Rat Zfhep (Zinc Finger Homeodomain Enhancer-binding Protein) is a transcription factor that contains two zinc finger domains and one homeodomain, and which was isolated as a TRE-binding protein by expression screening of a GH3 cell cDNA library. Electrophoretic mobility shift assays showed that the protein binds to the a-subunit glycoprotein, GH, and TSHb gene TREs. Homologous proteins from other species have been shown to repress transcription by binding E box-like sequences (Mol Cell Biol 14:6153, 1994). In this study, we investigated whether Zfhep can regulate transcription from the rat GH gene promoter. Initial experiments used a plasmid with the first 250 bp of the GH promoter fused to the luciferase gene (GH250Luc) to measure promoter activity in transient transfections of GH3 cells, by a chemiluminescent luciferase assay. Either a Zfhep expression plasmid (pcDNAI/Zfhep), or vector alone, were cotransfected with GH250Luc, as was RSV-b-galactosidase to allow for correction of differences in transfection efficiency. Cotransfection of pcDNAI/Zfhep caused a significant 34% decrease in luciferase expression from the GH250 promoter. The degree of repression was dependent on the amount of Zfhep cDNA transfected. To determine whether the TRE was sufficient for this response, two copies of the GH TRE (base -162 to base -193) were subcloned into the pT109Luc reporter vector (containing the thymidine kinase gene promoter) to generate the GH2Luc reporter plasmid. Cotransfection of pcDNAI/Zfhep, or vector alone, with pT109Luc indicates no effect of Zfhep on this reporter vector. However, luciferase expression from GH2Luc was repressed 58% by cotransfection with pcDNAI/Zfhep (P<0.05; n=3 experiments) in the presence of thyroid hormone (T3). Transfections in the presence or absence of 100nM T3 demonstrated a 44.8% decrease of the T3-response of GH2Luc when pcDNAI/Zfhep was included. Cotransfections of GH2Luc with a TRb expression vector (CDMTRb) and with either pcDNAI/Zfhep, or vector alone, demonstrated that Zfhep can inhibit the T3-response of the GH TRE even in the presence of additional TRb. Conclusions: 1.) Zfhep represses the activity of the native GH gene promoter. 2.) The GH TRE and flanking sequences are sufficient for mediating inhibition by Zfhep. 3.) Zfhep decreases the responsiveness of the GH TRE to T3. These results suggest that Zfhep may play a role in the mechanism of action of thyroid hormones at the GH gene.
