Unliganded Thyroid Hormone Receptor Research Articles

Thyroid hormones regulate Zfp423 expression in regionally distinct adipose depots through direct and cell-autonomous action.

The hypothalamic pituitary thyroid axis is a major regulator of many differentiation processes, including adipose tissue. However, it remains unclear whether and how thyroid hormone (TH) signaling contributes to preadipocyte commitment and differentiation into mature adipocytes. Here, we show a cell-autonomous effect of TH on the transcriptional regulation of zinc finger protein 423 (Zfp423), an early adipogenic determination factor, in murine adipose depots. Mechanistically, binding of the unliganded TH receptor to a negative TH responsive element within the Zfp423 promoter activates transcriptional activity that is reversed upon TH binding. Zfp423 upregulation is associated with increased GFP+ preadipocyte recruitment in stromal vascular fraction isolated from white fat of hypothyroid Zfp423GFP reporter mice. RNA sequencing identified Zfp423-driven gene programs that are modulated in response to TH during adipogenic differentiation. Collectively, we identified Zfp423 as a key molecule that integrates TH signaling into the regulation of adipose tissue plasticity.

The roles of thyroid hormone receptor and T3 in metamorphosis of Haliotis diversicolor

Thyroid hormone is a kind of important hormone which regulates metamorphosis. Its role is well described in amphibian metamorphosis. Thyroid hormones (T3 and T4) have also been demonstrated to play a role in metamorphosis of marine invertebrates. However, the mechanism of thyroid hormone in metamorphosis of marine invertebrates remains unknown. A homolog of vertebrate thyroid hormone receptor (TR) was cloned and identified in abalone Haliotis diversicolor and was named HdTR. The mRNA expressions of HdTR, thyroid peroxidase ( TPO ), thyroid peroxidase 1 ( TPO1 ), idothyronine deiodinase III ( IDIII ) and integrin alpha-V ( ITGAV ) had significant difference in metamorphosis of H. diversicolor. Metamorphosis rate and mortality rate were significantly different in HdTR RNAi experiment and T3 inducing experiment. In RNAi experiment, ITGAV and CCND1 (cyclin D1) expression of dsRNA HdTR exposing group were significantly lower than those of blank control and negative control. But CTNNB (catenin beta) expression of dsRNA HdTR exposing group was higher than that those of blank control and negative control. ERK (extracellular signal regulated kinases) and PI3K (phosphoinositide-3-kinase) had no significant difference in RNAi experiment. Moreover, ITGAV of 1 μmol/L T3 group was significantly lower than that of 0 μmol/L T3 group, PI3K expression of 10 μmol/L T3 group was higher than that of 0 μmol/L T3 group, and the other genes expression had no significant difference in T3 inducing experiment. The data of genes expression suggested that CCND1 might be an effector gene of TR genomic action, while CTNNB might be regulated by unliganded TR. CCND1 and CTNNB may be involved in cell proliferation of metamorphosis. T3 might regulate the expression level of PI3K via nongenomic way. These results shed light on the mechanism of thyroid hormone in abalone metamorphosis.

A Possible Novel Mechanism of Action of Genistein and Daidzein for Activating Thyroid Hormone Receptor-Mediated Transcription

Thyroid hormone receptors (TRs) are members of the nuclear receptor superfamily that regulate their target genes for controlling organ development and functional maintenance. Soybean isoflavones, especially genistein and daidzein, modulate various hormone-mediated pathways. However, their effects on TRs have not yet been extensively studied. In this study, the effects of these isoflavones on TR action were evaluated using transient transfection-based reporter gene assays and molecular docking studies. Genistein and daidzein augmented T3-liganded TR-mediated transcription in a concentration-dependent manner. In the mammalian 2-hybrid study, these isoflavones augmented the recruitment of steroid receptor coactivator-1 and nuclear corepressor to liganded or unliganded TRs. Using a series of mutant TRs, we also showed that the activation function-2 domain of TRs was responsible for the augmentation by these isoflavones. CV-1 cells had expressed TRα, TRβ1, and ERα mRNAs. However, neither the overexpression nor the knocking down of ERα altered the augmentation of TR action by isoflavones, indicating that the effects of isoflavones are exerted through their direct action on TRs. In silico molecular docking studies showed that genistein and daidzein can directly bind to the TR-ligand-binding domain. These findings indicate that the augmentation of the TR-mediated transcription by genistein and daidzein is due to their direct binding to TR-ligand-binding domain to induce the recruitment of steroid receptor coactivator-1. Our study reports a novel mode of action of soybean isoflavones on TR function. The biological effects and the relevance of these isoflavones to human health may be partially attributable to the activation of thyroid hormone signaling.

