T3 Receptor Alpha Research Articles

Thyroid Hormone Regulation of Gene Expression in Primary Cerebrocortical Cells: Role of Thyroid Hormone Receptor Subtypes and Interactions with Retinoic Acid and Glucocorticoids

The effects of thyroid hormone on brain development and function are largely mediated by the binding of 3,5,3′-triiodo-L-thyronine (T3) to its nuclear receptors (TR) to regulate positively or negatively gene expression. We have analyzed by quantitative polymerase chain reaction the effect of T3 on primary cultured cells from the embryonic mouse cerebral cortex, on the expression of Hr, Klf9, Shh, Dio3, Aldh1a1, and Aldh1a3. In particular we focused on T3 receptor specificity, and on the crosstalk between T3, retinoic acid and dexamethasone. To check for receptor subtype specificity we used cerebrocortical cells derived from wild type mice and from mice deficient in thyroid hormone receptor subtypes. Receptor subtype specificity was found for Dio3 and Aldh1a1, which were induced by T3 only in cells expressing the T3 receptor alpha 1 subtype. Interactions of T3 with retinoic acid signaling through the control of retinoic acid metabolism are likely to be important during development. T3 had opposing influences on retinoic acid synthesizing enzymes, increasing the expression of Aldh1a1, and decreasing Aldh1a3, while increasing the retinoic acid degrading enzyme Cyp26b1. Dexamethasone increased Klf9 and Aldh1a1 expression. The effects of T3 and dexamethasone on Aldh1a1 were highly synergistic, with mRNA increments of up to 20 fold. The results provide new data on thyroid hormone regulation of gene expression and underscore the importance of thyroid hormone interactions with retinoic acid and glucocorticoids during neural development.

Low-temperature arrest of the triiodothyronine-dependent transcription in Rana catesbeiana red blood cells.

We examined possible molecular mechanisms for the low-temperature arrest of T3-induced Rana catesbeiana metamorphosis. Scatchard plots revealed that the ratios of maximum binding capacity/dissociation constant for high-affinity sites of tadpole serum proteins for T3 at 20 and 28 C was 3.3-4.6 times less than that at 4 C, due to the decrease in maximum binding capacity values. Kinetic studies of T3 uptake into tadpole red blood cells demonstrated that the ratio of maximum uptake rate/Michaelis constant at 23 C was approximately 13 times greater than that at 4 C. The process of intracellular transport of T3 into the nucleus was not arrested at 4 C. The ratio of T3 incorporated into nuclei to that taken up into red blood cells was not significantly different at 4, 20, and 28 C, indicating the absence of temperature-sensitive sites in this process. T3 binding to the T3 receptors alpha and beta were not temperature sensitive at least at 4 and 20 C. Transcription of the tr genes, early primary T3 response genes, was activated by 10 nM T3 at 20 and 28 C but was barely detected at 4 C. These results indicate that the major molecular event causing the low-temperature arrest of amphibian metamorphosis occurs after T3 entry into the nucleus but before or during the transcriptional activation of the tr genes. Plasma proteins binding T3 and the cellular thyroid hormone uptake system on the plasma membrane may contribute to the slowing of the incorporation of T3 into nucleus at 4 C by decreasing the uptake velocity of T3.

Expression of Thyroid Hormone Receptor Isoforms in the Oligodendrocyte Lineage

Thyroid hormone (T3) regulates brain development and function and in particular ensures normal myelination. Animal models and in vitro systems have been employed to demonstrate the effects of T3, which acts via nuclear hormone receptors. T3 receptors (TRs) are transcription factors that activate or suppress target gene expression, such as myelin basic protein (MBP), in a hormone-dependent or -independent fashion. Two distinct genes, TR alpha and TR beta, encode several receptor isoforms with specific functions. This overview summarizes current knowledge on the cellular expression and the role of these isoforms and also examines the action of T3 on oligodendrocyte lineage cell types at defined developmental stages. Re-expression of TRs and also that of other transcription factors in oligodendrocytes may constitute some of the metabolic changes required for succesfull remyelination in the adult central nervous system after demyelinating lesions.

