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Early life maternal care received has a profound effect on later-life behaviour in adult offspring, and previous studies have suggested epigenetic mechanisms are involved. Changes in thyroid hormone receptor signalling may be related to differences in maternal care received and DNA methylation modifications. We investigated the effects of variations in temperature exposure (a proxy of maternal contact) and licking-like tactile stimulation on these processes in week-old female rat pups. We assessed thyroid hormone receptor signalling by measuring circulating triiodothyronine and transcript abundance of thyroid hormone receptors and the thyroid hormone-responsive genes DNA methyltransferase 3a and oxytocin in the paraventricular nucleus of the hypothalamus. DNA methylation of the oxytocin promoter was assessed in relation to changes in thyroid hormone receptor binding. Repeated room temperature exposure was associated with a decrease in thyroid hormone receptor signalling measures relative to nest temperature exposure, while acute room temperature exposure was associated with an increase. Repeated room temperature exposure also increased thyroid hormone receptor binding and DNA methylation at the oxytocin promoter. These findings suggest that repeated room temperature exposure may affect DNA methylation levels as a consequence of alterations in thyroid hormone receptor signalling.

Early-life maternal care received has a profound effect on later-life behaviour in adult offspring and previous studies have suggested epigenetic mechanisms (e.g., DNA methylation) are involved. Changes in thyroid hormone receptor signaling might be related to differences in maternal care received and DNA methylation modifications. We investigated the effects of two factors in the maternal environment, temperature exposure (a proxy of maternal contact) and licking-like tactile stimulation, on these processes in week-old female rat pups. We assessed thyroid hormone receptor signaling by measuring circulating triiodothyronine and transcript abundance of thyroid hormone receptors and the thyroid hormone-responsive genes DNA methyltransferase 3a and oxytocin in the paraventricular nucleus of the hypothalamus. DNA methylation of the oxytocin promoter was assessed in relation to changes in thyroid hormone receptor binding. Repeated room temperature exposure was associated with a decrease in thyroid hormone receptor signaling measures relative to nest temperature exposure, while acute room temperature exposure was associated with an increase. Repeated room temperature exposure also increased thyroid hormone receptor binding and DNA methylation at the oxytocin promoter. These findings suggest that repeated room temperature exposure may affect DNA methylation levels as a consequence of alterations in thyroid hormone receptor signaling.

Thyroid hormone (TH) possesses the ability to lower cholesterol and improve cardiac performance, which have prompted the efforts to design analogs that can utilize the cholesterol-lowering property without adversely affecting heart function. In order to gain insights into the interaction mechanism for agonists at the active site of thyroid hormone receptor β (TRβ), quantitative structure–activity relationship (QSAR) models have been developed on TRβ agonists, significant statistical coefficients were obtained (CoMFA, R2cv, .732), (CoMSIA, R2cv, .853), indicating the internal consistency of the models, the obtained models were further validated using the test set, the acquired R2pred values .7054 and .7129 were in good agreement with the experimental results. The key amino acids affecting ligand binding were identified by molecular docking, and the detailed binding modes of the compounds with different activities were also determined. Furthermore, molecular dynamics (MD) simulations were conducted to assess the reliability of the derived models and the docking results. Moreover, TH exerts significant physiological effects through modulation of the two human thyroid hormone receptor subtypes. Because TRβ and TRα locate in different target cells, selective TR ligands would target specific tissues regulated by one receptor without affecting the other. Thus, the 3D information was analyzed to reveal the most relevant structural features involved in selectivity. The findings serve as the basis for further investigation into selective TRβ/TRα agonists.

Thyroid hormone plays important roles in development, differentiation, and metabolic homeostasis by binding to nuclear thyroid hormone receptors, which regulate target gene expression by interacting with DNA response elements and coregulatory proteins. We show that thyroid hormone receptors also are single-stranded RNA binding proteins and that this binding is functionally significant. By using a series of deletion mutants, a novel RNA-binding domain was localized to a 41-amino acid segment of thyroid hormone receptor alpha1 between the second zinc finger and the ligand-binding domain. This RNA-binding domain was necessary and sufficient for thyroid hormone receptor binding to the steroid receptor RNA activator (SRA). Although SRA does not bind directly to steroid receptors, it has been identified as a steroid receptor coactivator, and was thought not to be a coactivator for thyroid hormone receptors. However, transfection studies revealed that SRA enhances thyroid hormone induction of appropriate reporter genes and that the thyroid hormone receptor RNA-binding domain is important for this enhancement. We conclude that thyroid hormone receptors bind RNA through a novel domain and that the interaction of this domain with SRA, and perhaps other RNAs, enhances thyroid hormone receptor function.

