3 DISTINCT REGIONS WITHIN THE CONSTITUTIVE ACTIVATION DOMAIN OF CAMP REGULATORY ELEMENT-BINDING PROTEIN (CREB) ARE REQUIRED FOR TRANSCRIPTION ACTIVATION

The transcription factor cAMP regulatory element-binding protein (CREB ) mediates both constitutive and cAMP-induced gene expression through distinct, independently acting domains. The constitutive activation do main (CAD) (amino acids (aa) 165-252) encompasses and overlaps exon 9 of the CREB gene (E9, aa 180-243). In the present study, deletion of e ither the CAD or exon 9 from CREB-GAL4 (CRG) reduced constitutive acti vity to less than 2-fold, without affecting kinase inducible activity. However, fusion of the CAD to the GAL4 DNA binding domain (CAD-G4) st imulated transcription, whereas fusion of exon 9 sequences did not. De letion of the amino-terminal flanking region of exon 9 (aa 165-180), b ut not COOH-terminal flanking sequences (aa 243-252), decreased consti tutive activation in either the CAD-G4 or CRG background. Deletion of the previously characterized glutamine-rich region (Q3, aa 218-252) or of a region containing a hydrophobic cluster of amino acids (HC, aa 1 80-218) also reduced constitutive activation by either CAD-G4 or CRG. No single mutation of hydrophobic residues within HC impaired activity of the CAD, but double and triple mutations did, suggesting that mult iple weak interactions are involved in function of the HC region. Thus , exon 9 of the CREB gene is necessary but not sufficient for constitu tive activation. The CAD requires three distinct regions for function, suggesting that CREB may interact with multiple targets in the RNA po lymerase II complex.