Reversal of TR-T 3 Inhibition of the hTRH Gene by Excess TR Ligand-Binding Domain: Evidence for Novel Accessory Protein

Abstract

The thyrotropin-releasing hormone (TRH) gene is regulated negatively at the transcriptional level by thyroid hormone (T₃). T₃ positive regulatory effects on other target genes, such as the growth hormone gene, are mediated through heterodimerization of thyroid hormone receptors (TRs) with RXR or other auxiliary nuclear protein(s). To explore whether an accessory co-suppressor protein(s) may be involved in T₃ inhibitory regulation of human TRH gene transcription, transient gene expression studies have been carried out using a hTRH-luciferase (TRH-Luc) chimeric reporter construct, an hTRβ₁ expressionssion construct, and pABgal-hTRβ1 ligand-binding domain (LBD) fusion constructs, cotransfected into a human neuroblastoma cell line (HTB-11,ATCC). Results herein indicate that T₃-dependent inhibitory regulation (48-60% of control) of the hTRH gene promoter by HTRβ1-T₃ complexes could be abrogated completely by cotransfection of a 10 × excess of hTRβ1-LBD (TR 168–456 aa) in a pABgal94 vector. In striking contrast, cotransfection of a 10 × excess of highly truncated hTRβ₁-LBD (TR 452–456 aa) failed to reverse T₃-mediated TRH promoter inhibition. This squelching effect by excessive intact TR-LBD, moreover, could not be reversed by raising T₃ concentration 100-fold (from 10^-8 to IO^-6 M), thus excluding a squelching effect of T₃ itself by excess LBD. These results suggest that negative regulation of the hTRH gene promoter activity by TRβ1-T₃ complexes involves interactions with an accessory co-suppressor protein, which may bridge DNA-bound TRβ1-T₃ complexes to the transcriptional initiation complex.