I. INTRODUCTION

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Thyroid hormones (THs) play critical roles in differentiation, growth, and metabolism. Indeed, TH is required for the normal function of nearly all tissues, with major effects on oxygen consumption and metabolic rate (375). Disorders of the thyroid gland are among the most common endocrine maladies. Furthermore, endemic cretinism due to iodine deficiency remains a public health problem in developing countries at the advent of the third millennium. Thus the study of TH action has important biological and medical implications.

The story of TH action is interwoven with many of the major advances in biomedical science during the past century. Contributions from clinical medicine, physiology, biochemistry, and molecular genetics have had major impacts on our understanding of TH action (376, 551). The following outline sketches only some of the many early contributions to our knowledge.

In 1888, the Clinical Society of London published the definitive report that first linked cretinism and adult hypothyroidism to the destruction of the thyroid gland (97a). Soon afterward, thyroid extracts from sheep were used for the treatment of hypothyroidism. Around this time, Emil Kocher performed some of his pioneering studies on the pathology and surgery of the thyroid gland for which he was awarded the Nobel prize in medicine in 1909. In 1914, Kendall (241) isolated 3,5,3',5'-tetraiodo-L-thyronine (T₄) from thyroid extracts, and almost 40 years later, Gross and Pitt-Rivers (178) synthesized 3,5,3'-triiodo-L-thyronine (T₃) and demonstrated its presence in human plasma and its ability to prevent goiter in thiouracil-treated rats. Over the ensuing years, the metabolic and oxygen consumption effects of THs as well as its effects on development, particularly in amphibians, were appreciated (59, 79, 463).

In the 1960s, Tata and co-workers (509, 510) first suggested that THs might be involved in the transcriptional regulation of target genes. These investigators observed that T₃ treatment of hypothyroid rats induced a rapid increase in RNA synthesis in the liver which preceded new protein formation and mitochondrial oxidation (509, 510). The groups of Oppenheimer (372) and Samuels (441) then used radiolabeled TH to demonstrate specific nuclear binding sites in different T₃-sensitive tissues, and thus provided the first evidence for TH receptors (TRs). Moreover, T₃ binding was observed in almost all tissues (377). Attempts to purify these receptors biochemically were only partially successful; however, photoaffinity labeling of nuclear extracts demonstrated different-sized receptors and raised the possibility of multiple TR isoforms (123, 384). Studies on T₃ induction of the rat growth hormone (GH) gene transcription suggested that TRs recognized enhancer sequences or TH response elements (TREs), similar to steroid hormone receptors (101, 281, 282, 440). Thus TRs behaved similar to steroid hormone receptors with respect to nuclear site of action, recognition of specific DNA sequences, and ligand-dependent regulation of transcription. In 1985, the glucocorticoid receptor was cloned and, surprisingly, had homology with a known viral oncogene product, v-erbA, that in conjunction with v-erbB, can cause erythroblastosis in chicks (209). Subsequent cloning of the estrogen receptor suggested that there was a family of nuclear hormone receptors (174). A year later, the laboratories of Evans (545) and Vennstrom (444) ushered in the molecular era of TH action when they cloned two different TR isoforms and showed they were the cellular homologs of v-erbA.

Since the molecular cloning of TRs 15 years ago, there has been an explosion of information on the molecular mechanisms of TR action. The power of molecular genetics has greatly aided our understanding of the roles of unliganded and liganded TRs in regulating target genes. We have learned that there are multiple TR isoforms that bind to TREs with variable orientation, spacing, and sequences for TRE half-sites. TRs also interact with other nuclear proteins such as corepressors or coactivators to form complexes that regulate local histone acetylation and interact with the basal transcriptional machinery. Additionally, the solution of the crystal structures of the TR ligand-binding domain (LBD) and other nuclear hormone receptors have provided insight into some of these complex interactions at the molecular level. The development of transgenic and knockout mouse models have shed light on the roles of TRs in the regulation of specific target genes and development. These findings have greatly aided our understanding of the molecular mechanisms of TH action in normal and disease states. In particular, much has been learned about the pathogenesis of the human genetic disorder of resistance to thyroid hormone (RTH). We review some of the major advances in these areas by initially focusing on what is known about the molecular mechanisms of TH action and then discuss their implications for TH action in specific tissues, RTH, and genetically engineered mouse models.

