Reversal of TR-T3 inhibition of the hTRH gene by excess TR ligand-binding domain: evidence for novel accessory protein.
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) chimetric reporter construct, an hTR beta 1 expression construct, and pABgal-hTR beta 1 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 hTR beta 1-T3 complexes could be abrogated completely by cotransfection of a 10 x excess of hTR beta 1-LBD (TR 168-456 aa) in a pABgal94 vector. In striking contrast, cotransfection of a 10 x excess of highly truncated hTR beta 1-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 10(-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 TR beta 1-T3 complexes involves interactions with an accessory co-suppressor protein, which may bridge DNA-bound TR beta 1-T3 complexes to the transcriptional initiation complex.
Li
Q L
QL
Department of Medicine, University of Maryland School of Medicine, Baltimore 21201, USA.
Feng
P
P
Koch
C
C
Shi
Z X
ZX
Wilber
J F
JF
D001665:Binding Sites; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D011401:Promoter Regions, Genetic; D011988:Receptors, Thyroid Hormone / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins; D013973:Thyrotropin-Releasing Hormone / Q000235:genetics*; D014162:Transfection; D014284:Triiodothyronine / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D011988:Receptors, Thyroid Hormone; D011993:Recombinant Fusion Proteins; D014284:Triiodothyronine; D013973:Thyrotropin-Releasing Hormone