Nuclear receptors mediate gene activation through ligand-dependent interaction with coactivators. We previously cloned and characterized thyroid hormone receptor-binding protein, TRBP (NcoA6: AIB3/ASC-2/RAP250/PRIP/TRBP/NRC), as an LXXLL-containing coactivator that associates with coactivator complexes through its C terminus. To search for protein factors involved in TRBP action, we identified a distinct set of proteins from HeLa nuclear extract that interacts with the C terminus of TRBP. Analysis by mass spectrometric protein sequencing revealed a DNA-dependent protein kinase (DNA-PK) complex including its catalytic subunit and regulatory subunits, Ku70 and Ku86. DNA-PK is a heterotrimeric nuclear phosphatidylinositol 3-kinase that functions in DNA repair, recombination, and transcriptional regulation. DNA-PK phosphorylates TRBP at its C-terminal region, which directly interacts with Ku70 but not Ku86 in vitro. In addition, in the absence of DNA, TRBP itself activates DNA-PK, and the TRBP-stimulated DNA-PK activity has an altered phosphorylation pattern from DNA-stimulated activity. An anti-TRBP antibody inhibits TRBP-induced kinase activity, suggesting that protein content of TRBP is responsible for the stimulation of DNA-independent kinase activity. Furthermore, in DNA-PK-deficient scid cells, TRBP-mediated transactivation is significantly impaired, and nuclear localization of TRBP is altered. The activation of DNA-PK in the absence of DNA ends by the coactivator TRBP suggests a novel mechanism of coactivator-stimulated DNA-PK phosphorylation in transcriptional regulation.

The adverse effects of chemicals exerted via estrogen receptors (ER) and androgen receptors (AR) have been studied extensively in recent years. However, those occurring via thyroid hormone receptors (TR) have not been studied enough. We examined the TR-binding activities of thyronine derivatives and alkylphenol derivatives (bisphenol A, parabens and antioxidants with o-t-butylphenol residue) using a yeast two-hybrid assay. In this assay system, the TR-binding activity of 3,5,3′-triiodo-L-thyronine (T3) was detectable at concentrations as low as 3.0 × 10-8 M and reached a plateau at 1.0 × 10-6 M. The concentration of T3 producing 10% of the activity stimulated at 1.0 × 10-6 M (10% relative effective concentration, REC10) was 2.1 × 10-8 M. 3,5,3′,5′-tetraiodo-L-thyronine (T4), 3,3′,5′-triiodo-L-thyronine (rT3) and 3, 5-diiodo-L-thyronine (T2) also exhibited TR-binding activities. The REC10 values of these chemicals were 4.2 × 10-8, 5.0 × 10-7 and 1.0 × 10-5 M, respectively. o-Isopropylphenol and o-t-butylphenol exhibited TR-binding activities with REC10 values of 3.1 × 10-4 and 4.8 × 10-5 M, respectively, whereas t-butylbenzene, isopropylbenzene and the other chemicals tested had no detectable TR-bindng activity. These results suggest that a phenolic hydroxyl group and the ortho-substituents may play important roles in the TR-binding activities of these chemicals. This assay system will be a useful tool for screening the TR-binding activities of chemicals.

Overproduction of zinc-α2-glycoprotein by adipose tissue is crucial in accounting for the lipolysis occurring in cancer cachexia of certain malignant tumors. The main aim of this study was to explore whether thyroid hormone could enhance zinc-α2-glycoprotein production in adipose tissue. In addition, the regulation of zinc-α2-glycoprotein by thyroid hormone in the liver was investigated. We performed in vitro (HepG2 cells and primary human adipocytes) and in vivo (C57BL6/mice) experiments addressed to examine the effect of thyroid hormone on zinc-α2-glycoprotein production (mRNA and protein levels) in liver and visceral adipose tissue. We also measured the zinc-α2-glycoprotein serum levels in a cohort of patients before and after controlling their hyperthyroidism. Our results showed that thyroid hormone up-regulates zinc-α2-glycoprotein production in HepG2 cells in a dose-dependent manner. In addition, the zinc-α2-glycoprotein proximal promoter contains functional thyroid hormone receptor binding sites that respond to thyroid hormone treatment in luciferase reporter gene assays in HepG2 cells. Furthermore, zinc-α2-glycoprotein induced lipolysis in HepG2 in a dose-dependent manner. Our in vivo experiments in mice confirmed the up-regulation of zinc-α2-glycoprotein induced by thyroid hormone in the liver, thus leading to a significant increase in zinc-α2-glycoprotein circulating levels. However, thyroid hormone did not regulate zinc-α2-glycoprotein production in either human or mouse adipocytes. Finally, in patients with hyperthyroidism a significant reduction of zinc-α2-glycoprotein serum levels was detected after treatment but was unrelated to body weight changes. We conclude that thyroid hormone up-regulates the production of zinc-α2-glycoprotein in the liver but not in the adipose tissue. The neutral effect of thyroid hormones on zinc-α2-glycoprotein expression in adipose tissue could be the reason why zinc-α2-glycoprotein is not related to weight loss in hyperthyroidism.

