Brown, Andrew Dickson; (1996) Identification and analysis of alternative and aberrant forms of the transcription factor ATF1. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The CREB/ATF transcription factor family form dimers which regulate a diverse array of genes through the recognition of related promoter elements. The factors CREB, CREM and ATF1 are particularly homologous and define a sub-family within which heterodimerisation and alternative splicing generates further complexity and therefore increased transcriptional versatility. Transcriptional responses involving CREB are mediated via direct interactions with additional transcription factors to confer cell or promoter- specific patterns of regulation. To clone CREB binding proteins, a cDNA expression library was screened with a radio-labelled CREB protein probe. This 'Far-Western' screen proved remarkably powerful and identified a novel alternatively spliced ATF1 isoform termed vATF1. A chromosomal translocation associated with the tumour malignant melanoma of soft parts (MMSP) links the N-terminus of the Ewing's sarcoma oncogene (EWS) to the C-terminus (including the DNA binding domain) of ATF1. The resulting chimeric protein EWS-ATF1 is predicted to de-regulate genes containing ATF binding sites resulting in tumo rigenesis. Consistent with this, exogenous expression of EWS-ATF1 in JEG3 cells, influences the activity of test promoters containing ATF binding sites. Specific effects range from strong activation to repression. Furthermore an identical pattern of test promoter activity is observed in two MMSP-derived cell lines, DTC1 and SU- CCS-1, which express endogenous EWS-ATF1. The identification of promoters that are selectively active in MMSP cell lines enabled a drug targeting strategy to be tested in vitro. Sequence encoding Herpes Simplex Virus thymidine kinase, which converts ganciclovir into a cytotoxic agent, was linked to an EWS-ATF1 activatable promoter. As expected DTC1 cells incorporating this 'suicide gene' are rendered sensitive to ganciclovir. It is anticipated that this approach which exploits the transcriptional properties of EWS-ATF1 could form the basis for a selective gene therapy strategy for MMSP.

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