Hypothalamic Thyroid Hormone Receptor α1 Signaling Controls Body Temperature.

Background: The importance of thyroid hormones (THs) for peripheral body temperature regulation has been long recognized, as medical conditions such as hyper- and hypothyroidism lead to alterations in body temperature and energy metabolism. In the past decade, the brain actions of THs and their respective nuclear receptors, thyroid hormone receptor α1 (TRα1) and thyroid hormone receptor beta (TRß), coordinating body temperature regulation have moved into focus. However, the exact roles of the individual TR isoforms and their precise neuroanatomical substrates remain poorly understood. Methods: Here we used mice expressing a mutant TRα1 (TRα1+m) as well as TRß knockouts to study body temperature regulation using radiotelemetry in conscious and freely moving animals at different ambient temperatures, including their response to oral 3,3',5-triiodothyronine (T3) treatment. Subsequently, we tested the effects of a dominant-negative TRα1 on body temperature after adeno-associated virus (AAV)-mediated expression in the hypothalamus, a region known to be involved in thermoregulation. Results: While TRß seems to play a negligible role in body temperature regulation, TRα1+m mice had lower body temperature, which was surprisingly not entirely normalized at 30°C, where defects in facultative thermogenesis or tail heat loss are eliminated as confounding factors. Only oral T3 treatment fully normalized the body temperature profile of TRα1+m mice, suggesting that the mutant TRα1 confers an altered central temperature set point in these mice. When we tested this hypothesis more directly by expressing the dominant-negative TRα1 selectively in the hypothalamus via AAV transfection, we observed a similarly reduced body temperature at room temperature and 30°C. Conclusion: Our data suggest that TRα1 signaling in the hypothalamus is important for maintaining body temperature. However, further studies are needed to dissect the precise neuroanatomical substrates and the downstream pathways mediating this effect.

Resistance to thyroid hormone induced tachycardia in RTHα syndrome.

Mutations in thyroid hormone receptor α1 (TRα1) cause Resistance to Thyroid Hormone α (RTHα), a disorder characterized by hypothyroidism in TRα1-expressing tissues including the heart. Surprisingly, we report that treatment of RTHα patients with thyroxine to overcome tissue hormone resistance does not elevate their heart rate. Cardiac telemetry in male, TRα1 mutant, mice indicates that such persistent bradycardia is caused by an intrinsic cardiac defect and not due to altered autonomic control. Transcriptomic analyses show preserved, thyroid hormone (T3)-dependent upregulation of pacemaker channels (Hcn2, Hcn4), but irreversibly reduced expression of several ion channel genes controlling heart rate. Exposure of TRα1 mutant male mice to higher maternal T3 concentrations in utero, restores altered expression and DNA methylation of ion channels, including Ryr2. Our findings indicate that target genes other than Hcn2 and Hcn4 mediate T3-induced tachycardia and suggest that treatment of RTHα patients with thyroxine in high dosage without concomitant tachycardia, is possible.

Single-cell RNA-based phenotyping reveals a pivotal role of thyroid hormone receptor alpha for hypothalamic development.

Thyroid hormone and its receptor TRα1 play an important role in brain development. Several animal models have been used to investigate this function, including mice heterozygous for the TRα1R384C mutation, which confers receptor-mediated hypothyroidism. These mice display abnormalities in several autonomic functions, which was partially attributed to a developmental defect in hypothalamic parvalbumin neurons. However, whether other cell types in the hypothalamus are similarly affected remains unknown. Here, we used single-nucleus RNA sequencing to obtain an unbiased view on the importance of TRα1 for hypothalamic development and cellular diversity. Our data show that defective TRα1 signaling has surprisingly little effect on the development of hypothalamic neuronal populations, but it heavily affects hypothalamic oligodendrocytes. Using selective reactivation of the mutant TRα1 during specific developmental periods, we find that early postnatal thyroid hormone action seems to be crucial for proper hypothalamic oligodendrocyte maturation. Taken together, our findings underline the well-known importance of postnatal thyroid health for brain development and provide an unbiased roadmap for the identification of cellular targets of TRα1 action in mouse hypothalamic development.

Thyroid receptor ß involvement in the effects of acute nicotine on hippocampus-dependent memory.

