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Mol Cell Biol. 1989 October; 9(10): 4272-4281

Multiple sequence elements of a single functional class are required for cyclic AMP responsiveness of the mouse c-fos promoter.

Cold Spring Harbor Laboratory, New York 11724.

ABSTRACT

Agents that elevate the intracellular concentration of cyclic AMP (cAMP) rapidly and transiently induce expression of the c-fos proto-oncogene in BALB/c 3T3 cells. We show that the mouse c-fos promoter-enhancer region contains multiple elements that contribute to cAMP responsiveness of the promoter in transient expression assays. The most potent element was found to correspond to a previously mapped basal promoter element and protein-binding site located 65 base pairs upstream of the transcriptional initiation site. This element and two less potent sites contained a match to the cAMP response element (CRE) core sequence defined in several mammalian genes. The relative potencies of these elements corresponded with their relative affinities for cellular factors that bound to the CRE in vitro. Mutation of all three elements failed to abolish completely cAMP responsiveness of the c-fos promoter in the transient expression assay. However, we present evidence that this residual responsiveness may have been due to sequences present in vector DNA. Finally, we show, by using a new microinjection competition assay, that a double-stranded oligonucleotide carrying the major c-fos CRE is sufficient to block induction of the endogenous c-fos gene by cAMP. Therefore, induction of the endogenous gene requires positively acting cellular factors that interact with a single functional class of regulatory sites in the c-fos gene. Unrelated regulatory elements, such as the serum response element and putative AP-2 sites, are not by themselves sufficient to mediate the cAMP response.

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