Nuclear hormone receptors activate gene transcription through ligand-dependent association with coactivators. Specific LXXLL sequence motifs present in these cofactors are sufficient to mediate these ligand-induced interactions. A thyroid hormone receptor (TR)-binding protein (TRBP) was cloned by a Sos-Ras yeast two-hybrid system using TRβ1-ligand binding domain as bait. TRBP contains 2063 amino acid residues, associates with TR through a LXXLL motif, and is ubiquitously expressed in a variety of tissues and cells. TRBP strongly transactivates through TRβ1 and estrogen receptor in a dose-related and ligand-dependent manner, and also exhibits coactivation through AP-1, CRE, and NFκB-response elements, similar to the general coactivator CBP/p300. The C terminus of TRBP binds to CBP/p300 and DRIP130, a component of the DRIP/TRAP/ARC complex, which suggests that TRBP may activate transcription by means of such interactions. Further, the association of TRBP with the DNA-dependent protein kinase (DNA-PK) complex and DNA-independent phosphorylation of TRBP C terminus by DNA-PK point to a potential connection between transcriptional control and chromatin architecture regulation.

The increased type 1 iodothyronine deiodinase expression in hyperthyroid patients increases the fraction of plasma T3 generated from T4 by the propylthiouracil-sensitive pathway. In this study, we extend our analysis of the thyroid hormone response elements (TREs) in the 5' flanking region of the human dio1 gene. The 5' TRE (TRE2), a direct repeat separated by 4 bp (DR+4) at -660 bp, arises from an A to G substitution in an Alu sequence, the first example of this phenomenon. An SP1 binding site immediately 5' to TRE2 increases basal expression of a 430-bp dio1 promoter-chloramphenicol acetyltransferase construct in the presence of unliganded thyroid hormone receptor, thus decreasing T3 responsiveness, but does not do so when this complex is placed in its more 5' wild-type location. The two octameric binding sites of TRE1, a retinoid X-receptor independent DR+10 structure at -90, can be exchanged or inverted without loss of T3 response potency, despite significant changes in thyroid hormone receptor binding, as assessed by gel shift assays. However, the retinoic acid response of the 716-bp dio1 5' flanking region is unaffected by elimination of TRE2 but is lost with mutations in TRE1. These findings indicate the importance of functional analyses of potential ligand-responsive transcription factors, as well as the influence of position, on TRE function and interaction with basal transcription factors. The unusual features of these TREs emphasize the need to consider alternatives to canonical half-site arrangements of receptor binding sites and contexts in the evaluation of T3- and retinoic acid-responsive genes.

Thyroid hormone receptors are ligand-inducible transcription factors that can potentially interact with thyroid hormone response elements as homodimers or heterodimers with the retinoid X receptor. It has generally been felt, however, that the heterodimer is responsible for induction of gene expression. We have demonstrated previously that the optimal thyroid hormone receptor binding sequence is not the consensus hexamer half-site AGGTCA but is an octamer, TAAGGTCA. Based upon these findings, we hypothesize that thyroid hormone response elements composed of optimal half-sites (TAAGGTCA) will bind thyroid hormone receptors readily and activate gene expression independently of the retinoid X receptor. In contrast, response elements composed of suboptimal half-sites (e.g. GCAGGTCA) will require the retinoid X receptor to facilitate thyroid hormone receptor-mediated gene expression. To test this hypothesis, we have reconstituted thyroid hormone receptor-mediated gene expression in yeast. Our studies confirm the hypothesis that the retinoid X receptor is required for gene expression from response elements composed of suboptimal half-sites, whereas thyroid hormone receptors are sufficient to activate gene expression maximally from response elements containing optimal half-sites. Furthermore, coexpression of steroid receptor coactivator-1 is required for ligand-dependent gene activation from single response elements. Surprisingly, however, coexpression of the retinoid X receptor decreases the steroid receptor coactivator-1-dependent thyroid hormone induction. Overall these data demonstrate that the architecture of the thyroid hormone response element dictates the nuclear receptor requirements for gene activation. The studies suggest that different coactivators may be required for gene activation depending upon the response element architecture and the nature of the bound thyroid hormone receptor complex (homo- versus heterodimer).

