Negative regulation of transcription in vitro by a glucocorticoid response element is mediated by a trans-acting factor.
In vitro experiments with cell extracts prepared from a mouse mammary epithelial cell line demonstrated that a cis-acting glucocorticoid response element (GRE) of the mouse mammary tumor virus represses transcription from its homologous promoter. Competition transcription experiments, in which a molar excess of a restriction fragment that contains the GRE is added to the cell-free assay, revealed that a nuclear factor mediates in trans the negative regulation of mammary tumor virus transcription in vitro. Gel retention assays indicated that a factor in the extracts specifically recognizes the GRE. One unusual result of the gel retention studies was that heating the GRE probe to 65 degrees C before addition to a binding assay increases the formation of the specific protein-DNA complex 20-fold. Exonuclease III footprinting demonstrated that the sequences recognized by the factor are identical for either untreated or heat-treated probe. The footprinting also demonstrated that this factor recognizes sequences that are distinct from those recognized by the glucocorticoid receptor. A synthetic oligonucleotide based on the sequences identified by the footprinting experiments repressed the activity of a heterologous enhancer-promoter in vivo, as assayed by transient expression assays. We propose that this negative transcription element may control the basal level of expression of some glucocorticoid-modulated genes and may explain the insensitivity of certain tumor cells to steroid hormone action.
Langer
S J
SJ
Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710.
Ostrowski
M C
MC
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D004262:DNA Restriction Enzymes; D005786:Gene Expression Regulation / Q000187:drug effects*; D005809:Genes, Regulator; D005938:Glucocorticoids / Q000494:pharmacology*; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013095:Spermidine / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection
D005938:Glucocorticoids; D004262:DNA Restriction Enzymes; D013095:Spermidine