Differentially expressed genes in hypertensive rats developing cerebral ischemia

The molecular events occuring after cerebral ischemia in hypertension may include de novo expression of numerous genes. Receptor genes are predominantely involved in the process of cell death, neuroprotection and reconstruction after ischemic injury. Ischemic stroke was observed in the non-genetic, non-surgical model of hypertension, the cold-induced hypertensive rat. In hypertensive rats suppression subtractive hybridization analysis was used to identify differentially expressed receptor genes in stroke-tissue compared to normal rat brain. We found 76 genes predominantely expressed in hypertensive rat stroke-tissue. These predominantely expressed genes included genes involved in energy metabolism, signal transduction/cell regulation, and replication/transcription/translation. For example, the T3 receptor alpha was predominantely expressed in stroke-tissue, indicating that regeneration of nerves in stroke tissue may be facilitated by increased T3 receptor alpha expression.

The Homeodomain Proteins PBX and MEIS1 Are Accessory Factors That Enhance Thyroid Hormone Regulation of the Malic Enzyme Gene in Hepatocytes

Triiodothyronine (T3) stimulates a robust increase (>40-fold) in transcription of the malic enzyme gene in chick embryo hepatocytes. Previous work has shown that optimal T3 regulation of malic enzyme transcription is dependent on the presence of an accessory element (designated as region E) that immediately flanks a cluster of five T3 response elements in the malic enzyme gene. Here, we have analyzed the binding of nuclear proteins to region E and investigated the mechanism by which region E enhances T3 responsiveness. In nuclear extracts from hepatocytes, region E binds heterodimeric complexes consisting of the homeodomain proteins PBX and MEIS1. Region E contains four consecutive PBX/MEIS1 half-sites. PBX-MEIS1 heterodimers bind the first and second half-sites, the third and fourth half-sites, and the first and fourth half-sites. The configuration conferring the greatest increase in T3 responsiveness consists of the first and fourth half-sites that are separated by 7 nucleotides. Stimulation of T3 response element functions by region E does not require the presence of additional malic enzyme sequences. In pull-down experiments, PBX1a and PBX1b specifically bind the nuclear T3 receptor-alpha, and this interaction is enhanced by the presence of T3. A T3 receptor-alpha region containing the DNA binding domain plus flanking sequences (amino acids 21-157) is necessary and sufficient for binding to PBX1a and PBX1b. These results indicate that PBX-MEIS1 complexes interact with nuclear T3 receptors to enhance T3 regulation of malic enzyme transcription in hepatocytes.

Localization of a Negative Thyroid Hormone-Response Region in Hepatic Stearoyl-CoA Desaturase Gene 1

The effect of thyroid hormone on stearoyl-CoA desaturase gene 1 (SCD1) expression was investigated in mouse liver. Daily injections of 15 μg triiodothyronine (T3)/100 g body weight to hypothyroid mice resulted in repression of SCD1 mRNA levels by more than 50% in 48 hours and up to 65% in 6 days. Transient cotransfections were performed with an expression vector for T3receptor alpha (T3Rα) in HepG2 cells using chimeric reporter gene constructs of the SCD1 5′-flanking region. Transcriptional repression of the SCD1 putative promoter was observed upon treatment with 100 nM T3when cotransfected with T3Rα, but not without cotransfection of receptor. Transient gene expression studies localized a T3response region to a 70-bp sequence in the SCD1 putative promoter. Eliminating the TATA box and an AP-2 binding site, DNA mobility shift analysis demonstrated specific binding ofin vivonuclear protein from mouse liver nuclear extract to a 43-bp sequence. DNA mobility shift with purified T3Rα confirmed the presence of a T3receptor binding site in this thyroid hormone-responsive region. These data indicate that SCD1 contains a negative T3response region in its proximal promoter.

Hormone-dependent and -independent transcriptional activation by thyroid hormone receptors are mediated by different mechanisms.

Transcriptional activation by thyroid hormone (T3) receptor (T3R) generally requires the binding of its high affinity ligand. However, we reported previously that chicken T3R alpha (cTaR alpha) and human T3R beta 1 (hT3R beta 1) could activate transcription from several promoters containing T3R response elements (TREs) in a hormone-independent fashion when expressed in rat anterior pituitary GH4C1 cells. In this study we show that rat T3R alpha 1 also activates transcription without T3 in GH4C1 cells and that the oncoprotein v-erbA that is derived from cT3R alpha but does bind T3 is not a constitutive activator in these cells. Increased expression of T3R results in transcriptional activation of both native and minimal promoters, and this activation does not appear to require a defined TRE in the promoter. Because hormone-independent activity is not observed in several other cell lines, this activity may involve specific factors present in GH4C1 cells. Three mutants with single amino acid changes in a 20-amino acid region of the ligand-binding domain of cT3R alpha do not mediate hormone-independent activity. This region is highly conserved within the nuclear receptor family and has been implicated in interactions with other proteins, suggesting participation of other transcription or accessory factors in the hormone-independent activity of T3R. Two of these mutants mediate hormone-dependent transcriptional activation similar to wild-type cT3R alpha. All three mutants interact in vitro with retinoid X receptor beta similar to wild-type cT3R alpha. Our findings suggest that hormone-dependent and hormone-independent transactivation proceed by distinct mechanisms.

