CG is encoded by separate alpha- and beta-subunit genes. Expression of both genes is stimulated by cAMP, but the kinetics of activation are different, with cAMP stimulation of the alpha-gene preceding that of the beta-gene. The cAMP response element (CRE) in the alpha-gene contains a palindromic DNA sequence, TGACGTCA, that binds the transcription factor CREB, a nuclear phosphoprotein that is activated by protein kinase-A. Previously, detailed characterization of a CRE in the CG-beta-gene had been difficult due to low levels of expression in transfected cells. In this study the 5'-flanking sequence of the CG-beta-gene was fused to a sensitive luciferase (LUC) reporter gene, allowing delineation of a CG-beta-CRE in transient expression assays performed in JEG-3 choriocarcinoma cells. The full-length CG-beta-promoter, -3700 to 362 basepairs (bp), was stimulated 8- to 14-fold by treatment with 1 mM 8-bromo-cAMP. Analyses of a series of deletion mutants in the CG-beta-promoter demonstrated that -311 CG-beta-LUC retained nearly complete cAMP stimulation, but deletion to -187 bp eliminated cAMP responsiveness. Overlapping DNA fragments between -311 and -30 bp were fused to a heterologous promoter (-99-alpha-LUC) to further define the locations of basal elements and CREs. Basal expression required a combination of at least two distinct elements between -311 and -30 bp, whereas cAMP responsiveness was conferred by sequences between -311 and -202 bp. Shorter DNA sequences within this region were insufficient for cAMP stimulation, suggesting that more than one element may be required. DNase-I footprinting and gel mobility shift studies demonstrated at least three distinct protein-binding sites within the CG-beta-CRE sequence. Recombinant CREB (expressed in E. coli) did not bind to these sites, and they share no sequence homology with the alpha-gene CRE, indicating that a cAMP-responsive transcription factor other than CREB interacts with the CG-beta-promoter.