Transducin β-like 1, X-linked and nuclear receptor co-repressor cooperatively augment the ligand-independent stimulation of TRH and TSHβ gene promoters by thyroid hormone receptors.

Mutations in TBL1X, a component of the nuclear receptor co-repressor (N-CoR) and silencing mediator of retinoic acid and thyroid hormone receptor co-repressor complexes, have recently been implicated in isolated central hypothyroidism (CeH). However, the mechanisms by which TBL1X mutations affect negative feedback regulation in the hypothalamus-pituitary-thyroid axis remain unclear. N-CoR was previously reported to paradoxically enhance the ligand-independent stimulation of TRH and TSHβ gene promoters by thyroid hormone receptors (TR) in cell culture systems. We herein investigated whether TBL1X affects the unliganded TR-mediated stimulation of the promoter activities of genes negatively regulated by T3 in cooperation with N-CoR. In a hypothalamic neuronal cell line, the unliganded TR-mediated stimulation of the TRH gene promoter was significantly enhanced by co-transfected TBL1X, and the co-transfection of TBL1X with N-CoR further enhanced promoter activity. In contrast, the knockdown of endogenous Tbl1x using short interfering RNA significantly attenuated the N-CoR-mediated enhancement of promoter activity in the presence of unliganded TR. The co-transfection of N365Y or Y458C, TBL1X mutants identified in CeH patients, showed impaired co-activation with N-CoR for the ligand-independent stimulation of the TRH promoter by TR. In the absence of T3, similar or impaired enhancement of the TSHβ gene promoter by the wild type or TBL1X mutants, respectively, was observed in the presence of co-transfected TR and N-CoR in CV-1 cells. These results suggest that TBL1X is needed for the full activation of TRH and TSHβ gene promoters by unliganded TR. Mutations in TBL1X may cause CeH due to the impaired up-regulation of TRH and/or TSHβ gene transcription despite low T3 levels.

NCoR1-independent mechanism plays a role in the action of the unliganded thyroid hormone receptor

Nuclear receptor corepressor 1 (NCoR1) is considered to be the major corepressor that mediates ligand-independent actions of the thyroid hormone receptor (TR) during development and in hypothyroidism. We tested this by expressing a hypomorphic NCoR1 allele (NCoR1ΔID), which cannot interact with the TR, in Pax8-KO mice, which make no thyroid hormone. Surprisingly, abrogation of NCoR1 function did not reverse the ligand-independent action of the TR on many gene targets and did not fully rescue the high mortality rate due to congenital hypothyroidism in these mice. To further examine NCoR1's role in repression by the unliganded TR, we deleted NCoR1 in the livers of euthyroid and hypothyroid mice and examined the effects on gene expression and enhancer activity measured by histone 3 lysine 27 (H3K27) acetylation. Even in the absence of NCoR1 function, we observed strong repression of more than 43% of positive T3 (3,3',5-triiodothyronine) targets in hypothyroid mice. Regulation of approximately half of those genes correlated with decreased H3K27 acetylation, and nearly 80% of these regions with affected H3K27 acetylation contained a bona fide TRβ1-binding site. Moreover, using liver-specific TRβ1-KO mice, we demonstrate that hypothyroidism-associated changes in gene expression and histone acetylation require TRβ1. Thus, many of the genomic changes mediated by the TR in hypothyroidism are independent of NCoR1, suggesting a role for additional signaling modulators in hypothyroidism.

Thyroid Hormone Regulation of Adult Neurogenesis.