A shift in the ligand responsiveness of thyroid hormone receptor alpha induced by heterodimerization with retinoid X receptor alpha.

Thyroid hormone (T3) receptors (T3Rs) are ligand-modulated transcription factors that bind to thyroid hormone response elements (T3REs) and mediate either positive or negative transcriptional regulation of target genes. In addition, in response to ligand binding, T3Rs can interfere with AP-1 activity and thereby inhibit transcription of AP-1-responsive genes. T3Rs were recently shown to form heterodimers with retinoid X receptors (RXRs), leading to increased binding to T3REs in vitro and potentiation of transcriptional responses in vivo. Here we demonstrate that T3R alpha forms stable heterodimers with RXR alpha in living cells. Most important, we describe a new role for RXR alpha in modulating ligand-dependent T3R alpha activity: heterodimerization with RXR alpha greatly increases transcriptional interference with AP-1 activity, augments T3-dependent transcriptional activation, and potentiates the reversal of ligand-independent activation by T3R alpha. In all three cases, the responses occur at substantially lower T3 concentrations when elicited by T3R alpha plus RXR alpha than by T3R alpha alone. In vitro, the binding of T3 decreases the DNA-binding activity of T3R alpha homodimers but does not affect DNA binding by T3R alpha:RXR alpha heterodimers. We provide evidence that increased activities of T3R alpha at lower T3 concentrations are not due to changes in its T3 binding properties. Instead, the altered response could be mediated by either RXR alpha-induced conformational changes, increased stability of heterodimers over homodimers, especially following T3 binding, or both.

Effect of perinatal hypothyroidism on the developmental regulation of rat pituitary growth hormone and thyrotropin genes

We studied the effects of thyroid hormone (T3) on GH, TSH, and T3 receptor (TR) gene expression as well as deiodinase activities during rat pituitary development. By reverse transcriptase-polymerase chain reaction, GH and TSH beta transcripts were detectable on fetal day 15. Although with certain differences, the expression of both GH and TSH beta genes was under T3 control during fetal and neonatal life. Differences in plasma, but not pituitary, TSH concentrations were observed between control and hypothyroid animals throughout the period studied. Both TR alpha and TR beta genes were expressed in the fetal pituitary. TR alpha 1, TR beta 2, and c-erbA alpha 2 transcripts displayed a developmental profile different from that of TR beta 1. Thyroid hormone repressed TR alpha 1, TR beta 2, and c-erbA alpha 2 and stimulated TR beta 1. Type I and type II deiodinase activities (5'DI and 5'DII, respectively) had different ontogenic patterns; 5'D-II was the predominant activity in fetuses, with levels similar to those in adults, whereas the level of 5'D-I was low and increased with age. T3 stimulated 5'D-I and decreases 5'D-II. These results demonstrate that in somatotroph and thyrotroph cells, the mechanisms responsible for T3 action are mature and active very early in development and suggest an involvement of this hormone in the establishment and/or maintenance of the somatotroph and thyrotroph phenotype.

Effect of perinatal hypothyroidism on the developmental regulation of rat pituitary growth hormone and thyrotropin genes.

