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Nucleic Acids Research, 1993, Vol. 21, No. 23 5351-5359
© 1993

MOLECULAR BIOLOGY

Identification of positive and negative regulatory elements involved in the retinoic acid/cAMP induction of Fgf-3 transcription in F9 cells

Akira Murakami, Daniel Grinberg+, Jane Thurlow and Clive Dickson*

Imperial Cancer Research Fund Laboratories PO Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, UK

*To whom correspondence should be addressed

Received September 1, 1993. Revised October 11, 1993. Accepted October 11, 1993.

The proto-oncogene Fgf-3 has been implicated as an important signalling molecule in vertebrate development. In the mouse, it is expressed for a limited time at a multitude of sites from embryonic day 7 to birth. Transcription of Fgf-3 initiates at three promoter regions resulting in the generation of various mRNAs which nevertheless all encode the same protein products. A 1.7kb DNA fragment which encompasses these regions was joined to the CAT reporter gene and shown to function as a promoter in embryonal carcinoma cells. In stable transfectants the promoter retains its retinoic acid inducibility, initiating transcription at the same cap-sites as the endogenous gene. In differentiated F9 cells, transient transfection of progressive and targeted deletion mutants of the promoter region has revealed at least two positive and three negative regulatory elements. With one exception, loss of these elements was shown to dramatically affect promoter activity in stable transfectants of F9 cells. However the promoter remained inducible by retinoic acid to differing degrees, apart from deletions encompassing PS-4A which essentially abolished promoter activity in both undifferentiated and differentiated cells. The sequences of these potential regulatory regions were further defined using DNase-l footprinting, revealing some similarities to consensus binding sites for known transcription factors.

+Present address: Department de Genetica, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal, 08071 Barcelona, Spain


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