Thyroid hormone is classically known to play a crucial role in neurodevelopment. The potent effects that thyroid hormone exerts on the adult mammalian brain have been uncovered relatively recently, including an important role in the modulation of progenitor development in adult neurogenic niches. This chapter extensively reviews the current understanding of the influence of thyroid hormone on distinct stages of adult progenitor development in the subgranular zone (SGZ) of the hippocampus and subventricular zone (SVZ) that lines the lateral ventricles. We discuss the role of specific thyroid hormone receptor isoforms, in particular TRα1, which modulates cell cycle exit in neural stem cells, progenitor survival, and cell fate choice, with both a discrete and overlapping nature of regulation noted in SGZ and SVZ progenitors. The balance between liganded and unliganded TRα1 can evoke differing consequences for adult progenitor development, and the relevance of this to conditions such as adult-onset hypothyroidism, wherein unliganded thyroid hormone receptors (TRs) dominate, is also a focus of discussion. Although a detailed molecular understanding of the specific thyroid hormone target genes that contribute to the neurogenic actions of thyroid hormone is currently lacking, we highlight the current state of knowledge and discuss avenues for future investigation. The goal of this chapter is to provide a comprehensive and detailed analysis of the effects of thyroid hormone on adult neurogenesis, to discuss putative molecular mechanisms that mediate these effects, and the behavioral, functional, and clinical implications of the neurogenic actions of thyroid hormone.

Regulation of growth rate and developmental timing by Xenopus thyroid hormone receptor α.

Thyroid hormone (TH) is critical for vertebrate postembryonic development, a period around birth in mammals when plasma TH levels are high. Interestingly, TH receptors (TRs), especially TRα, are expressed prior to the synthesis and secretion of zygotic TH, suggesting the existence of unliganded TR during development. However, the role of unliganded TR during mammalian development has been difficult to study, in part due to the relatively weak phenotype of TR knockout mice. Amphibian metamorphosis resembles postembryonic development in mammals and is controlled by TH via TRs. Like in mammals, TRα gene is highly activated and is the major TR expressed prior to the synthesis of endogenous TH. By using TALEN (transcriptional activator like effector nucleases)-mediated gene editing approach, we and others have now shown that unliganded TRα has two independent functions during Xenopus premetamorphosis, i.e. inhibiting growth rate and slowing development. Furthermore, molecular and transgenic studies have shown that unliganded TRα accomplishes these via the recruitment of histone deacetylase (HDAC)-containing corepressor complexes to repress the expression of TH-inducible genes.

Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling.

A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand.

The Optimal Corepressor Function of Nuclear Receptor Corepressor (NCoR) for Peroxisome Proliferator-activated Receptor γ Requires G Protein Pathway Suppressor 2

Repression of peroxisome proliferator-activated receptor γ (PPARγ)-dependent transcription by the nuclear receptor corepressor (NCoR) is important for homeostatic expression of PPARγ target genes in vivo. The current model states that NCoR-mediated repression requires its direct interaction with PPARγ in the repressive conformation. Previous studies, however, have shown that DNA-bound PPARγ is incompatible with a direct, high-affinity association with NCoR because of the inherent ability of PPARγ to adopt the active conformation. Here we show that NCoR acquires the ability to repress active PPARγ-mediated transcription via G protein pathway suppressor 2 (GPS2), a component of the NCoR corepressor complex. Unlike NCoR, GPS2 can recognize and bind the active state of PPARγ. In GPS2-deficient mouse embryonic fibroblast cells, loss of GPS2 markedly reduces the corepressor function of NCoR for PPARγ, leading to constitutive activation of PPARγ target genes and spontaneous adipogenesis of the cells. GPS2, however, is dispensable for repression mediated by unliganded thyroid hormone receptor α or a PPARγ mutant unable to adopt the active conformation. This study shows that GPS2, although dispensable for the intrinsic repression function of NCoR, can mediate a novel corepressor repression pathway that allows NCoR to directly repress active PPARγ-mediated transcription, which is important for the optimal corepressor function of NCoR for PPARγ. Interestingly, GPS2-dependent repression specifically targets PPARγ but not PPARα or PPARδ. Therefore, GPS2 may serve as a unique target to manipulate PPARγ signaling in diseases.

Unliganded thyroid hormone receptor α regulates developmental timing via gene repression in Xenopus tropicalis.