Tumor necrosis factor (TNF) is a powerful pro-inflammatory cytokine and immuno-regulatory molecule, and\ud modulates susceptibility to musculoskeletal diseases. Several meta-analyses and replicated association studies have\ud implicated the minor ‘A’ variant within the TNF promoter single nucleotide polymorphism (SNP) rs361525 (-238A/G)\ud as a risk allele in joint related disorders, including psoriatic and juvenile idiopathic arthritis, and osteolysis after joint\ud arthroplasty. Here we characterized the effect of this variant on TNF promoter function. A transcriptional reporter,\ud encoding the -238A variant of the TNF promoter, resulted in 2.2 to 2.8 times greater transcriptional activation versus\ud the ‘G’ variant in murine macrophages when stimulated with pro-inflammatory stimuli. Bioinformatic analysis\ud predicted a putative binding site for thyroid hormone receptor (TR) for the -238A but not the -238G allele.\ud Overexpression of TR-α induced promoter expression 1.8-fold in the presence of the ‘A’ allele only. TR-α expression\ud both potentiated and sensitized the -238A response to LPS or a titanium particulate stimulus, whilst siRNA\ud knockdown of either THRA or THRB impaired transcriptional activation for the -238A variant only. This effect was\ud independent of receptor-ligand binding of triiodothyronine. Immunohistochemical analysis of osteolysis interface\ud membranes from patients undergoing revision surgery confirmed expression of TR-α within osteoclast nuclei at the\ud resorption surface. The ‘A’ allele at rs361525 confers increased transcriptional activation of the TNF promoter and\ud influences susceptibility to several arthritic conditions. This effect is modulated, at least in part, by binding of TR,\ud which both sensitizes and potentiates transcriptional activation of the ‘A’ variant independent of its endogenous\ud ligand.\ud

Thyrotropin, by binding its specific receptor on the plasma membrane of the thyrocyte, regulates thyroid function and differentiation. In FRTL5 cells, thyrotropin down-regulates the thyrotropin receptor (TSHr) promoter activity and induces the transcription of the alpha form of thyroid hormone receptor (TR-alpha 1). In this study we show that the thyrotropin receptor down-regulation, induced by thyrotropin, is mediated by TR-alpha 1. The thyroid hormone receptor binds, in vitro, the thyrotropin receptor minimal promoter and inhibits promoter activity in cotransfections experiments in CV 1 cells. The inhibition is achieved only in the presence of the thyroid hormone. The TSHr promoter mutated in the thyroid hormone receptor binding site does not bind TR, in vitro, and its activity is not inhibited, in cotransfection experiments, in CV 1 cells. The same mutation abolishes the TSH mediated down-regulation of the TSHr promoter activity in FRTL5 thyroid cells. These results support the hypothesis of a regulatory short loop of thyroid hormone in thyroid cells.

The human Thyroid Hormone Receptor (TR) is a member of the nuclear receptor (NR) protein family. Members of this family of proteins are responsible for propagating the signals from specific hormone ligands by binding to specific DNA response elements and regulating the transcription of target genes. Like many NRs, TR is composed of multiple domains. The N-terminal region contains two zinc-coordinating modules that can bind DNA. The C-terminal region contains a ligand binding domain that can bind thyroid hormones, co-regulator proteins, and is also able to dimerize in solution. Depending on the DNA sequence and ligation status, TR can bind different DNA response elements and function as either a heterodimer, a homodimer, or a monomer. The DNA associated dimerization capability has a major impact on the NR function as a transcription regulator. We have investigated the structural basis behind the cooperativity observed for TR binding to different DNA response elements. We have found specific interactions between the DNA binding domains are able to facilitate the cooperative binding at certain DNA response elements. The DNA mediated cooperative binding is observed even in the isolated DNA binding domains of TR and is thus not a result of the dimerization interface in the ligand binding domain.

