Transcription of the rat glucagon gene by the cyclic AMP response element-binding protein CREB is modulated by adjacent CREB-associated proteins.

The cyclic AMP (cAMP) response element (CRE) of the rat glucagon gene (Glu-CRE, 5'-TGACGTCA-3') mediates transcriptional responses to 8-bromo-cAMP and protein kinase A (PKA) in a glucagon-producing hamster islet cell line (InR1G9). By several different DNA-protein binding assays, we show that the transcription factor CREB binds to the CRE octamer and that additional nuclear proteins bind to sequences adjacent to the CRE. Mutation of the Glu-CRE octamer attenuates both the binding of CREB and cAMP-dependent PKA-stimulated transcriptional activity in transient transfection experiments but does not affect the binding of adjacent CREB-associated proteins. Progressive deletions and clustered point mutations of the sequences flanking the Glu-CRE identify sequences (5'-TCATT-3') located both 5' and 3' to the core CRE octamer that bind several proteins. Two proteins with molecular masses of 80 and 100 kDa bind to each of the 5' and 3' TCATT sites. Formation of additional protein-DNA complexes containing 45- and 20-kDa proteins depends upon the integrity of both TCATT sequences. Deletion or point mutation of the TCATT motif located on the 3' side of the CRE octamer results in enhanced transcriptional responses to PKA, suggesting that the CREB-associated proteins decrease the ability of CREB to mediate PKA-stimulated transcription. Results from these studies demonstrate that nucleotides flanking the core CRE octamer can influence the activity of the CRE by serving as binding sites for proteins that modulate the function of CREB and suggest a mechanism to explain why some consensus palindromic CREs are less responsive to cAMP stimulation than others.