We studied the effects of thyroid hormone (T3) on GH, TSH, and T3 receptor (TR) gene expression as well as deiodinase activities during rat pituitary development. By reverse transcriptase-polymerase chain reaction, GH and TSH beta transcripts were detectable on fetal day 15. Although with certain differences, the expression of both GH and TSH beta genes was under T3 control during fetal and neonatal life. Differences in plasma, but not pituitary, TSH concentrations were observed between control and hypothyroid animals throughout the period studied. Both TR alpha and TR beta genes were expressed in the fetal pituitary. TR alpha 1, TR beta 2, and c-erbA alpha 2 transcripts displayed a developmental profile different from that of TR beta 1. Thyroid hormone repressed TR alpha 1, TR beta 2, and c-erbA alpha 2 and stimulated TR beta 1. Type I and type II deiodinase activities (5'DI and 5'DII, respectively) had different ontogenic patterns; 5'D-II was the predominant activity in fetuses, with levels similar to those in adults, whereas the level of 5'D-I was low and increased with age. T3 stimulated 5'D-I and decreases 5'D-II. These results demonstrate that in somatotroph and thyrotroph cells, the mechanisms responsible for T3 action are mature and active very early in development and suggest an involvement of this hormone in the establishment and/or maintenance of the somatotroph and thyrotroph phenotype.

A novel retinoid X receptor-independent thyroid hormone response element is present in the human type 1 deiodinase gene.

We identified two thyroid hormone response elements (TREs) in the 2.5-kb, 5'-flanking region of the human gene encoding type 1 iodothyronine deiodinase (hdio1), an enzyme which catalyses the activation of thyroxine to 3,5,3'-triiodothyronine (T3). Both TREs contribute equally to T3 induction of the homologous promoter in transient expression assays. The proximal TRE (TRE1), which is located at bp -100, has an unusual structure, a direct repeat of the octamer YYRGGTCA hexamer that is spaced by 10 bp. The pyrimidines in the -2 position relative to the core hexamer are both essential to function. In vitro binding studies of TRE1 showed no heterodimer formation with retinoid X receptor (RXR) beta or JEG nuclear extracts (containing RXR alpha) and bacterially expressed chicken T3 receptor alpha 1 (TR alpha) can occupy both half-sites although the 3' half-site is dominant. T3 causes dissociation of TR alpha from the 5' half-site but increases binding to the 3' half-site. Binding of a second TR to TRE1 is minimally cooperative; however, no cooperativity was noted for a functional mutant in which the half-sites are separated by 15 bp, implying that TRs bind as independent monomers. Nonetheless, T3 still causes TR dissociation from the DR+15, indicating that dissociation occurs independently of TR-TR contact and that rebinding of a T3-TR complex to the 3' half-site occurs because of its slightly higher affinity. A distal TRE (TRE2) is found at bp -700 and is a direct repeat of a PuGGTCA hexamer spaced by 4 bp. It has typical TR homodimer and TR-RXR heterodimer binding properties. The TRE1 of hdio1 is the first example of a naturally occurring TRE consisting of two relatively independent octamer sequences which do not require the RXR family of proteins for function.

Interactions of thyroid hormone receptor with the human immunodeficiency virus type 1 (HIV-1) long terminal repeat and the HIV-1 Tat transactivator

Thyroid hormone (T3) receptor (T3R) regulates the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by binding to and activating thyroid hormone response elements (TREs) embedded within the viral NF-kappa B and Sp1 motifs. The TREs within the NF-kappa B sites are necessary for activation by T3 in the absence of Tat, while those in the Sp1 motifs function as TREs only when Tat is expressed, suggesting that Tat and T3R interact in the cell. Transactivation of the HIV-1 LTR by T3R alpha and several receptor mutants revealed that the 50-amino-acid N-terminal A/B region of T3R alpha, known to interact with the basal transcription factor TFIIB, is critical for activation of both Tat-dependent and Tat-independent responsive sequences of the LTR. A single amino acid change in the highly conserved tau 1 region in the ligand-binding domain of T3R alpha eliminates Tat-independent but not Tat-dependent activation of the HIV-1 LTR by T3. Ro 5-3335 [7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepin-2(H)-one], which inhibits Tat-mediated transactivation of HIV-1, also inhibits the functional interaction between Tat and T3R alpha. Binding studies with glutathione-S-transferase fusion proteins and Western (immunoblot) analysis indicate that T3R alpha interacts with Tat through amino acids within the DNA-binding domain of T3R alpha. Mutational analysis revealed that amino acid residues in the basic and C-terminal regions of Tat are required for the binding of Tat to T3R alpha, while the N terminus of Tat is not required. These studies provide functional and physical evidence that stimulation of the HIV-1 LTR by T3 involves an interaction between T3R alpha and Tat. Our results also suggest a model in which multiple domains of T3R alpha interact with Tat and other factors to form transcriptionally important complexes.

