DNA binding and interaction with the nuclear receptor corepressor of thyroid hormone receptor are required for ligand-independent stimulation of the mouse preprothyrotropin-releasing hormone gene

A negative thyroid hormone response element (TRE) in the mouse preprothyrotropin-releasing hormone (TRH) gene was previously mapped within the proximal promoter element between - 83 and + 53 that contained a TRE half-site motif at - 57 ((-57)TGACCT(-52)). In transfection experiments, the promoter activity is stimulated by unliganded thyroid hormone receptor (TR) and T-3 reverses the basal promoter stimulation. In this study, we determined whether the direct binding of TR to the TRE half-site in the mouse TRH gene is required for the ligand-independent stimulation using a transient transfection assay into CV-1 cells and electrophoretic mobility shift assays (EMSA). In addition, the role of a corepressor protein for the ligand-independent stimulation was examined using a putative splicing variant of the nuclear receptor corepressor (N-CoRI). Point mutations introduced into the TRE half-site at - 57 eliminated the binding of TR and the stimulatory effect of unliganded TR. Two mutant TRs lacking DNA-binding activity and two CoR box mutant TRs showed no stimulation in the wild-type TRH promoter. The cotransfected N-CoRI potentiated the ligand-independent stimulation by the wild-type TR, but did not compensate for the impaired function of the CoR box mutant TR. In EMSA, TR strongly bound as homodimers and weakly as heterodimers with retinoid X receptor (RXR) to the element containing the TRE half-site at - 57. Binding of TR to the TRE half-site was essential to form homo- and heterodimers, and the RXR binding site appeared to be located downstream of the TRE half-site. In vitro translated N-CoRI preferentially bound TR homodimers over TR/RXR heterodimers. These results collectively suggest that the DNA-bound TR/corepressor complex might be directly involved in the ligand-independent stimulation of the mouse TRH gene promoter. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.