The human thyrotropin-releasing hormone gene is regulated by thyroid hormone through two distinct classes of negative thyroid hormone response elements.

Abstract

TRH is the principal positive regulator of TSH synthesis and secretion in man. T3 is able to control TRH synthesis through feedback inhibition at the transcriptional level, presumably by binding to its receptor which interacts with one or more negative thyroid hormone response elements (TREs) present within the human TRH promoter. In the present study we have identified the specific negative TREs within the TRH promoter and characterized their ability to interact with thyroid hormone receptors (TRs), and the retinoid X receptor (RXR). Our analysis demonstrates that ligand-independent and dependent regulation of the human TRH promoter is restricted to the TR beta 1 isoform. Deletional analysis of the TRH promoter identified two discrete regions that are responsible for mediating ligand-dependent negative regulation of the TRH promoter. Mutagenesis of potential TR binding half-sites within these regions 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 act in combination to allow for negative regulation. Mutation and/or deletion of each of these sites leads to a loss of negative regulation of the TRH promoter by T3. Gel-mobility shift assays of site 4 and its surrounding nucleotides revealed that this region of the promoter is capable of binding TR monomers, homodimers, and TR-RXR heterodimers. Mutagenesis 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 addition of RXR to the binding reaction leads to a loss of specific monomeric binding. To assess the functional importance of site 4 and its surrounding nucleotides we cotransfected RXR isoforms along with TR beta with TRH promoter constructs containing either site 4 or its mutant. In the presence of wild type site 4 sequence, cotransfected RXR enhanced negative regulation of the TRH promoter. Mutation and or deletion of site 4 leads to a loss of this enhancement. These data demonstrate that two structurally different negative TREs cooperate to allow for negative regulation of the human TRH promoter and that negative regulation is TR isoform-specific and modulated by the RXR-signaling pathway through a novel negative TRE.

Thyroid Hormone Receptor DNA Binding Is Required for Both Positive and Negative Gene Regulation

“…A DRϩ4 probe was employed as a pTRE and a TRH site 4 probe was utilized as a nTRE. The site 4 sequence from human TRH promoter is a well established nTRE (13). As shown in Fig.…”

“…The human TRH promoter contains three nTREs, including separate half-sites which are responsible for T 3 -dependent negative regulation and binding of TR. Site 4 in the TRH gene, which is capable of binding not only TR monomers but also TR homodimers and TR-RXR heterodimers, may mediate the enhancement of negative regulation by the RXR (13).…”

“…Electrophoretic Mobility Shift Assay-The oligonucleotide sequences of DRϩ4 and TRH site 4 probe were reported previously (13,37). Hybrid TR/GR-response elements, TR/GRE 1, 2, and 3-DRϩ4 were designed to contain an intact 5Ј half-site and a mutant 3Ј half-site separated by a four-nucleotide gap.…”

“…A number of artificial and natural positive thyroid hormone-response elements (pTREs) have been described, and they can be classified based on the half-site arrangement in the element: DRϩ4, PAL, and LAP (LYS) (9 -11). On target genes negatively regulated by T 3 , response elements (nTREs) have been described in the TRH and TSH-␤ subunit genes (12)(13)(14)(15)(16)(17). In these nTREs, TR bound as a monomer to widely spaced half-sites or in the case of TRH with retinoid X receptor (RXR) to an unusual element (13, 18 -20).…”

Increased sensitivity to thyroid hormone in mice with complete deficiency of thyroid hormone receptor alpha

“…In vitro DNA binding studies and functional assays in transfected cells have generated conflicting results concerning the specific effect of TR isoforms in gene regulation (12,13). Interpretation is complicated, however, because data are derived from artificial systems using the overexpression of chimeric gene constructs that may not be faithful models of events occurring in the intact animal.…”