lung carcinomas encoded proteins which failed to bind DRTF1/E2F suggesting that the growth suppressing properties of pRb may be mediated at the transcriptional level through DRTF1/E2F. Cyclin-dependent kinases are believed to exert their control by regulating the activity protein substrates through phosphorylation at key points during cell cycle progression. Cyclin A binds to two kinase subunits, p33cdk2 and p34cdc2, but was only able to direct p33cdk2 to DRTF1/E2F. The presence of a cdc2-like kinase in DRTF1/E2F implies that this transcription factor may play a role in cell cycle control by coupling cell cycle events with the initiation of transcription. Recently two distinct DNA binding components of DRTF1/E2F have been isolated termed E2F-1 and DP-1. DP-1 is a common component of all E2F site DNA-binding complexes in certain cell types whereas, based on other studies, E2F-1 is restricted to a proportion of DRTF1/E2F complexes. I have shown that DP-1 and E2F-1 are able to form heterodimers with greater DNA binding activity than either homodimer, an interaction which requires a region of similarity between both proteins. During cell cycle progression DRTF1/E2F complexes are regulated with "free" transcriptionally active DRTF1/E2F appearing in S-phase. DP-1 was found to be a component of all DRTF1/E2F complexes during the cell cycle in NIH 3T3 cells and is likely to form heterodimers with other E2F-1-like molecules during cell cycle progression. A number of genes expressed in S-phase contain DRTF1/E2F binding sites in their promoters, such as the DHFR, DNA polymerase α, B-myb and cdc2 genes, suggesting that this transcription factor may co-ordinate the expression of these genes during the cell cycle. These results imply that DRTF1/E2F plays a pivotal role in regulating cell cycle progression by enabling cell cycle events to be integrated with transcription.

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