REGULATION OF THE HUMAN CHORIONIC-GONADOTROPIN ALPHA-SUBUNIT AND BETA-SUBUNIT PROMOTERS BY AP-2

Production of the placental hormone, chorionic gonadotropin (CG), incr eases dramatically as cytotrophoblasts fuse to form syncytiotrophoblas ts. The CG alpha- and beta-promoters are both responsive to cAMP, alth ough the kinetics of cAMP stimulation are different, In an effort to u nderstand 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 promoter regions of the alph a and CG beta genes, AP-2 bound to the upstream regulatory element (-1 86 to -156 base pairs (bp)) in the alpha-promoter and to several diffe rent regions of the CG beta promoter, including footprints 2 and 4B (F P2, -311 to -279 bp; FP4B, 221 to -200 bp). AP-2 antibodies induced su pershifts of these complexes, confirming the identity of the protein-D NA complex. In JEG-3 cells, which contain abundant AP-2, mutations in these CG beta AP-2 sites reduced basal activity and decreased cAMP sti mulation. In AP-2-deficient Hep-G2 cells, co-transfection of AP-2 stim ulated expression of the CG beta promoter 10-20-fold, and the alpha-pr omoter was induced by 3-6-fold. Mutations that eliminate AP-2 binding to CG beta FP4B reduced AP-2 stimulation by more than 80%, whereas mut ations in FP2 reduced AP-2 stimulation by less than 50%, Analyses of A P-2 mutants revealed a requirement for the DNA binding/dimerization do main and the amino terminal proline-rich and acid-rich transactivatio n domains for stimulation of the CGP promoter. Primary cultures of pla cental cytotrophoblasts were differentiated into syncytiotrophoblasts in vitro to examine AP-2 expression by reverse transcriptase-polymeras e chain reaction. AP-2 mRNA levels increased by day 2 and continued to rise in parallel with a marked increase in alpha and CG beta gene exp ression. We conclude that both the alpha and CG beta promoters contain binding sites for AP-2 and suggest that this transcription factor pro vides a mechanism for coordinating the induction of these genes during placental cell differentiation.