A panel of anti-thyroid hormone receptor (TR) antisera were generated to allow direct assay of the concentrations of the alpha 1 and beta 1 receptor isoforms in nuclear extracts from adult rat liver, kidney, brain and heart, and fetal brain. An antiserum, immunoglobulin G (IgG)-beta 1, raised against amino acid sequence 62-92 of the rat TR-beta 1 specifically precipitated only TR-beta 1 in vitro translation products. A second antiserum, IgG-alpha 1/beta, generated against a sequence that is identical in the ligand binding region of rat TR-alpha 1 and TR-beta isoforms immunoprecipitated both TR-alpha 1 and -beta 1 translation products. These IgG preparations were used to specifically immunoprecipitate thyroid hormone receptor binding activity from nuclear extracts. IgG-beta 1 cleared almost 80%, and the IgG-alpha 1/beta immunoprecipitated nearly all binding from hepatic nuclear extracts. This distribution of TR protein, 80% beta 1 and 20% alpha 1, is the same as previously reported for their respective mRNAs in liver. In heart, kidney, and brain IgG-beta 1 cleared 45, 43, and 28% of total binding, respectively, and IgG-alpha 1/beta cleared all T3 binding activity from these tissues. In agreement with an earlier study, marked variations in specific protein/mRNA ratios were noted among these tissues. Consistent with our earlier report of the presence of only very low levels of TR-beta 1 mRNA in fetal brain, IgG-beta 1 cleared just 5% of binding in this tissue. Studies using an antiserum (IgG-ch) generated against homologous segments of the hinge region in both TR-alpha 1 and -beta 1 yielded results which contrasted sharply with those of IgG-alpha 1/beta. Whereas IgG-ch could also immunoprecipitate virtually all binding from hepatic extracts it cleared only 40-50% of binding from the other tissues, including fetal brain in which TR-alpha 1 accounts for greater than 90% of binding protein. The data suggest the presence of posttranslational modification of the TR-alpha 1 protein in the hinge region, consistent with the presence in this segment of potential phosphorylation sites.

The epidermal growth factor (EGF) receptor (EGFR) promoter is negatively regulated by thyroid hormone and retinoic acid. This regulation can be mapped to a 36-basepair GC-rich region of the promoter (EGFR P/E) that functions autonomously as a promoter and an enhancer when placed in front of the thymidine kinase gene TATA element. Direct high affinity binding of the thyroid hormone receptor (T3R) to this element requires a nuclear protein. Through ion exchange chromatography and gel filtration of HeLa nuclear extract, this activity was identified as a protein of approximately 67 kilodaltons. This protein did not bind to DNA alone, but greatly augmented T3R binding to the EGFR P/E sequence in gel mobility shift and DNA precipitation assays. When combined with the T3R auxillary protein (TRAP), the T3R migrated as a larger complex on the DNA. Chemical cross-linking identified this complex as a heterodimer between T3R and TRAP. T3R-TRAP binds to a 7-basepair site in the EGFR P/E (GGGACTC) that has weak homology to a consensus thyroid response element half-site. Thus, on this element, T3R-TRAP heterodimers contact the DNA primarily on a single site that comprises an inhibitory thyroid response element.

The conserved 11 zinc-finger protein CTCF is involved in several transcriptional mechanisms, including insulation and enhancer blocking. We had previously identified two composite elements consisting of a CTCF and a TR binding site at the chicken lysozyme and the human c-myc genes. Using these it has been demonstrated that thyroid hormone mediates the relief of enhancer blocking even though CTCF remains bound to its binding site. Here we wished to determine whether CTCF and TR combined sites are representative of a general feature of the genome, and whether such sites are functional in regulating enhancer blocking. Genome wide analysis revealed that about 18% of the CTCF regions harbored at least one of the four different palindromic or repeated sequence arrangements typical for the binding of TR homodimers or TR/RXR heterodimers. Functional analysis of 10 different composite elements of thyroid hormone responsive genes was performed using episomal constructs. The episomal system allowed recapitulating CTCF mediated enhancer blocking function to be dependent on poly (ADP)-ribose modification and to mediate histone deacetylation. Furthermore, thyroid hormone sensitive enhancer blocking could be shown for one of these new composite elements. Remarkably, not only did the regulation of enhancer blocking require functional TR binding, but also the basal enhancer blocking activity of CTCF was dependent on the binding of the unliganded TR. Thus, a number of composite CTCF/TR binding sites may represent a subset of other modular CTCF composite sites, such as groups of multiple CTCF sites or of CTCF/Oct4, CTCF/Kaiso or CTCF/Yy1 combinations.

Thyroid hormone (TH) is critical to normal brain development, but the mechanisms operating in this process are poorly understood. We used chromatin immunoprecipitation to enrich regions of DNA bound to thyroid receptor beta (TRbeta) of mouse cerebellum sampled on post natal day 15. Enriched target was hybridized to promoter microarrays (ChIP-on-chip) spanning -8 kb to +2 kb of the transcription start site (TSS) of 5000 genes. We identified 91 genes with TR binding sites. Roughly half of the sites were located in introns, while 30% were located within 1 kb upstream (5') of the TSS. Of these genes, 83 with known function included genes involved in apoptosis, neurodevelopment, metabolism and signal transduction. Two genes, MBP and CD44, are known to contain TREs, providing validation of the system. This is the first report of TR binding for 81 of these genes. ChIP-on-chip results were confirmed for 10 of the 13 binding fragments using ChIP-PCR. The expression of 4 novel TH target genes was found to be correlated with TH levels in hyper/hypothyroid animals providing further support for TR binding. A TRbeta binding site upstream of the coding region of myelin associated glycoprotein was demonstrated to be TH-responsive using a luciferase expression system. Motif searches did not identify any classic binding elements, indicating that not all TR binding sites conform to variations of the classic form. These findings provide mechanistic insight into impaired neurodevelopment resulting from TH deficiency and a rich bioinformatics resource for developing a better understanding of TR binding.

