CCAAT/enhancer-binding protein beta is an accessory factor for the glucocorticoid response from the cAMP response element in the rat phosphoenolpyruvate carboxykinase gene promoter

The cyclic AMP response element (CRE) of the rat phosphoenolpyruvate carbox ykinase (PEPCK) gene promoter is required for a complete glucocorticoid res ponse. Proteins known to bind the PEPCK CRE include the CRE-binding protein (CREB) and members of the CCAAT/enhancer-binding protein (C/EBP) family We took two different approaches to determine which of these proteins provide s the accessory factor activity for the glucocorticoid response from the PE PCK CRE. The first strategy involved replacing the CRE of the PEPCK promote r/chloramphenicol acetyltransferase reporter plasmid (pPL32) with a consens us C/EBP-binding sequence. This construct, termed p Delta CREC/EBP, binds C /EBP alpha and beta but not CREB, yet it confers a nearly complete glucocor ticoid response when transiently transfected into H4IIE rat hepatoma cells. These results suggest that one of the C/EBP family members may be the acce ssory factor. The second strategy involved cotransfecting H4IIE cells with a pPL32 mutant, in which the CRE was replaced with a GAL4-binding sequence (p Delta CREGAL4), and various GAL4 DNA-binding domain (DBD) fusion protein expression vectors. Although chimeric proteins consisting of the GAL4 DBD fused to either CREB or C/EBPa: are able to confer an increase in basal tra nscription, they do not facilitate the glucocorticoid response. In contrast , a fusion protein consisting of the GAIA DBD and amino acids 1-118 of C/EB P beta provides a significant glucocorticoid response. Additional GAIA fusi on studies were done to map the minimal domain of C/EBP beta needed for acc essory factor activity to the glucocorticoid response. Chimeric proteins co ntaining amino acid regions 1-84, 52-118, or 85-118 of C/EBP beta fused to the GAL4 DBD do not mediate a glucocorticoid response. We conclude that the amino terminus of C/EBP beta contains a multicomponent domain necessary to confer accessory factor activity to the glucocorticoid response from the C RE of the PEPCK gene promoter.