Thyroid hormone (TH) receptor (TR) expression begins early in development in all vertebrates when circulating TH levels are absent or minimal, yet few developmental roles for unliganded TRs have been established. Unliganded TRs are expected to repress TH-response genes, increase tissue responsivity to TH, and regulate the timing of developmental events. Here we examined the role of unliganded TRα in gene repression and development in Xenopus tropicalis. We used transcription activator-like effector nuclease gene disruption technology to generate founder animals with mutations in the TRα gene and bred them to produce F1 offspring with a normal phenotype and a mutant phenotype, characterized by precocious hind limb development. Offspring with a normal phenotype had zero or one disrupted TRα alleles, and tadpoles with the mutant hind limb phenotype had two truncated TRα alleles with frame shift mutations between the two zinc fingers followed by 40–50 mutant amino acids and then an out-of-frame stop codon. We examined TH-response gene expression and early larval development with and without exogenous TH in F1 offspring. As hypothesized, mutant phenotype tadpoles had increased expression of TH-response genes in the absence of TH and impaired induction of these same genes after exogenous TH treatment, compared with normal phenotype animals. Also, mutant hind limb phenotype animals had reduced hind limb and gill responsivity to exogenous TH. Similar results in methimazole-treated tadpoles showed that increased TH-response gene expression and precocious development were not due to early production of TH. These results indicate that unliganded TRα delays developmental progression by repressing TH-response genes.

Genome-Wide Binding Patterns of Thyroid Hormone Receptor Beta

Thyroid hormone (TH) receptors (TRs) play central roles in metabolism and are major targets for pharmaceutical intervention. Presently, however, there is limited information about genome wide localizations of TR binding sites. Thus, complexities of TR genomic distribution and links between TRβ binding events and gene regulation are not fully appreciated. Here, we employ a BioChIP approach to capture TR genome-wide binding events in a liver cell line (HepG2). Like other NRs, TRβ appears widely distributed throughout the genome. Nevertheless, there is striking enrichment of TRβ binding sites immediately 5′ and 3′ of transcribed genes and TRβ can be detected near 50% of T3 induced genes. In contrast, no significant enrichment of TRβ is seen at negatively regulated genes or genes that respond to unliganded TRs in this system. Canonical TRE half-sites are present in more than 90% of TRβ peaks and classical TREs are also greatly enriched, but individual TRE organization appears highly variable with diverse half-site orientation and spacing. There is also significant enrichment of binding sites for TR associated transcription factors, including AP-1 and CTCF, near TR peaks. We conclude that T3-dependent gene induction commonly involves proximal TRβ binding events but that far-distant binding events are needed for T3 induction of some genes and that distinct, indirect, mechanisms are often at play in negative regulation and unliganded TR actions. Better understanding of genomic context of TR binding sites will help us determine why TR regulates genes in different ways and determine possibilities for selective modulation of TR action.

The adenovirus 55 residue E1A protein is a transcriptional activator and binds the unliganded thyroid hormone receptor

The early region 1A (E1A) of human adenovirus types 2 and 5 is differentially spliced to yield five distinct mRNAs that encode different proteins. The smallest E1A RNA transcript encodes a 55 residue (55R) protein that shares only 28 amino acid residues with the other E1A proteins. Even though it is the most abundant E1A transcript at late times post-infection, little is known about the functions of this E1A isoform. In this study, we show that the E1A 55R protein interacts with, and modulates the activity of the unliganded thyroid hormone receptor (TR). We demonstrate that E1A 55R contains a signature motif known as the CoRNR box that confers interaction with the unliganded TR; this motif was originally identified in cellular corepressors. Using a system reconstituted in the yeast Saccharomyces cerevisiae, which lack endogenous TR and TR coregulators, we show that E1A 55R nonetheless differs from cellular corepressors as it functions as a strong co-activator of TR-dependent transcription and that it possesses an intrinsic transcriptional activation domain. These data indicate that the E1A 55R protein functions as a transcriptional regulator.

Tetrabromobisphenol-A disrupts thyroid hormone receptor alpha function in vitro: Use of fluorescence polarization to assay corepressor and coactivator peptide binding