Cigarette smoking is common despite adverse health effects. Nicotine's effects on learning may contribute to addiction by enhancing drug-context associations. Effects of nicotine on learning could be direct or could occur by altering systems that modulate cognition. Because thyroid signaling can alter cognition and nicotine/smoking may change thyroid function, nicotine could affect learning through changes in thyroid signaling. These studies investigate the functional contributions of thyroid receptor (TR) subtypes ß and α1 to nicotine-enhanced learning and characterize the effects of acute nicotine and learning on thyroid hormone levels. We conducted a high throughput screen of transcription factor activity to identify novel targets that may contribute to the effects of nicotine on learning. Based on these results, which showed that combined nicotine and learning uniquely acted to increase TR activation, we identified TRs as potential targets of nicotine. Further analyses were conducted to determine the individual and combined effects of nicotine and learning on thyroid hormone levels, but no changes were seen. Next, to determine the role of TRß and TRα1 in the effects of nicotine on learning, mice lacking the TRß or TRα1 gene and wildtype littermates were administered acute nicotine prior to fear conditioning. Nicotine enhanced contextual fear conditioning in TRα1 knockout mice and wildtypes from both lines but TRß knockout mice did not show nicotine-enhanced learning. This finding supports involvement of TRß signaling in the effect of acute nicotine on hippocampus-dependent memory. Acute nicotine enhances learning and these effects may involve processes regulated by the transcription factor TRß.

TR alpha 2 exerts dominant negative effects on hypothalamic Trh transcription in vivo.

Mammalian thyroid hormone receptors (TRs) have multiple isoforms, including the bona fide receptors that bind T3 (TRα1, TRß1 and TRß2) and a non-hormone-binding variant, TRα2. Intriguingly, TRα2 is strongly expressed in the brain, where its mRNA levels exceed those of functional TRs. Ablation of TRα2 in mice results in over-expression of TRα1, and a complex phenotype with low levels of free T3 and T4, without elevated TSH levels, suggesting an alteration in the negative feedback at the hypothalamic-pituitary level. As the hypothesis of a potential TRH response defect has never been tested, we explored the functional role of TRα2 in negative feedback on transcription of hypothalamic thyrotropin, Trh. The in vivo transcriptional effects of TRα2 on hypothalamic Trh were analysed using an in vivo reporter gene approach. Effects on Trh-luc expression were examined to that of two, T3 positively regulated genes used as controls. Applying in vivo gene transfer showed that TRα2 over-expression in the mouse hypothαlamus abrogates T3-dependent repression of Trh and T3 activation of positively regulated promoters, blocking their physiological regulation. Surprisingly, loss of function studies carried out by introducing a shTRα2 construct in the hypothalamus also blocked physiological T3 dependent regulation. Thus, modulating hypothalamic TRα2 expression by either gain or loss of function abrogated T3 dependent regulation of Trh transcription, producing constant transcriptional levels insensitive to feedback. This loss of physiological regulation was reflected at the level of the endogenous Trh gene, were gain or loss of function held mRNA levels constant. These results reveal the as yet undescribed dominant negative role of TRα2 over TRα1 effect on hypothalamic Trh transcription.

A histone deacetylase inhibitor improves hypothyroidism caused by a TRα1 mutant.

Mutations of the thyroid hormone receptor α gene (THRA) cause hypothyroidism in patients with growth and developmental retardation, and skeletal dysplasia. Genetic evidence indicates that the dominant negative activity of TRα1 mutants underlies pathological manifestations. Using a mouse model of hypothyroidism caused by a dominant negative TRα1PV mutant and its derived mouse model harboring a mutated nuclear receptor corepressor (NCOR1ΔID) (Thra1(PV/+)Ncor1(ΔID/ΔID) mice), we recently showed that aberrant release of TRα1 mutants from the NCOR1 repressor complex mediates dominant negative actions of TRα1 mutants in vivo. We tested the hypothesis that deacetylation of nucleosomal histones associated with aberrant recruitment of corepressors by TRα1 mutants underlies pathological phenotypic expression. We treated Thra1(PV/+)and Thra1(PV/+)Ncor1(ΔID/ΔID) mice with a histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxyamic acid (SAHA). SAHA significantly ameliorated the impaired growth, bone development and adipogenesis of Thra1(PV/+) mice. In Thra1(PV/+)Ncor1(ΔID/ΔID) mice, SAHA improved these abnormalities even further. We focused our molecular analyses on how SAHA improved the impaired adipogenesis leading to the lean phenotype. We found that SAHA reverted the impaired adipogenesis by de-repressing the expression of the two master regulators of adipogenesis, C/ebpα and Pparγ, as well as other adipogenic genes at both the mRNA and protein levels. Chromatin immunoprecipitation analyses indicated SAHA increased the extent of acetylation of nucleosomal H4K5 and H3 to re-activate adipogenic genes to reverting adipogenesis. Thus, HDAC confers in vivo aberrant actions of TRα1 mutants. Importantly, for the first time, the present studies show that HDAC inhibitors are clearly beneficial for hypothyroidism and could be therapeutics for treatment.