Purpose of review Proper thyroid hormone signaling is essential for brain development and adult brain function. Signaling can be disrupted at many levels due to altered thyroid hormone secretion, conversion or thyroid hormone receptor binding. Recent findings Mutated genes involved in thyroid hormone signaling in patients and animal models have increased the understanding of the (patho-)physiological consequences of altered thyroid hormone signaling. Neuroanatomical studies have provided more insight in the underlying neuroanatomical pathways. Summary A number of thyroid hormone signaling pathways in the hypothalamus have been proposed, which may be involved in the adaptation of the thyroid axis, not only to hypo- and hyperthyroidism, but also to inflammation, critical illness and fasting. Studies in knockout and transgenic mouse models have shown that the individual characteristics of mutations in thyroid hormone receptors can cause striking differences in the observed phenotypes.

Silencer elements, by analogy to enhancer elements, function independently of their position and orientation. We show that the chicken lysozyme silencer S-2.4 kb has many other characteristics in common with enhancer elements. The silencer is comprised of modules that independently repress gene activity-repression being increased synergistically when different or identical modules are combined. Repression is effective both on a complete and on a minimal promoter consisting of a TATA box only. One silencer module is bound in vitro by a 75–93 kd protein, termed NeP1; the other can be bound either by the product of the oncogene v- erbA or by the thyroid hormone receptor. This erbA binding site is unusual in that the palindromic sequence is inverted.

Direct repeats of the hexamer AGGTCA can serve as response elements for vitamin D, thyroid hormone, or retinoic acid. The specificity of the response appears to reside in the spacing between the hexamers, with response elements for vitamin D restricted to direct repeats separated by a 3-base pair (bp) spacer, thyroid hormone a 4-bp spacer, and retinoic acid a 5-bp spacer (3-4-5 rule). Recently we have shown that the optimum thyroid hormone receptor binding site consists of an 8-bp sequence (TAAGGTCA), not a hexamer. Therefore we tested whether the 3-4-5 rule is valid for octamer sequence direct repeats. In transfection experiments octamer direct repeats with 3-, 4-, or 5-bp spacers conferred equivalently strong thyroid hormone responses, although a repeat with a 9-bp spacer was substantially weaker. For the 4- and 5-bp spacer constructs, the 5' half-site octamer had as strong an influence on thyroid hormone induction as did the 3' half-site octamer, although for the 3-bp spacer construct the 5' octamer was marginally less potent than the 3' octamer. Transfection and gel shift experiments did not suggest a simple correlation between the binding of thyroid hormone receptor-retinoid X receptor heterodimers and thyroid hormone induction from these response elements. We conclude that half-site sequence can override the effect of spacing in determining the hormone responsiveness of a direct repeat response element. In addition, the thyroid hormone response may not be due simply to the binding of thyroid hormone receptor-retinoid X receptor heterodimers to the DNA.

There is a substantial clinical overlap between chronic renal failure (CRF) and hypothyroidism, suggesting the presence of hypothyroidism in uremic patients. Although CRF patients have low T3 and T4 levels with normal thyroid-stimulating hormone (TSH), they show a higher prevalence of goiter and evidence for blunted tissue responsiveness to T3 action. However, there are no studies examining whether thyroid hormone receptors (TRs) play a role in thyroid hormone dysfunction in CRF patients. To evaluate the effects of an uremic environment on TR function, we investigated the effect of uremic plasma on TRβ1 binding to DNA as heterodimers with the retinoid X receptor alpha (RXRα) and on T3-dependent transcriptional activity. We demonstrated that uremic plasma collected prior to hemodialysis (Pre-HD) significantly reduced TRβ1-RXRα binding to DNA. Such inhibition was also observed with a vitamin D receptor (VDR) but not with a peroxisome proliferator-activated receptor gamma (PPARγ). A cell-based assay confirmed this effect where uremic pre-HD ultrafiltrate inhibited the transcriptional activation induced by T3 in U937 cells. In both cases, the inhibitory effects were reversed when the uremic plasma and the uremic ultrafiltrate were collected and used after hemodialysis (Post-HD). These results suggest that dialyzable toxins in uremic plasma selectively block the binding of TRβ1-RXRα to DNA and impair T3 transcriptional activity. These findings may explain some features of hypothyroidism and thyroid hormone resistance observed in CRF patients.