TH synthesis and secretion is exquisitely regulated by a negative-feedback system that involves the hypothalamus, pituitary, and thyroid gland [hypothalamic/pituitary/thyroid (HPT) axis] (467). Thyrotropin releasing hormone (TRH) is a tripeptide (PyroGlu-His-Pro) synthesized in the paraventricular nucleus of the hypothalamus. It is transported via axons to the median eminence and then to the anterior pituitary via the portal capillary plexus. TRH binds to TRH receptors in pituitary thyrotropes, a subpopulation of pituitary cells that secrete thyroid stimulating hormone (TSH). TRH receptors are members of the seven-transmembrane spanning receptor family and are coupled to G_q11. TRH stimulation leads to release and synthesis of new TSH in thyrotropes. TSH is a 28-kDa glycoprotein composed of - and -subunits designated as glycoprotein hormone - and TSH -subunits. The -subunit also is shared with other hormones such as luteinizing hormone, follicle stimulating hormone, and chorionic gonadotropin. Both TRH and TSH secretion are negatively regulated by TH. An important mechanism for the negative regulation of TSH may be the intrapituitary conversion of circulating T₄ to T₃ by type II deiodinase. Additionally, somatostatin and dopamine from the hypothalamus can negatively regulate TSH secretion.

TSH is the primary regulator of TH release and secretion. It also has a critical role in thyroid growth and development. TSH binds to the TSH receptor (TSHr), which also is a seven-transmembrane spanning receptor coupled to G_s (255, 382). Activation of TSHr by TSH or autoantibodies in Graves' disease leads to an increase in intracellular cAMP and stimulation of protein kinase A-mediated pathways. A number of thyroid genes, including Na⁺/I symporter (NIS), thyroglobulin (Tg), and thyroid peroxidase (TPO), are stimulated by TSH and promote the synthesis of TH. Of note, activating mutations in TSHr and G_s have been described in autonomously functioning thyroid nodules and familial congenital hyperthyroidism (381, 383).

The THs, T₄ and the more potent T₃, are synthesized in the thyroid gland (Fig. 1). Iodide is actively transported and concentrated into the thyroid by NIS (102, 475). The trapped iodide is oxidized by TPO in the presence of hydrogen peroxide and incorporated into the tyrosine residues of a 660-kDa glycoprotein, Tg. This iodination of specific tyrosines located on Tg yields monoiodinated and diiodinated residues (MIT, monoiodo-tyrosines; DIT, diiodo-tyrosines) that are enzymatically coupled to form T₄ and T₃. The iodinated Tg containing MIT, DIT, T₄, and T₃, then is stored as an extracellular storage polypeptide in the colloid within the lumen of thyroid follicular cells. Genetic defects along the synthetic pathway of THs have been described in humans and are major causes of congenital hypothyroidism in iodine-replete environments (117, 261).

View larger version (8K): [in this window] [in a new window]   Fig. 1. Structure of thyroid hormones.

The secretion of THs requires endocytosis of the stored iodinated Tg from the apical surface of the thyroid follicular cell (511). The internalized Tg is incorporated in phagolysosomes and undergoes proteolytic digestion, recapture of MIT and DIT, and release of T₄ and T₃ into the circulation via the basal surface. The majority of released TH is in the form of T₄, as total serum T₄ is 40-fold higher than serum T₃ (90 vs. 2 nM). Only 0.03% of the total serum T₄ is free (unbound), with the remainder bound to carrier proteins such as thyroxine binding globulin (TBG), albumin, and thyroid binding prealbumin. Approximately 0.3% of the total serum T₃ is free, with the remainder bound to TBG and albumin. It is the free TH that enters target cells and generates a biological response.

