Regulation of the human chorionic gonadotropin α- and β-subunit promoters by AP-2

Production of the placental hormone, chorionic gonadotropin (CG), increases dramatically as cytotrophoblasts fuse to form syncytiotrophoblasts. The CG α- and β-promoters are both responsive to cAMP, although the kinetics of cAMP stimulation are different. In an effort to understand the mechanisms of coordinate induction of these genes, AP-2 binding sites were identified in the promotor regions of the α and CGβ genes. AP-2 bound to the upstream regulatory element (-180 to -150 base pairs (bp)) in the α- promotor and to several different regions of the CGβ promoter, including footprints 2 and 4B (FP2, -311 to -279 bp; FP4B, 221 to -200 bp). AP-2 antibodies induced supershifts of these complexes, confirming the identity of the protein-DNA complex. In JEG-3 cells, which contain abundant AP-2, mutations in these CGβ AP-2 sites reduced basal activity and decreased cAMP stimulation. In AP-2-deficient Hep-G2 cells, co-transfection of AP-2 stimulated expression of the CGβ promoter 10-20-fold, and the α-promoter was induced by 3-6-fold. Mutations that eliminate AP-2 binding to CGβFP4B reduced AP-2 stimulation by more than 80%, whereas mutations in FP2 reduced AP-2 stimulation by less than 50%. Analyses of AP-2 mutants revealed a requirement for the DNA binding/dimerization domain and the amino-terminal proline-rich and acid-rich transactivation domains for stimulation of the CGβ promoter. Primary cultures of placental cytotrophoblasts were differentiated into syncytiotrophoblasts in vitro to examine AP-2 expression by reverse transcriptase-polymerase chain reaction. AP-2 mRNA levels increased by day 2 and continued to rise in parallel with a marked increase in α and CGβ gene expression. We conclude that both the α and CGβ promoters contain binding sites for AP-2 and suggest that this transcription factor provides a mechanism for coordinating the induction of these genes during placental cell differentiation.