Thyroid hormone receptors (TRs) recruit corepressor or coactivator factors to the promoters of target genes to regulate their transcription. Corepressors such as nuclear hormone receptor corepressor (NCoR) are recruited by unliganded TRs, whereas coactivators such as steroid receptor coactivator-2 (SRC2) are recruited when triiodothyronine (T3) is bound to TRs. These coregulator proteins interact with the ligand binding domain (LBD) of TRs via short, conserved peptide sequences that can be used to probe the conformational changes induced in TR LBD by TR ligands. Recombinant LBD of the human TRα1 isoform (hTRα1 LBD) was produced as a fusion with glutathione S-transferase, and used to develop assays based on fluorescence polarization to quantify the binding of either NCoR- or SRC2-derived fluorescent peptides to the hTRα1 LBD. The optimum concentrations of recombinant hTRα1 LBD, and of peptide probes were adjusted in order to produce the greatest possible T3-dependent signal variations in fluorescence polarization. Under these conditions, T3 induced a dose-dependent decrease in NCoR peptide binding, and a reciprocal dose-dependent increase in SRC2 peptide binding, in both cases at similar 50%-effective doses. The TR agonists triiodothyroacetic acid and thyroxine were also effective in preventing NCoR peptide binding and increasing SRC2 peptide binding, whereas reverse-triiodothyronine was less efficient and the biologically inactive thyronine had no effect on either process. These experiments validate cell-free assays based on the use of binding of corepressor or coactivator peptide probes, as measured by fluorescence polarization, for investigating the conformational changes of TRα1 LBD induced by potentially TR-interfering compounds. Both these methods were used to elucidate the mechanism of the disrupting effects of tetrabromobisphenol-A (TBBPA) on the hTRα1 LBD conformation related to the transcriptional activity of the receptor. TBBPA is a flame retardant that is released into the environment, and is a suspected disrupter of thyroid homeostasis. The present results indicate that TBBPA did indeed interfere with the ability of the hTRα1 LBD to bind both NCoR and SRC2. TBBPA behaved similarly to T3 in promoting the release of NCoR from LBD, whereas it failed to promote LBD interactions with SRC2. However, it did reduce the T3-induced interactions between LBD and the coactivator peptide. This study therefore suggests that TBBPA in the micromolar range can affect the regulation of transcription by both the apo- and the holo-TRα1, with potential disruption of the expression of genes that are either up- or down-regulated by T3.

Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)-based Quantitative Proteomics Study of a Thyroid Hormone-regulated Secretome in Human Hepatoma Cells

The thyroid hormone, 3, 3',5-triiodo-l-thyronine (T(3)), regulates cell growth, development, differentiation, and metabolism via interactions with thyroid hormone receptors (TRs). However, the secreted proteins that are regulated by T(3) are yet to be characterized. In this study, we used the quantitative proteomic approach of stable isotope labeling with amino acids in cell culture coupled with nano-liquid chromatography-tandem MS performed on a LTQ-Orbitrap instrument to identify and characterize the T(3)-regulated proteins secreted in human hepatocellular carcinoma cell lines overexpressing TRα1 (HepG2-TRα1). In total, 1742 and 1714 proteins were identified and quantified, respectively, in three independent experiments. Among these, 61 up-regulated twofold and 11 down-regulated twofold proteins were identified. Eight proteins displaying increased expression and one with decreased expression in conditioned media were validated using Western blotting. Real-time quantitative RT-PCR further disclosed induction of plasminogen activator inhibitor-1 (PAI-1), a T(3) target, in a time-course and dose-dependent manner. Serial deletions of the PAI-1 promoter region and subsequent chromatin immunoprecipitation assays revealed that the thyroid hormone response element on the promoter is localized at positions -327/-312. PAI-1 overexpression enhanced tumor growth and migration in a manner similar to what was seen when T(3) induced PAI-1 expression in J7-TRα1 cells, both in vitro and in vivo. An in vitro neutralizing assay further supported a crucial role of secreted PAI-1 in T(3)/TR-regulated cell migration. To our knowledge, these results demonstrate for the first time that proteins involved in the urokinase plasminogen activator system, including PAI-1, uPAR, and BSSP4, are augmented in the extra- and intracellular space of T(3)-treated HepG2-TRα1 cells. The T(3)-regulated secretome generated in the current study may provide an opportunity to establish the mechanisms underlying T(3)-associated tumor progression and prognosis.