Excess folate during adolescence suppresses thyroid function with permanent deficits in motivation and spatial memory.

Cognitive and memory deficits can be caused or exacerbated by dietary folate deficiency, which has been combatted by the addition of folate to grains and dietary supplements. The recommended dose of the B9 vitamin folate is 400 µg/day for adolescents and non-pregnant adults, and consumption above the recommended daily allowance is not considered to be detrimental. However, the effects of excess folate have not been tested in adolescence when neuro and endocrine development suggest possible vulnerability to long-term cognitive effects. We administered folate-supplemented (8.0 mg folic acid/kg diet) or control lab chow (2.7 mg folic acid/kg diet) to rats ad libitum from 30 to 60 days of age, and subsequently tested their motivation and learning and memory in the Morris water maze. We found that folate-supplemented animals had deficits in motivation and spatial memory, but they showed no changes of the learning- and memory-related molecules growth-associated protein-43 or Gs-α subunit protein in the hippocampus. They had decreased levels of thyroxine (T4) and triiodothyronine (T3) in the periphery and decreased protein levels of thyroid receptor-α1 and -α2 (TRα1 and TRα2) in the hippocampus. The latter may have been due to an observed increase of cytosine-phosphate-guanosine island methylation within the putative thyroid hormone receptor-α promoter, which we have mapped for the first time in the rat. Overall, folate supplementation in adolescence led to motivational and spatial memory deficits that may have been mediated by suppressed thyroid hormone function in the periphery and hippocampus.

Changes of thyroid hormone levels and related gene expression in zebrafish on early life stage exposure to triadimefon.

In this study, zebrafish was exposed to triadimefon. Thyroid hormones levels and the expression of related genes in the hypothalamic-pituitary-thyroid (HPT) axis, including thyroid-stimulating hormone (TSH-beta), deiodinases (dio1 and dio2) and the thyroid hormone receptor (thraa and thrb) were evaluated. After triadimefon exposure, increased T4 can be explained by increased thyroid-stimulating hormone (TSH-beta). The conversion of T4 to T3 (deiodinase type I-dio1) was decreased, which reduced the T3 level. Thyroid hormone receptor beta (thrb) mRNA levels were significantly down-regulated, possibly as a response to the decreased T3 levels. The overall results indicated that triadimefon exposure could alter gene expression in the HPT axis and that mechanisms of disruption of thyroid status by triadimefon could occur at several steps in the synthesis, regulation, and action of thyroid hormones.

Thyroid hormones regulate selenoprotein expression and selenium status in mice.

Impaired expression of selenium-containing proteins leads to perturbed thyroid hormone (TH) levels, indicating the central importance of selenium for TH homeostasis. Moreover, critically ill patients with declining serum selenium develop a syndrome of low circulating TH and a central downregulation of the hypothalamus-pituitary-thyroid axis. This prompted us to test the reciprocal effect, i.e., if TH status would also regulate selenoprotein expression and selenium levels. To investigate the TH dependency of selenium metabolism, we analyzed mice expressing a mutant TH receptor α1 (TRα1+m) that confers a receptor-mediated hypothyroidism. Serum selenium was reduced in these animals, which was a direct consequence of the mutant TRα1 and not related to their metabolic alterations. Accordingly, hyperthyroidism, genetically caused by the inactivation of TRß or by oral TH treatment of adult mice, increased serum selenium levels in TRα1+m and controls, thus demonstrating a novel and specific role for TRα1 in selenium metabolism. Furthermore, TH affected the mRNA levels for several enzymes involved in selenoprotein biosynthesis as well as serum selenoprotein P concentrations and the expression of other antioxidative selenoproteins. Taken together, our results show that TH positively affects the serum selenium status and regulates the expression of several selenoproteins. This demonstrates that selenium and TH metabolism are interconnected through a feed-forward regulation, which can in part explain the rapid parallel downregulation of both systems in critical illness.