The ability of a retinoic acid (RA) response element (RARE) in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter to mediate effects of either RA or thyroid hormone (T3) on gene expression was studied. Fusion gene constructs consisting of PEPCK promoter sequences ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were used for this analysis. While T3 induced CAT expression to a small degree (about twofold) when such constructs were transiently transfected into H4IIE rat hepatoma cells, along with an expression vector encoding the alpha subtype of the T3 receptor (TR), this effect was mediated by promoter sequences distinct from the PEPCK RARE. Although TRs were capable of binding the PEPCK RARE in the form of putative monomers, dimers, and heterodimers with RA receptors (RARs), this element failed to mediate any positive effect of T3 on gene expression. In contrast, the PEPCK RARE mediated six- to eightfold induction of CAT expression by RA. When TRs were coexpressed along with RARs in transfected H4IIE cells, this RA induction was substantially blunted in a T3-independent manner. This inhibitory effect may be due to the binding of nonfunctional TRs or TR-RAR heterodimers to the PEPCK RARE. A model is proposed to explain the previously observed in vivo effects of T3 on PEPCK gene expression.

Photoaffinity label probes of iodothyronines can interact with nuclear receptors in intact cells and in solubilized receptor preparations. These probes have certain advantages over a chemical affinity label in analyzing receptor structure. First, a photoaffinity label probe covalently cross-links only after photoactivation. Therefore, it is possible to demonstrate with appropriate competitive inhibition studies that the photoaffinity label probe associates with the receptor in question. Secondly, since cross-linking only occurs after photolysis, it is possible to adjust the concentration of the photoaffinity label to maximize association with "specific" binding sites relative to "non-specific" associations prior to covalent linkage by photoactivation. The different [125I]iodothyronine-PAL analogues may be useful as probes of the thyroid hormone receptor binding domain since PAL compounds with different affinities for receptor may photocouple to different receptor residues within or proximate to the hormone binding region. These probes may also be useful as an adjunct to receptor purification and in probing the organization of the receptor in chromatin. Lastly, they may provide insights into possible alterations of receptor structure in patients with partial end organ resistance to thyroid hormone (Refetoff et al., 1967; Eil et al., 1982).

Gel shift assays have been employed to examine the association of the thyroid hormone receptor with specific DNA sequences in the 5′-flanking DNA of the rat growth hormone (rGH) gene. This DNA is known to have structure(s) that mediate thyroid hormone effects on the rGH promoter. The receptors used were obtained from preparations purified 300-500-fold from rat liver nuclear extracts and contained about 1% pure receptors. Thyroid hormone receptor binding to DNA was assessed by monitoring protein-bound 32P-labeled restriction endonuclease fragments in parallel with L-tri[125I]iodothyronine-labeled protein-DNA complexes. The receptors were found to bind specifically to four different regions of the rGH 5′-flanking DNA (nucleotides -1730 to -1230, -530 to -230, -181 to -149, and -149 to +12) numbered with respect to the transcriptional start site. The specificity of the binding was documented by the finding that the receptor did not bind to other rGH 5′-flanking DNA sequences or to several other DNAs and by the fact that only the DNAs exhibiting specific binding could block the binding of radiolabeled DNA. The binding was also detected in NaCl concentrations up to 140 mM, reduced by Mg2+ concentrations up to 5 mM, and inhibited by 1 mM zinc. The DNA sequence-specific binding of the receptor was found to require occupancy of the receptor by the hormone (L-triiodothyronine) and could also be observed when the receptor was occupied by the thyroid hormone antagonist amiodarone. These results indicate that thyroid hormone receptors interact specifically with several sites on the 5′-flanking DNA of the rGH gene and that hormone occupancy is not required for the binding. Thus, thyroid hormone may act by stimulating a transcriptional activation function of the receptor rather than by stimulating DNA binding per se.

