Cooperative Mechanism of Transcriptional Activation by a Cyclic AMP-response Element Modulator α Mutant Containing a Motif for Constitutive Binding to CREB-binding Protein

Abstract

Cyclic AMP-response element modulator α (CREMα) is a transcription factor that is highly related to cAMP-response element-binding protein (CREB) but represses cAMP-induced gene expression from simple artificial promoters containing a cAMP-response element (CRE). CREMα lacks two glutamine-rich Q regions that, in CREB, are thought to be necessary for transcriptional activation. Nevertheless, protein kinase A stimulation induces CREMα to activate the complex native promoter in the phosphoenolpyruvate carboxykinase (PEPCK) gene. To study this phenomenon in the absence of protein kinase A stimulation, we introduced a mutation into CREMα to allow constitutive binding to the coactivator CREB-binding protein. This mutant, CREMαDIEDML, constitutively activated the PEPCK promoter. By engineering the leucine zipper regions of CREMαDIEDML and CREBDIEML to direct their patterns of dimerization, we found that only CREMαDIEDML homodimers fully activated the PEPCK promoter. By using a series of deletion and block mutants of the PEPCK promoter, we found that activation by CREMα DIEDML depended on the CRE and two CCAAT/enhancer-binding protein (C/EBP) sites. A dominant negative inhibitor of C/EBP, A-C/EBP, suppressed activation by CREMαDIEDML. Furthermore, a GAL4-C/EBPα fusion protein and CREMαDIEDML cooperatively activated a promoter containing three GAL4 sites and the PEPCK CRE. Thus, we propose that the C/EBP sites in the PEPCK promoter allow CREMα to activate transcription despite its lack of Q regions.