T3 differentially regulates TRH expression in developing hypothalamic neurons in vitro.

Triiodothyronine (T3) plays an important role during development of the central nervous system. T3 effects on gene expression are determined in part by the type of thyroid hormone receptors (TRs) expressed in a given cell type. Previous studies have demonstrated that thyrotropin releasing hormone (TRH) transcription in the adult hypothalamus is subjected to negative regulation by thyroid hormones. However, the role of T3 on the development of TRH expression is unknown. In this study we used primary cultures derived from 17-day-old fetal rat hypothalamus to analyze the effects of T3 on TRH gene expression during development. T3 increased TRH mRNA expression in immature cultures, but decreased it in mature cultures. In addition, T3 up-regulated TRalpha1 and TRbeta2 mRNA expression. TRalpha1 expression coincided chronologically with that of TRH in the rat hypothalamus in vivo. Maturation of TRH expression in the hypothalamus may involve T3 acting through TRalpha1.

Characterization of a novel thyroid hormone receptor alpha variant involved in the regulation of myoblast differentiation.

The regulation of gene expression by thyroid hormone (T3) involves binding of the hormone to nuclear receptors [thyroid hormone receptor (TR)] acting as T3-dependent transcription factors encoded by TRalpha (NR1A1) and TRbeta (NR1A2) genes. Several TRalpha variants have already been characterized, but only some of them display T3 binding activity. In this study, we have identified another transcript, TRalpha-DeltaE6, produced by alternative splicing with microexon 6b instead of exon 6. This splicing leads to the synthesis of a protein devoid of a hinge domain. The TRalpha-DeltaE6 transcript is detected in all mouse tissues tested. Although TRalpha-DeltaE6 did not bind DNA, its expression induced a TRalpha1 sequestration in the cytoplasm. Functional studies demonstrated that TRalpha-DeltaE6 inhibits the transcriptional activity of TRalpha1 and retinoic X receptor-alpha, but not of retinoic acid receptor-alpha. We also found that TRalpha-DeltaE6 efficiently decreased the ability of TRalpha to inhibit MyoD transcriptional activity during myoblast proliferation. Consequently, when overexpressed in myoblasts, it stimulated terminal differentiation. We suggest that this novel TRalpha variant may act as down regulator of overall T3 receptor activity, including its ability to repress MyoD transcriptional activity during myoblast proliferation.

Analysis of thyroid hormone receptor betaA mRNA expression in Xenopus laevis tadpoles as a means to detect agonism and antagonism of thyroid hormone action.

Amphibian metamorphosis represents a unique biological model to study thyroid hormone (TH) action in vivo. In this study, we examined the utility of thyroid hormone receptors alpha (TRalpha) and betaA (TRbetaA) mRNA expression patterns in Xenopus laevis tadpoles as molecular markers indicating modulation of TH action. During spontaneous metamorphosis, only moderate changes were evident for TRalpha gene expression whereas a marked up-regulation of TRbetaA mRNA occurred in hind limbs (prometamorphosis), head (late prometamorphosis), and tail tissue (metamorphic climax). Treatment of premetamorphic tadpoles with 1 nM 3,5,3'-triiodothyronine (T3) caused a rapid induction of TRbetaA mRNA in head and tail tissue within 6 to 12 h which was maintained for at least 72 h after initiation of T3 treatment. Developmental stage had a strong influence on the responsiveness of tadpole tissues to induce TRbetaA mRNA during 24 h treatment with thyroxine (0, 1, 5, 10 nM T4) or T3 (0, 1, 5, 10 nM). Premetamorphic tadpoles were highly sensitive in their response to T4 and T3 treatments, whereas sensitivity to TH was decreased in early prometamorphic tadpoles and strongly diminished in late prometamorphic tadpoles. To examine the utility of TRbetaA gene expression analysis for detection of agonistic and antagonistic effects on T3 action, mRNA expression was assessed in premetamorphic tadpoles after 48 h of treatment with the synthetic agonist GC-1 (0, 10, 50, 250 nM), the synthetic antagonist NH-3 (0, 40, 200, 1000 nM), and binary combinations of NH-3 (0, 40, 200, 1000 nM) and T3 (1 nM). All tested concentrations of GC-1 as well as the highest concentration of NH-3 caused an up-regulation of TRbetaA expression. Co-treatment with NH-3 and T3 revealed strong antagonistic effects by NH-3 on T3-induced TRbetaA mRNA up-regulation. Results of this study suggest that TRbetaA mRNA expression analysis could serve as a sensitive molecular testing approach to study effects of environmental compounds on the thyroid system in X. laevis tadpoles.