We have determined the concentration of thyroid hormone receptor binding sites in nuclear extracts derived from rat fetal organs throughout gestation and the postnatal period. Before day 14 of gestation nuclear extracts were obtained from whole fetuses. No receptor binding activity could be detected at day 12 of gestational age, and small amounts were detected at day 13 (maximum binding capacity less than 50 fmol/mg DNA). The receptor could be measured in pools of individual organs from day 14 (brain) or from day 16 (heart, liver, and lung) onwards. The order of analog binding affinity at 14 days was triiodothyroacetic acid = T3 greater than T4 greater than rT3, suggesting that at 14 days of fetal age the receptor has the same binding specificity as the receptor from mature tissues. In brain, the concentration of binding sites increased from 77 fmol/mg DNA at 14 days to 210 fmol/mg DNA at 17 days, remaining at this level until birth. Receptor concentration was identical whether the binding assays were performed on purified nuclei or nuclear extracts. There was no effect of maternofetal hypothyroidism on receptor concentration in the brain at 21 days of gestational age. Lung concentrations of receptor also remained constant during the fetal period. During the postnatal period, there was an increase in receptor concentration in brain and lung, with maximum levels at day 6. The pattern of receptor development in heart and liver was different, since its concentration increased progressively throughout the fetal and postnatal periods towards the levels found in adult rat tissues. The results suggest that the appearance of the thyroid hormone receptor coincides with that of the first fetal thyroid gland structures, but that it occurs much before thyroid function is fully established. As far as the receptor is concerned, fetal tissues have the potential to respond to thyroid hormone as early as the 13th day of gestational age.

Thyroid-stimulating hormone alpha and beta subunit genes are negatively regulated by thyroid hormone at the transcriptional level. Transient gene expression studies were used to demonstrate that the erbA beta form of the thyroid hormone receptor mediates negative regulation of the alpha-subunit promoter in a hormone-dependent manner. In JEG-3 choriocarcinoma cells, which are deficient in thyroid hormone receptors, coexpression of erbA beta with alpha CAT reporter genes markedly suppressed alpha CAT expression after treatment with thyroid hormone, whereas a reporter gene containing a known positive thyroid response element was induced. Thus, a single form of thyroid hormone receptor mediates both positive and negative responses to thyroid hormone in this system. Transient expression analyses of alpha gene 5' flanking sequence deletion mutants localized the negative thyroid response element to the proximal region of the promoter between -100 and +4 base pairs. The location of the negative thyroid response element in the alpha gene is therefore distinct from that of previously identified regulatory elements including the tissue-specific upstream regulatory elements, the cAMP response elements, and the glucocorticoid response elements. Overlapping segments of the alpha promoter were examined for potential thyroid hormone receptor binding sites by using gel shift assays and biotinylated DNA-binding studies. A specific thyroid hormone receptor binding site was identified between -22 and -7 base pairs, which is immediately downstream from the TATA box. This region of the alpha promoter interacts with erbA beta receptor synthesized in vitro as well as with endogenous nuclear thyroid hormone receptors, and it competes for receptor binding to a known positive thyroid response element. These studies suggest a mechanism for negative regulation in which the thyroid hormone receptor interacts with the alpha gene promoter immediately downstream of the TATA box to inhibit transcription.

Background Thyroid hormones play an essential role in early vertebrate development as well as other key processes. One of its modes of action is to bind to the thyroid hormone receptor (TR) which, in turn, binds to thyroid response elements (TREs) in promoter regions of target genes. The sequence motif for TREs remains largely undefined as does the precise chromosomal location of the TR binding sites. A chromatin immunoprecipitation on microarray (ChIP-chip) experiment was conducted using mouse cerebellum post natal day (PND) 4 and PND15 for the thyroid hormone receptor (TR) beta 1 to map its binding sites on over 5000 gene promoter regions. We have performed a detailed computational analysis of these data. Results By analysing a recent spike-in study, the optimal normalization and peak identification approaches were determined for our dataset. Application of these techniques led to the identification of 211 ChIP-chip peaks enriched for TR binding in cerebellum samples. ChIP-PCR validation of 25 peaks led to the identification of 16 true positive TREs. Following a detailed literature review to identify all known mouse TREs, a position weight matrix (PWM) was created representing the classic TRE sequence motif. Various classes of promoter regions were investigated for the presence of this PWM, including permuted sequences, randomly selected promoter sequences, and genes known to be regulated by TH. We found that while the occurrence of the TRE motif is strongly correlated with gene regulation by TH for some genes, other TH-regulated genes do not exhibit an increased density of TRE half-site motifs. Furthermore, we demonstrate that an increase in the rate of occurrence of the half-site motifs does not always indicate the specific location of the TRE within the promoter region. To account for the fact that TR often operates as a dimer, we introduce a novel dual-threshold PWM scanning approach for identifying TREs with a true positive rate of 0.73 and a false positive rate of 0.2. Application of this approach to ChIP-chip peak regions revealed the presence of 85 putative TREs suitable for further in vitro validation. Conclusions This study further elucidates TRβ gene regulation in mouse cerebellum, with 211 promoter regions identified to bind to TR. While we have identified 85 putative TREs within these regions, future work will study other mechanisms of action that may mediate the remaining observed TR-binding activity.