trans-acting factors interact with a cyclic AMP response element to modulate expression of the human gonadotropin alpha gene.
The alpha subunit of the placental hormone chorionic gonadotropin is regulated by cyclic AMP (cAMP) at the transcriptional level. A cAMP-responsive fusion gene (alpha-CAT) containing 1.5 kilobases of the alpha gene 5'-flanking sequence linked to the chloramphenicol acetyltransferase (CAT) gene was used as a transcriptional reporter in competition assays in transfected JEG-3 choriocarcinoma cells. Expression of the alpha-CAT fusion gene increased linearly with increasing amounts of transfected plasmid and was maximal at the same amount of alpha-CAT DNA (2 micrograms) with or without cAMP treatment. Various amounts of different competitor DNA sequences were cotransfected with the alpha-CAT reporter plasmid to examine the interactions of intracellular trans-acting factors with the regulatory elements of the alpha gene promoter. An 800-base-pair fragment of alpha gene 5'-flanking sequence inhibited both basal and cAMP-stimulated transcription of the alpha-CAT reporter plasmid in a dose-dependent manner, indicative of interactions with one or more trans-acting factors that activate alpha gene expression. The alpha gene sequences that interact with intracellular regulatory factors were defined by using several discrete regions of the 5'-flanking sequence as competitors for alpha-CAT expression. A proximal promoter sequence (-99 to +44) containing the CCAAT box, TATA box, and transcriptional initiation site was a relatively ineffective competitor of alpha-CAT transcription. In contrast, an upstream sequence between -236 and -100 was an effective competitor for transcriptional activators of alpha-CAT expression. Competition for alpha-CAT expression by this regulatory sequence did not require cis interactions with downstream promoter elements and was equally effective with or without cAMP treatment. An 18-base-pair repeated sequence within this region of the alpha gene (-146 to -111) greatly enhanced both basal gene expression and cAMP responsivity and also competed for limiting cellular transcription factors. These findings suggest that JEG-3 cells contain trans-acting factors that interact with a cAMP response element to activate alpha gene transcription. The chorionic gonadotropin beta gene 5'-flanking sequence also competed for alpha-CAT expression, suggesting that a common trans-acting factor is shared by the regulatory sequences of the alpha and beta genes.
Jameson
J L
JL
Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Boston.
Deutsch
P J
PJ
Gallagher
G D
GD
Jaffe
R C
RC
Habener
J F
JF
D002822:Choriocarcinoma; D006063:Chorionic Gonadotropin / Q000235:genetics*; D000242:Cyclic AMP / Q000502:physiology*; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D006801:Humans; D046911:Macromolecular Substances; D011953:Receptors, Cyclic AMP / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014407:Tumor Cells, Cultured
D006063:Chorionic Gonadotropin; D046911:Macromolecular Substances; D011953:Receptors, Cyclic AMP; D014157:Transcription Factors; D000242:Cyclic AMP