Regulation of regional expression in rat brain PC2 by thyroid hormone/characterization of novel negative thyroid hormone response elements in the PC2 promoter.

The prohormone convertases (PCs) PC1 and PC2 are involved in the tissue-specific endoproteolytic processing of neuropeptide precursors within the secretory pathway. We previously showed that changes in thyroid status altered pituitary PC2 mRNA and that this regulation was due to triiodothyronine-dependent interaction of the thyroid hormone receptor (TR) with negative thyroid hormone response elements (nTREs) contained in a large proximal region of the human PC2 promoter. In the current study, we examined the in vivo regulation of brain PC2 mRNA by thyroid status and found that 6-n-propyl-2-thiouracil-induced hypothyroidism stimulated, whereas thyroxine-induced hyperthyroidism suppressed, PC2 mRNA levels in the rat hypothalamus and cerebral cortex. To address the mechanism of T3 regulation of the PC2 gene, we used human PC2 (hPC2) promoter constructs transiently transfected into GH3 cells and found that triiodothyronine negatively and 9-cis-retinoic acid positively regulated hPC2 promoter activity. EMSAs, using purified TRalpha1 and retinoid X receptor-beta (RXRbeta) proteins demonstrated that TRalpha bound the distal putative nTRE-containing oligonucleotide in the PC2 promoter, and RXR bound to both nTRE-containing oligonucleotides. EMSAs with oligonucleotides containing deletion mutations of the nTREs demonstrated that the binding to TR and RXR separately is reduced, but specific binding to TR and RXR together persists even with deletion of each putative nTRE. We conclude that there are two novel TRE-like sequences in the hPC2 promoter and that these regions act in concert in a unique manner to facilitate the effects of thyroid hormone and 9-cis-retinoic acid on PC2.

Dietary soy protein isolate and isoflavones modulate hepatic thyroid hormone receptors in rats.

Thyroid hormone receptors (TRs) are regulators of many genes involved in cholesterol and lipid metabolism. The purpose of this study was to examine the effect of soy protein isolate (SPI) and isoflavones on hepatic TRs in rats. In Expt. 1, Sprague-Dawley rats were fed diets containing either casein or alcohol-washed SPI with or without isoflavone supplementation (5-1250 mg/kg diet) for 70, 190, and 310 d. The offspring (F1) were fed the same diets as their parents (F0). In Expt. 2, Sprague-Dawley rats were fed diets containing casein or casein plus isoflavones (50-400 mg/kg diet) for 120 d. The mRNA and protein contents of the hepatic TRs were measured by semiquantitative RT-PCR and Western blot, respectively. TRalpha1, TRalpha2, and TRbeta2 contents were not affected by SPI. However, the content of the 52-kDa TRbeta1 protein, the major isoform present in the liver, was markedly increased by dietary SPI in both sexes of F0 and F1 compared with casein. The supplemental isoflavones had no effect on TRbeta1, whereas the high doses of isoflavones (250 and 1250 mg/kg diet) reduced the hepatic TRalpha1 protein content in F1 male rats on d 28. SPI had no effect on total T3 and T4 levels. However, higher dose of supplemental isoflavones markedly increased T4 level in female rats. Overall, this study demonstrates for the first time that SPI upregulates hepatic TRbeta1 expression, and that isoflavones reduce the hepatic TRalpha1 level in young male rats. The SPI-induced TRbeta1 may play a role in mediating the hypocholesterolemic and lipid-lowering actions of soy protein.

Molecular cloning and developmental expression patterns of thyroid hormone receptors and T3 target genes in the turbot (Scophtalmus maximus) during post-embryonic development.