A 10-Amino-Acid Sequence in the N-Terminal A/B Domain of Thyroid Hormone Receptor α Is Essential for Transcriptional Activation and Interaction with the General Transcription Factor TFIIB

The effects of the thyroid hormone (3,5,3'-triiodo-L-thyronine [T3]) on gene transcription are mediated by nuclear T3 receptors (T3Rs). alpha- and beta-isoform T3Rs (T3R alpha and -beta) are expressed from different genes and are members of a superfamily of ligand-dependent transcription factors that also includes the receptors for steroid hormones, vitamin D, and retinoids. Although T3 activates transcription by mediating a conformational change in the C-terminal approximately 220-amino-acid ligand-binding domain (LBD), the fundamental mechanisms of T3R-mediated transcriptional activation remain to be determined. We found that deletion of the 50-amino-acid N-terminal A/B domain of chicken T3R alpha (cT3R alpha) decreases T3-dependent stimulation of genes regulated by native thyroid hormone response elements about 10- to 20-fold. The requirement of the A/B region for transcriptional activation was mapped to amino acids 21 to 30, which contain a cluster of five basic amino acids. The A/B region of cT3R alpha is not required for T3 binding or for DNA binding of the receptor as a heterodimer with retinoid X receptor. In vitro binding studies indicate that the N-terminal region of cT3R alpha interacts efficiently with TFIIB and that this interaction requires amino acids 21 to 30 of the A/B region. In contrast, the LBD interacts poorly with TFIIB. The region of TFIIB primarily involved in the binding of cT3R alpha includes an amphipathic alpha helix contained within residues 178 to 201. Analysis using a fusion protein containing the DNA-binding domain of GAL4 and the entire A/B region of cT3R alpha suggests that this region does not contain an intrinsic activation domain. These and other studies indicate that cT3R alpha mediates at least some of its effects through TFIIB in vivo and that the N-terminal region of DNA-bound cT3R alpha acts to recruit and/or stabilize the binding of TFIIB to the transcription complex. T3 stimulation could then result from ligand-mediated changes in the LBD which may lead to the interaction of other factors with cT3R alpha, TFIIB, and/or other components involved in the initiation of transcription.

Functional regulation of thyroid hormone receptor variant TR alpha 2 by phosphorylation.

The thyroid hormone (T3) receptor (TR) variant TR alpha 2 is abundant in brain but does not bind T3 because of its unique C terminus. The only known function of TR alpha 2, inhibition of TR-dependent transactivation, involves competition for T3 response elements. Paradoxically, in vitro-translated TR alpha 2 bound poorly to these sites. We report here that dephosphorylation of TR alpha 2 restored its DNA binding. Mutation of C-terminal serine residues to alanine (TR alpha 2-SA) was equally effective. The C terminus of TR alpha 2 was phosphorylated in a human cell line, whereas that of TR alpha 2-SA was not. Conversely, TR alpha 2-SA was a much better inhibitor of T3 action than was wild-type TR alpha 2. The dominant negative activity of TR alpha 2-SA was less than stoichiometric with TR concentration, possibly because it was unable to heterodimerize with retinoid X receptor, which enhances the binding of other TRs. Purified casein kinase II as well as a reticulocyte casein kinase II-like activity phosphorylated TR alpha 2 on serines 474 and 475. Mutation of these two residues to alanine was sufficient to restore DNA binding. Thus, DNA binding by TR alpha 2 is regulated by phosphorylation at a site distant from the DNA-binding domain. The increased dominant negative activity of a nonphosphorylatable form of TR alpha 2 suggests that phosphorylation may provide a rapid, T3-independent mechanism for cell-specific modulation of the expression of T3-responsive genes.

Nuclear and Cytoplasmic Triiodothyronine‐Binding Sites in Primary Sensory Neurons and Schwann Cells: Radioautographic Study During Development

