Sodium butyrate-mediated induction of the glycoprotein hormone α- subunit gene: Requirement for continued protein synthesis, identification of a butyrate-responsive element, and inhibition of promoter activation by 2- deoxyglucose

Production of the glycoprotein hormone α-subunit (GPHα) was enhanced by sodium butyrate (Btr) in HeLa cells. Induction of the HeLa α-subunit gene by Btr was inhibited by the simultaneous addition of cycloheximide (CHX), indicating a requirement for continued protein synthesis. Transient expression assays using plasmids containing the GPHα gene promoter fused to the chloramphenicol acetyltransferase (CAT) reporter gene demonstrated that the GPHα promoter is inducible by Btr in HeLa cells, and this induction could be prevented by 2-deoxyglucose (dGlc). CAT production driven by the SV- 40 early promoter, the cytochrome P-450-IA1 promoter, and the Rous sarcoma virus long terminal repeat was also enhanced by Btr, but the augmented synthesis was not inhibited by the addition of dGlc, demonstrating that the effect is restricted only to some promoters. CAT synthesis could be induced by Btr when the GPHe promoter extended upstream to position -169 (relative to the transcription start site at + 1) but not when the promoter terminated at - 150, classifying the DNA between these termini as a Btr-responsive element (BRE). This region overlaps the composite trophoblast-specific enhancer. Inactivation of enhancer subdomains by site-directed mutagenesis confirmed the deletion analysis and ranked their response to Btr as CRE < TSE < URE < αACT. Electrophoretic mobility shift analysis failed to detect any significant difference among several enhancer binding proteins in nuclear extracts from untreated and Btr-treated cells. Together, these results suggest that Btr-mediated induction of the α-subunit gene in HeLa cells is manifest either through the synthesis of a new transcription factor(s), which is inhibited by CHX but required for increased transcription from the GPHα gene promoter, or through the activity of existing factors that may require glycosylation or phosphorylation by a modification system that is inducible by Btr and inhibited by dGlc and CHX. These results further suggest that the factor is not an enhancer-binding protein or that Btr increases its transactivation potential without altering its DNA-binding activity.