Regulation of the human chorionic gonadotropin alpha- and beta-subunit promoters by AP-2.
Production of the placental hormone, chorionic gonadotropin (CG), increases dramatically as cytotrophoblasts fuse to form syncytiotrophoblasts. The CG alpha- and beta-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 promoter regions of the alpha and CGbeta genes. AP-2 bound to the upstream regulatory element (-186 to -156 base pairs (bp)) in the alpha-promoter and to several different regions of the CGbeta 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 CGbeta 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 CGbeta promoter 10-20-fold, and the alpha-promoter was induced by 3-6-fold. Mutations that eliminate AP-2 binding to CGbeta FP4B 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 CGbeta 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 alpha and CGbeta gene expression. We conclude that both the alpha and CGbeta 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.
Johnson
W
W
Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Albanese
C
C
Handwerger
S
S
Williams
T
T
Pestell
R G
RG
Jameson
J L
JL
D002467:Cell Nucleus / Q000378:metabolism; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D010920:Placenta / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism*
D018997:Chorionic Gonadotropin, beta Subunit, Human; D004268:DNA-Binding Proteins; D015292:Glycoprotein Hormones, alpha Subunit; D050656:Transcription Factor AP-2; D014157:Transcription Factors