title: Repression of the human glycoprotein hormone alpha-subunit gene by glucocorticoids: evidence for receptor interactions with limiting transcriptional activators.
abstract: Expression of the glycoprotein hormone alpha gene is regulated divergently by glucocorticoids in different cell types. Coexpression of the glucocorticoid receptor (GR) with an alpha-CAT reporter gene caused activation of alpha promoter activity in fibroblasts, but repression in JEG-3 choriocarcinoma cells, indicating that cell-specific factors dictate positive vs. negative regulation of this promoter by GR. Cell-specific sequences and other enhancer elements in the the alpha gene have been relatively well characterized in JEG-3 cells, and this model was used to further examine the mechanism of transcriptional repression by glucocorticoids. Promoter mutagenesis indicated that the degree of GR-mediated repression was impaired by a variety of deletional and site-directed mutations between -171 and -111 bp, a region that includes both cell-specific and cAMP response elements (CREs). In an attempt to further localize a negative glucocorticoid response element (GRE) sequence, binding studies were used to assess GR interactions with alpha promoter DNA sequences. Using avidin-biotin complex DNA binding assays, a series of overlapping alpha promoter DNA sequences between -170 to 29 basepairs were tested, but each failed to bind GR, whereas a control GRE avidly bound receptor. Similarly, in competition assays in transfected CV-1 cells, the alpha gene 5'-flanking sequence did not compete for GR stimulation of a glucocorticoid responsive reporter gene, whereas a sequence that contains known GR-binding sites (murine mammary tumor virus) effectively inhibited GR-mediated expression. The absence of high affinity GR-binding sites in the alpha promoter suggested that mutations that affected GR inhibition may have eliminated recognition sites for transactivators, which are themselves targets for the GR, rather than altering specific negative GRE sites in the DNA sequence. To examine this possibility, GR repression was studied using chimeric transcription factors. The transcription-activating domains of several different proteins (CREB, thyroid hormone receptor, or VP16) were linked to the DNA-binding domain of Gal-4, and transcription was driven by the Gal-4 recognition site (UAS). GR markedly repressed transactivation by Gal-4-CREB and, to a lesser degree, the Gal-4-thyroid hormone receptor and Gal-4-VP16 chimeric proteins. Repression occurred when UAS was linked to either the alpha promoter or to the E1B promoter. Thus, inhibition occurs in the absence of either the CRE or the proximal alpha promoter. These results support a mechanism in which GR-mediated repression in JEG-3 cells occurs by receptor interference with the transactivating potential of enhancer-binding proteins or associated transcription factors.
authors.affiliation: Thyroid Unit, Massachusetts General Hospital, Harvard Medical School, Boston 02114.
authors.forename: V K
authors.initials: VK
authors.lastname: Chatterjee
authors.forename: L D
authors.initials: LD
authors.lastname: Madison
authors.forename: S
authors.initials: S
authors.lastname: Mayo
authors.forename: J L
authors.initials: JL
authors.lastname: Jameson
mesh_terms: D001483:Base Sequence; D001667:Binding, Competitive; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002822:Choriocarcinoma / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D005938:Glucocorticoids / Q000494:pharmacology*; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011965:Receptors, Glucocorticoid / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000494:pharmacology*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D005938:Glucocorticoids; D015292:Glycoprotein Hormones, alpha Subunit; D011965:Receptors, Glucocorticoid; D014157:Transcription Factors; D003907:Dexamethasone; D004247:DNA; D000242:Cyclic AMP; D015500:Chloramphenicol O-Acetyltransferase