Thyroid hormones (TH) are pleiotropic factors important for many developmental and physiological functions in vertebrates and particularly in amphibian metamorphosis. Their effects are mediated by two specific receptors (TRalpha and TRbeta), which are ligand-dependent transcription factors, members of the nuclear hormone receptor superfamily. Besides their pivotal role in amphibian metamorphosis, TH are also critical for fish metamorphosis. As this later role of TH is less studied, we analyzed their action in the turbot (Scophtalmus maximus), a metamorphosing flat fish. We describe the isolation of sequences for the turbot orthologs of a number of Xenopus genes, which are induced during amphibian metamorphosis. Developmental expression of these genes during turbot metamorphosis was studied by several methods and the expression patterns of these genes compared with those in Xenopus and flounder. We find that the period between the onset and the end of eye migration (day 22 to day 30 post-hatching) most likely corresponds to the metamorphic climax with either high TRalpha or high TH levels. Our results show that in contrast to amphibians, it is TRalpha and not TRbeta mRNA that is up-regulated during metamorphosis. Our results highlight the notion that TH regulates, through a rise of TR expression, a genetic cascade during turbot metamorphosis. The fact that TH regulates metamorphosis in amphibian and teleost fishes suggests that TH-regulated metamorphosis is a post-embryonic process conserved in most vertebrates.

Cardiac expression and function of thyroid hormone receptor beta and its PV mutant.

Thyroid hormone (T3) influences cardiac function, and mice with deletion of thyroid hormone receptor (TR)alpha have diminished cardiac function. TR alpha 1 represents 70% and TR beta 1 represents the remaining 30% of TR in ventricular myocytes, and its role in cardiac function is not well established. To determine the role of TR beta 1 in detail, we compared contractility in isolated perfused hearts from wild-type (WT) and TR beta knockout mice under normal and increased work load. TR beta knockout hearts showed contractile function similar to WT hearts at baseline and under conditions of enhanced demand. To gain insight into the role of TR beta, we used mice with a homozygous mutation in exon 10 of TR beta encoding the dominant negative PV mutant (TR beta PV) expressed from the endogenous TR beta promoter. TR beta PV mice treated with 6-propyl-2-thiouracil and supplemented with T3 to make them euthyroid have decreased contractility with negative and positive rates of relaxation and contraction as well as peak systolic pressure diminished by 35 +/- 5, 34 +/- 6, and 35 +/- 6% in comparison with WT mice. Heart rate is diminished by 36 +/- 7%, which is accompanied by decreased expression of the pacemaker-related gene hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4). The expression of TR beta 1 in the pacemaker myocytes of the sinoatrial node was confirmed by quantitation of TR alpha 1 and TR beta 1 mRNA in sinoatrial node, which showed that TR beta 1 mRNA represents 27.5 +/- 1.6% of the ligand-binding isoforms of the TR. In summary, although TR beta is expressed at much lower levels in all regions of the heart than TR alpha 1, expression of the strong dominant negative TR beta PV mutant results in decreased contractile function and heart rate.

Feedback on hypothalamic TRH transcription is dependent on thyroid hormone receptor N terminus.

The beta thyroid hormone receptor (TRbeta), but not TRalpha1, plays a specific role in mediating T(3)-dependent repression of hypothalamic TRH transcription. To investigate the structural basis of isoform specificity, we compared the transcriptional regulation and DNA binding obtained with chimeric and N-terminally deleted TRs. Using in vivo transfection assays to follow hypothalamic TRH transcription in the mouse brain, we found that TRbeta1 and chimeras with the TRbeta1 N terminus did not affect either transcriptional activation or repression from the rat TRH promoter, whereas N-terminally deleted TRbeta1 impaired T(3)-dependent repression. TRalpha1 or chimeras with the TRalpha1 N terminus reduced T(3)-independent transcriptional activation and blocked T(3)-dependent repression of transcription. Full deletion of the TRalpha1 N terminus restored ligand-independent activation of transcription. No TR isoform specificity was seen after transcription from a positive thyroid hormone response element. Gel mobility assays showed that all TRs tested bound specifically to the main negative thyroid hormone response element in the TRH promoter (site 4). Addition of neither steroid receptor coactivator 1 nor nuclear extracts from the hypothalamic paraventricular nuclei revealed any TR isoform specificity in binding to site 4. Thus N-terminal sequences specify TR T(3)-dependent repression of TRH transcription but not DNA recognition, emphasizing as yet unknown neuron-specific contributions to protein-promoter interactions in vivo.