Negative regulation of the gene for the preprothyrotropin-releasing hormone from the mouse by thyroid hormone requires additional factors in conjunction with thyroid hormone receptors

To gain additional insights into the negative gene regulatory action by triiodothyronine (T-3), we isolated a 2-kilobase pair 5'-flanking region of the mouse preprothyrotropin-releasing hormone (ppTRH) gene and characterized the DNA elements mediating inhibitory regulation by T-3 in the promoter region. In GH(4)C(1) cells, the expression of the 2-kilobase pair mouse ppTRH 5'-flanking region fused to the luciferase reporter gene occurred by transfection and was significantly suppressed by T-3. In contrast, T-3 suppression was not observed in T-3 receptor (T(3)R)-deficient CV-1 cells, suggesting that T(3)Rs were required for the negative regulation. Cotransfected mouse T(3)R alpha 1, beta 1, and beta 2 possessed indistinguish able potency for the negative regulation. Deletion analysis localized the element mediating the negative regulation to the region between -83 and +46, and the sequence downstream of the transcription start site (TSS) between +12 and +46 was found to be essential for the inhibitory regulation. In mobility shift assays, only T(3)R monomers bound to the element containing a T-3 response element half-site at -57. No apparent T(3)R binding was observed to the element downstream of TSS. Neither the T-3 response element half-site nor the element downstream of the TSS confer T-3 suppression individually in heterologous promoters. These results indicate that the negative regulation of murine ppTRH gene by T-3 might be mediated by the cooperation of T(3)R monomers with unknown factor(s) interacting with the element downstream of the TSS.