The effects of the thyroid hormones on target cells are mediated through nuclear T3 receptors. In the peripheral nervous system, nuclear T3 receptors were previously detected with the monoclonal antibody 2B3 mAb in all the primary sensory neurons throughout neuronal life and in peripheral glia at the perinatal period only (Eur. J. Neurosci. 5, 319, 1993). To determine whether these nuclear T3 receptors correspond to functional ones able to bind T3, cryostat sections and in vitro cell cultures of dorsal root ganglion (DRG) or sciatic nerve were incubated with 0.1 nM [125I]-labeled T3, either alone to visualize the total T3-binding sites or added with a 10(3) fold excess of unlabeled T3 to estimate the part due to the non-specific T3-binding. After glutaraldehyde fixation, radioautography showed that the specific T3-binding sites were largely prevalent. The T3-binding capacity of peripheral glia in DRG and sciatic nerve was restricted to the perinatal period in vivo and to Schwann cells cultured in vitro. In all the primary sensory neurons, specific T3-binding sites were disclosed in foetal as well as adult rats. The detection of the T3-binding sites in the nucleus indicated that the nuclear T3 receptors are functional. Moreover the concomitant presence of both T3-binding sites and T3 receptors alpha isoforms in the perikaryon of DRG neurons infers that: 1) [125I]-labeled T3 can be retained on the T3-binding 'E' domain of nascent alpha 1 isoform molecules newly-synthesized on the perikaryal ribosomes; 2) the alpha isoforms translocated to the nucleus are modified by posttranslational changes and finally recognized by 2B3 mAb as nuclear T3 receptor. In conclusion, the radioautographic visualization of the T3-binding sites in peripheral neurons and glia confirms that the nuclear T3 receptors are functional and contributes to clarify the discordant intracellular localization provided by the immunocytochemical detection of nuclear T3 receptors and T3 receptor alpha isoforms.

The Molecular Basis of Thyroid Hormone Action

Progress has been made in understanding the molecular basis of a number of clinical manifestations of thyroid disease, yet many questions remain. Why are there two thyroid hormone-receptor genes? Is the function of each of the two receptors indeed unique? How T3 receptors interact with other nuclear proteins and DNA-binding sites and how these interactions are influenced by T3 is incompletely understood. The developmental regulatory role of T3 receptor alpha 1 and its non-T3-binding alpha 2 variant needs to be defined. Most T3-regulated processes, especially those related to metabolism, muscle contraction, and brain development, function in concert with a number of other regulatory factors. The therapeutic applications of knowledge gained about the basic mechanisms of thyroid hormone action should ultimately extend beyond thyroid disease to processes regulated or influenced by T3; these include cardiac function, lipid metabolism, pituitary hormone secretion, and neural development.

Thyroid hormone receptor modulates the expression of the rabbit cardiac sarco (endo) plasmic reticulum Ca(2+)-ATPase gene.

We have analyzed the effect of thyroid hormone (T3) on SERCA2 gene expression using fetal chicken primary cardiac myocytes and C2C12 skeletal muscle cells in culture. Northern blot analysis of both cell types demonstrated that T3 induced a 3-fold accumulation of SERCA2 mRNA compared with cells grown in medium lacking T3. We have engineered deletion constructs containing various lengths of the 5'-flanking region from the rabbit SERCA2 gene which we have cloned previously (Zarain-Herzberg, A., MacLennan, D. H., and Periasamy, M. (1990) J. Biol. Chem. 265, 4670-4677). A stable transfectant in C2C12 containing a chimeric SERCA2/CAT gene construct including -254 base pairs (bp) of SERCA2 5'-flanking region showed increased transcription activity upon the addition of 50 nM T3. We have analyzed the expression of several deletion constructs spanning 1,102 bp of the 5'-up-stream sequence of the SERCA2 gene by functional expression assays. Transient coexpression of the T3 receptor alpha 1 with various SERCA2/CAT deletion constructs showed trans-activation of chimeric constructs containing more than -267 bp, indicating that a thyroid hormone-responsive element was localized, at least in part, to the region -267 to -72 bp. T3 receptor-DNA binding assays demonstrated binding of the rat T3 receptor alpha 1 to a fragment containing a proposed T3 response element located between position -254 and -72 in the 5'-flanking region of the SERCA2 gene.

Dominant negative activity of an endogenous thyroid hormone receptor variant (alpha 2) is due to competition for binding sites on target genes.

