Cooperative mechanism of transcriptional activation by a cyclic AMP-response element modulator α mutant containing a motif for constitutive binding to CREB-binding protein

Cyclic AMP-response element modulator cu (CREM alpha) 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), CREM alpha lacks two glutamine-rich Q regions that, in CREB, are thought to be necessary for transcriptional activation. Nevertheless, protein kinase A stimulation induces CREM alpha 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 CREM alpha to allow constitutive binding to the coactivator CREB-binding protein. This mutant, CREM alpha (DIEDML) constitutively activated the PEPCK promoter. By engineering the leucine zipper regions of CREM alpha (DIEDML) and CREB alpha (DIEDML) to direct their patterns of dimerization, we found that only CREM alpha (DIEDML) homodimers fully activated the PEPCK promoter. By using a series of deletion and block mutants of the PEPCK promoter, we found that activation by CREM alpha (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 CREM alpha (DIEDML) Furthermore, a GAL4-C/EBP alpha fusion protein and CREM alpha (DIEDML) 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 CREMa to activate transcription despite its lack of Q regions.