Functional dissection in vitro of the human c-fos promoter

Abstract

Using internal and 5' deletions, the elements contributing to the in vitro activity of the human c-fos promoter have been identified. Wild type and mutant promoters were fused to the G-free cassette and tested, using HeLa nuclear and whole cell extracts, with the fos wild type promoter as the internal control. The proximal promoter domain, spanning from -124 to -58 in the fos promoter, is the primary determinant of activity. Two elements in this domain are important, the direct repeats and the -60 element, which contains overlapping MLTF/USF and CREB/ATF transcription factor binding sites. CREB/ATF appears to be dominant, since a canonical CRE functions well in place of the -60 element. Interestingly, the direct repeats appear to require the -60 element to exert their effect. Upstream elements do not Influence promoter activity in their normal position or adjacent to the TATA box, except the serum response element (SRE). Templates containing various lengths of the fos wild type SRE next to the TATA box are stimulated by adding purified serum response factor (SRF), while SRE mutants are not responsive. The stimulation is independent of small spacing differences between the SRE and TATA elements, and the CArG core of the fos SRE suffices to respond to added SRF in vitro.