THE HUMAN THYROTROPIN-RELEASING-HORMONE GENE IS REGULATED BY THYROID-HORMONE THROUGH 2 DISTINCT CLASSES OF NEGATIVE THYROID-HORMONE RESPONSE ELEMENTS

TRH is the principal positive regulator of TSH synthesis and secretion in man. T3 is able to control TRH synthesis through feedback inhibiti on at the transcriptional level, presumably by binding to its receptor which interacts with one or more negative thyroid hormone response el ements (TREs) present within the human TRH promoter. In the present st udy we have identified the specific negative TREs within the TRH promo ter and characterized their ability to interact with thyroid hormone r eceptors (TRs), and the retinoid X receptor (RXR). Our analysis demons trates that ligand-independent and dependent regulation of the human T RH promoter is restricted to the TR beta 1 isoform. Deletional analysi s of the TRH promoter identified two discrete regions that are respons ible for mediating ligand-dependent negative regulation of the TRH pro moter. Mutagenesis of potential TR binding half-sites within these reg ions identified three separate half-sites (site 4 from -55 to -60 base pairs (bp); site 5, +14 to +19 bp; and site 6, +37 to +42 bp) which a ct in combination to allow for negative regulation. Mutation and/or de letion of each of these sites leads to a loss of negative regulation o f the TRH promoter by T3. Gel-mobility shift assays of site 4 and its surrounding nucleotides revealed that this region of the promoter is c apable of binding Tn monomers, homodimers, and TR-RXR heterodimers. Mu tagenesis of site 4 leads to a loss of all binding to this region. The region encompassing sites 5 and 6 binds only TR monomer, and the addi tion of RXR to the binding reaction leads to a loss of specific monome ric binding. To assess the functional importance of site 4 and its sur rounding nucleotides we cotransfected RXR isoforms along with TRP with TRH promoter constructs containing either site 4 or its mutant. In th e presence of wild type site 4 sequence, cotransfected RXR enhanced ne gative regulation of the TRH promoter. Mutation and or deletion of sit e 4 leads to a loss of this enhancement. These data demonstrate that t wo structurally different negative TREs cooperate to allow for negativ e regulation of the human TRH promoter and that negative regulation is TR isoform-specific and modulated by the RXR-signaling pathway throug h a novel negative TRE.