trans-acting factors interact with a cyclic AMP response element to modulate expression of the human gonadotropin alpha gene.

Abstract

The a subunit of the placental hormone chorionic gonadotropin is regulated by cyclic AMP (cAMP) at the transcriptional level. A cAMP-responsive fusion gene (a-CAT) containing 1.5 kilobases of the a 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 a-CAT fusion gene increased linearly with increasing amounts of transfected plasmid and was maximal at the same amount of a-CAT DNA (2 ,ug) with or without cAMP treatment. Various amounts of different competitor DNA sequences were cotransfected with the a-CAT reporter plasmid to examine the interactions of intracellular trans-acting factors with the regulatory elements of the a gene promoter. An 800-base-pair fragment of a gene 5'-flanking sequence inhibited both basal and cAMP-stimulated transcription of the a-CAT reporter plasmid in a dose-dependent manner, indicative of interactions with one or more trans-acting factors that activate a gene expression. The a gene sequences that interact with intracellular regulatory factors were defined by using several discrete regions of the 5'-flanking sequence as competitors for a-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 a-CAT transcription. In contrast, an upstream sequence between -236 and -100 was an effective competitor for transcriptional activators of a-CAT expression. Competition for a-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 a 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 a gene transcription. The chorionic gonadotropin II gene 5'-flanking sequence also competed for a-CAT expression, suggesting that a common trans-acting factor is shared by the regulatory sequences of the a and ,I genes.

Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene.

“…This suggests that phosphorylation of the factor binding to the CRE modifies its functional activity rather than its gross affinity for specific DNA-binding sites. This is consistent with competition experiments which indicate that cAMP doe's not appear to alter the affinity of limiting transcription factors for the 5'-flanking sequence, including the CRE, of the aCG gene (19).…”

Characterization and differentiation of human first trimester placenta trophoblastic cells in culture

“…As a function of culture time, a parallel study was performed to determine the level of secreted intact hCG and its free a and J3 subunits, using specific monoclonal antibodies and radiometric assay (Bellet et al, 1986;Ozturk et al, 1987). In contrast to data obtained from either term placenta cultured cells (Kliman et al, 1986), or from first trimester explant cultures (Maruo et al, 1987) (Jameson et al, 1987;Zhou et al, 1987;Nulsen et al, 1988). Moreover, the addition of cAMP stimulated the secretion of hPL by cultured cells, in contrast to previous results obtained for term placenta (Zeitler et al, 1983).…”

Tissue-specific gene expression in the pituitary: the glycoprotein hormone alpha-subunit gene is regulated by a gonadotrope-specific protein.

“…In this report, we present analysis of the pituitary tissue specificity of the glycoprotein hormone at-subunit gene, using introduction of hybrid genes into transgenic mice and into the cell line of pituitary gonadotrope lineage derived from tumors generated in transgenic mice (55). We find that, as is the case in placental cells (10,25,46), deletion of the CREs destroys expression of the human at-subunit gene in pituitary cells. However, specification of expression of the human ao-subunit gene to the anterior pituitary gonadotrope does not involve TSEB (10), ao-ACT (49), or Pit-1/GHF-1 (2,31), which are all absent from this gonadotrope precursor cell.…”