Thyroid Hormone and COUP-TF1 Regulate Kallikrein-Binding Protein (KBP) Gene Expression

Kallikrein-binding protein (KBP) is a component of the kallikrein-kinin system that mediates vasodilation and inhibits tumor growth by antagonizing vascular endothelial growth factor-mediated angiogenesis. We demonstrate that KBP gene expression is repressed by T(3) and modulated by the orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1). In hypothyroid mice, KBP mRNA expression in the testis was increased 2.1-fold compared with euthyroid mice. We have identified two negative thyroid hormone response elements (nTREs) in the mouse KBP gene, nTRE1 located in the 5' flanking region (-53 to -29) and nTRE2, located in the first intron (104-132). We used functional assays, cofactor knockdown, and chromatin immunoprecipitation assays to characterize nTRE1 and nTRE2 in hepatic (HepG2) and testes (GC-1spg) cell lines. Reporter expression directed by both elements was enhanced with addition of thyroid hormone receptor and repressed with the addition of T(3). COUP-TF1 enhanced basal expression of both elements but blunted unliganded thyroid hormone receptor enhancement and T(3) repression of nTRE1 but not nTRE2. Both nTREs bound nuclear corepressor and binding increased in response to T(3). Nuclear corepressor knockdown resulted in loss of T(3) repression of both nTRE1 and nTRE2. COUP-TF1, which usually represses T(3) induction of positive thyroid hormone response elements, reverses T(3) repression mediated by nTRE1 in the mouse KBP gene. Endogenous KBP expression is repressed by T(3) and two functional nTREs, both of which are required, have been characterized in the KBP gene. COUP-TF1 may be an important factor to modulate expression of genes that are repressed by T(3).

Increased depressive behaviour in mice harboring the mutant thyroid hormone receptor alpha 1

Clinical evidence indicates that hypothyroidism contributes to mood disorders. The present study tested if the mutant thyroid hormone receptor alpha 1 (TRα1) that causes a receptor-mediated hypothyroidism in the brain affects depressive and anxious behaviour in mice. Mice heterozygous for the TRα1 allele (TRα1+/m), yielding a receptor protein with a 10-fold reduced affinity to triiodothyronine (T3), and wildtype (wt) mice were subjected to several paradigms specifically testing depressive and anxious behaviour. Mutant and wt mice were either treated with T3 or vehicle. Untreated TRα1+/m animals displayed reduced locomotion, higher rates of helplessness in the shuttle box-, greater levels of anxiety in the startle response- and dark light box behavioural paradigms when compared to wt mice. Continuous T3-substitution therapy was effective in alleviating anxious and depressive behaviour without affecting locomotion in mutant mice. Notably, continuous T3-substitution reduced overall locomotion and increased helpless behaviour in wt mice when compared to untreated wt mice. The data suggest that receptor-mediated hypothyroidism caused by an unliganded thyroid hormone receptor alpha 1 leads to a depressive and anxious phenotype in mice, which is responsive to continuous T3-substitution and that an iatrogeneously induced hyperthyreoidism by continuous T3-administration leads to a hypolocomotive and depressive phenotype.

Regulation of herpes simplex virus type 1 thymidine kinase gene expression by thyroid hormone receptor in cultured neuronal cells

Herpes simplex virus type 1 (HSV-1) undergoes acute infection in epithelial cells followed by establishment of latency in the neurons of trigeminal ganglia. The latent virus maintains a dormant state and can recurs spontaneously, suggesting transcriptional silencing and reactivation occur in neurons. Computer data mining identified a nuclear hormone response element (NRE), the binding site for the thyroid hormone receptor (TR) or other nuclear hormone receptor, in the promoter of HSV-1 thymidine kinase (TK). TRs are transcription factors whose activity is dependent on their ligand thyroid hormone (T(3); triiodothyronine). We hypothesize that TR and T(3) exert regulation on HSV-1 gene expression in neurons. A neuroblastoma cell line expressing the TR isoform beta (N2aTRbeta) was utilized for in vitro investigation. Results showed that liganded TR repressed TK promoter activity but unliganded TR relieved the inhibition. The mutagenesis study demonstrated that one nucleotide mutation at the NRE abolished the T(3)/TR-mediated regulation. N2aTRbeta cells treated with T(3) were suppressive to TK expression and virus release but the removal of T(3) de-repressed TK expression and increased virus release, confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and plaque assays, respectively. Chromatin immunoprecipitation (ChIP) assays showed that TRs were enriched at TK NRE in the presence of T(3). Additional results demonstrated that hyper acetylated histone H4 and monomethylated H3 modified at lysine 9 (H3K9me1) were enriched at transcriptionally active TK promoters but were dissociated from the NRE by T(3)/TR. These results suggest that T(3) could regulate HSV-1 gene expression through its receptor via histone modification in cultured neuronal cells.