Regulation of development and metabolism by thyroid hormone (T3) may be influenced by a non-T3 binding T3 receptor (TR) isoform, TR alpha 2, which can inhibit transcriptional activation by legitimate TRs. Numerous mechanisms have been postulated to explain the dominant negative actions of TR alpha 2, including competition for target genes, formation of inactive heterodimers, and squelching. We have found that excess TR alpha 2 was required to inhibit TR alpha 1-mediated transactivation from multiple T3 response elements (TREs). Inhibition of T3 action by TR alpha 2 was specific for TRE-containing genes, because a GAL4/TR alpha 1 chimera, which heterodimerized with the 9-cis-retinoic acid receptor (RXR) and activated transcription from the GAL4 binding site in the presence of T3, was not inhibited by TR alpha 2. In contrast, TR alpha 2 inhibited transactivation by TR alpha/VP16, a chimeric protein containing the N-terminal DNA binding domain (DBD) of TR alpha 1 fused to the transcriptional activation domain of VP16. Indeed, TR alpha 2 inhibited the binding of TR alpha 1 monomers, homodimers, and RXR-heterodimers to DNA in vitro, whereas the TR alpha 2 C terminus alone did not. Although TR alpha 2 bound to TREs with less affinity than TR alpha 1, it bound directly to target genes in the cell nucleus. Furthermore, a TR alpha 2 mutant which binds more avidly to TREs was a more effective inhibitor of T3 action than wild type TR alpha 2. Together these data indicate that TR alpha 2 inhibits T3 action by competing for binding to TREs.

The herpes simplex virus thymidine kinase gene promoter contains a novel thyroid hormone response element.

Thyroid hormone (T3) receptors (T3Rs) regulate transcription by binding to T3 response elements (TREs) located within promoter regions of T3-regulated genes. In rat pituitary GH4C1 cells, expression of a reporter containing herpes simplex virus thymidine kinase (TK) gene sequences (-105/+51) linked to the chloramphenicol acetyltransferase gene was stimulated 4- to 5-fold by T3. Linker scanning mutants of the TK promoter revealed that regions around -80 containing a CTF/NF-1 recognition sequence and around -10 are both required for regulation by T3. Endogenous T3Rs from GH4C1 cells labeled with [125I]T3 bound only to TK promoter DNA fragments containing the -10 region. The -22/-2 sequence (TK-TRE) contains half-sites oriented as an inverted repeat separated by 6 basepairs that are identical to and similar to an optimized TRE half-site. Purified chicken T3R alpha 1 forms apparent monomeric and dimeric complexes on the 32P-labeled TK-TRE, as found previously with an inverted repeat of the optimized TRE (TREp) with no basepair gap. T3 enhances the formation and alters the mobility of these complexes on both elements. When positioned up-stream of a heterologous promoter-chloramphenicol acetyltransferase reporter, the TK-TRE conferred T3 regulation by endogenous T3R in GH4C1 cells and by cotransfected chicken T3R alpha 1 in HeLa cells. The TK-TRE does not bind and is not activated by retinoic acid receptor. T3Rs and nuclear proteins from GH4C1, HeLa, and COS1 cells form heterodimers on the TK-TRE which differ in abundance and mobility from heterodimers formed on the TREp. The identification of a TRE in the TK promoter raises the possibility that T3R or related proteins may play important roles in regulating the life cycle of herpes simplex virus.

Oligomeric binding of T3 receptor is required for maximal T3 response.

Receptors in the thyroid-steroid hormone superfamily bind preferentially as dimers to palindromic response elements containing two hexameric half-sites. The 23-base pair rat growth hormone (rGH) T3 response element (T3RE), however, contains three hexameric binding domains, all of which are required for maximal T3 response. We examined the binding of purified T3 receptor alpha (T3R alpha), overexpressed in Escherichia coli, to wild-type and up and down mutations of the rGH T3RE to evaluate whether transcriptional potency correlates with changes in T3R binding. T3R binds to the rGH T3RE as a monomer, dimer, or higher order oligomer. Cooperative T3R dimer binding was demonstrated to two hexameric domains of the rGH T3RE arranged as either direct or inverted repeats. Decreased binding was seen with point mutations in each domain as well as with mutations which altered hexamer orientation and spacing within the site. These results demonstrate that all three hexamers of the rGH T3RE are involved in binding T3R. Occupancy of all three hexamers by T3R in the gel shift assay was observed with functional up mutations of the T3RE, increasing receptor concentration or addition of nuclear extract. The transcriptional response potencies of T3RE up or down mutants in a transient transfection assay correlated closely with T3R binding. These results confirm our earlier hypothesis that all three hexamers of the rGH T3RE bind T3R in a novel binding arrangement and provide a model for the interaction of T3R and other nuclear proteins with the DNA sequences of thyroid hormone-regulated genes.