Dual Functions of Thyroid Hormone Receptors in Vertebrate Development: The Roles of Histone-Modifying Cofactor Complexes

Thyroid hormone (TH) receptor (TR) plays critical roles in vertebrate development. Transcription studies have shown that TR activates or represses TH-inducible genes by recruiting coactivators or corepressors in the presence or absence of TH, respectively. However, the developmental roles of these TR cofactors remain largely unexplored. Frog metamorphosis is totally dependent on TH and mimics the postembryonic period in mammalian development during which TH levels are also high. We have previously proposed a dual function model for TR in the development of the anuran Xenopus laevis. That is, unliganded TR recruits corepressors to TH-inducible genes in premetamorphic tadpoles to repress these genes and prevent premature metamorphic changes and subsequently, when TH becomes available, liganded TR recruits coactivators to activate these same genes, leading to metamorphosis. Over the years, we and others have used molecular and genetic approaches to demonstrate the importance of the dual functions of TR in Xenopus laevis. In particular, unliganded TR has been shown to recruit histone deacetylase-containing corepressor complexes in premetamorphic tadpoles to control metamorphic timing. In contrast, metamorphosis requires TH-bound TR to recruit coactivator complexes containing histone acetyltransferases and methyltransferases to activate transcription. Furthermore, the concentrations of coactivators appear to regulate the rate of metamorphic progression. Studies in mammals also suggest that the dual function model for TR is conserved across vertebrates.

The adenoviral E1A protein displaces corepressors and relieves gene repression by unliganded thyroid hormone receptors in vivo

The human adenovirus type 5 early region 1A (E1A) is one of two oncogenes present in the adenovirus genome and functions by interfering with the activities of cellular regulatory proteins. The E1A gene is alternatively spliced to yield five products. Earlier studies have revealed that E1A can regulate the function of thyroid hormone (T3) receptors (TRs). However, analysis in yeast compared with transfection studies in mammalian cell cultures yields surprisingly different effects. Here, we have examined the effect of E1A on TR function by using the frog oocyte in vivo system, where the effects of E1A can be studied in the context of chromatin. We demonstrate that different isoforms of E1A have distinct effects on TR function. The two longest forms inhibit both the repression by unliganded TR and activation by T3-bound TR. We further show that E1A binds to unliganded TR to displace the endogenous corepressor nuclear receptor corepressor, thus relieving the repression by unliganded TR. On the other hand, in the presence of T3, E1A inhibits gene activation by T3-bound TR indirectly, through a mechanism that requires its binding domain for the general coactivator p300. Taken together, our results thus indicate that E1A affects TR function through distinct mechanisms that are dependent upon the presence or absence of T3.

Distinct Dysregulation of Lipid Metabolism by Unliganded Thyroid Hormone Receptor Isoforms

Thyroid hormone receptors (TRs) play critical roles in energy homeostasis. To understand the role of TRs in lipid homeostasis in vivo, we adopted the loss-of-function approach by creating knock-in mutant mice with targeted mutation in the TRalpha gene (TRalpha1PV mouse) or TRbeta gene (TRbetaPV mouse). The PV mutation, identified in a patient with resistance to thyroid hormone, exhibits potent dominant-negative activity. Here we show that in contrast to TRalpha1PV mouse, TRbetaPV mice exhibited no significant reduction in WAT but had significant increases in serum free fatty acids and total triglycerides. Moreover, the liver of TRbetaPV mice was markedly increased (33%) with excess lipid accumulation, but the liver mass of TRalpha1PV mouse was decreased (23%) with paucity of lipids. These results indicate that apo-TRbeta and apo-TRalpha1 exerted distinct abnormalities in lipid metabolism. Further biochemical analyses indicate that increased lipogenic enzyme expression, activated peroxisome proliferator-activated receptor gamma (Ppargamma) signaling, and decreased fatty acid beta-oxidation activity contributed to the adipogenic steatosis and lipid accumulation in the liver of TRbetaPV mice. In contrast, the expression of lipogenic enzymes and Ppargamma was decreased in the liver of TRalpha1PV mice. These results suggest that the regulation of genes critical for lipid metabolism by TRs in the liver is isoform dependent. These results indicate that apo-TRbeta and apo-TRalpha1 had different effects on lipid metabolism and that both TR isoforms contribute to the pathogenesis of lipid metabolism in hypothyroidism.