The major pathway for the production of T₃ is via 5'-deiodination of the outer ring of T₄ by deiodinases and accounts for the majority of the circulating T₃ (50, 256). Type I deioidinase is found in peripheral tissues such as liver and kidney and is responsible for the conversion of the majority of T₄ to T₃ in circulation. Type II deiodinase is found in brain, pituitary, and brown adipose tissue and primarily converts T₄ to T₃ for intracellular use. These deiodinases recently have been cloned and demonstrated to be selenoproteins (280). 5'-Deiodination by type I deiodinase and type III deioidinase, which is found primarily in placenta, brain, and skin, leads to the generation of rT₃, the key step in the inactivation of TH. rT₃ and T₃ can be further deiodinated in the liver and are sulfo- and glucuronide-conjugated before excretion in the bile (124). There also is an enterohepatic circulation of TH as intestinal flora deconjugates some of these compounds and promotes the reuptake of TH.

Although THs may exert their effects on a number of intracellular loci, their primary effect is on the transcriptional regulation of target genes. Early studies showed that the effects of THs at the genomic level are mediated by nuclear TRs, which are intimately associated with chromatin and bind TH with high affinity and specificity (375, 440). Similar to steroid hormones that also bind to nuclear receptors, TH enters the cell and proceeds to the nucleus (Fig. 2). It then binds to TRs, which may already be prebound to TREs located in promoter regions of target genes. The formation of ligand-bound TR complexes that are also bound to TREs is the critical first step in the positive or negative regulation of target genes and the subsequent regulation of protein synthesis. Given their abilities to bind both ligand and DNA as well as their ability to regulate transcription, TRs can be regarded as ligand-regulatable transcription factors.

View larger version (20K): [in this window] [in a new window]   Fig. 2. General model for thyroid hormone action in the nucleus. TR, thyroid hormone receptor; RXR, retinoid X receptors.

	III. MULTIPLE THYROID HORMONE RECEPTOR ISOFORMS

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In 1986, the laboratories of Vennstrom (444) and Evans (545) independently cloned cDNAs encoding two different TRs from embryonal chicken and human placental cDNA libraries. Several unexpected findings stemmed from their landmark work. First, they demonstrated by amino acid sequence comparison that TRs are the cellular homologs of the viral oncogene product v-erbA. Second, TRs were shown to have amino sequence homology with steroid hormone receptors. This was initially surprising since T₃ and cholesterol-derived steroids are structurally different ligands. However, in the ensuing years, TRs have been shown to belong to a large superfamily of nuclear hormone receptors that include the steroid, vitamin D, and retinoic acid receptors as well as "orphan" receptors for which there are no known ligand or function (35, 285). TRs share a similar domain organization with other family members as they have a central DNA-binding domain containing two "zinc fingers" and a carboxy-terminal LBD. These initial studies also suggested that there were multiple TR isoforms. Subsequent work by many groups has confirmed that there are two major TR isoforms encoded on separate genes, designated as TR and TR, encoded on human chromosomes 17 and 3, respectively (284). Moreover, these multiple isoforms exist in different species such as amphibians, chick, mouse, rat, and human (284). Both TR isoforms bind T₃ (reported dissociation constant values between 10⁹ and 10¹⁰ M) and mediate TH-regulated gene expression (148, 340, 449). In mammalian species, TR-1 and TR-1s range from 400 to slightly over 500 amino acids in size (284, 289) and contain highly homologous DNA-binding domains and LBD (Fig. 3).

View larger version (10K): [in this window] [in a new window]   Fig. 3. Comparison of amino acid homologies and their functional properties among TR isoforms. The length of receptors is indicated just above the receptor diagrams, and the percent amino acid homology with TR-2 is included in the receptor diagrams.

In addition to two separate genes that encode TRs, there is additional heterogeneity of TRs due to alternative splicing (220, 292, 330, 349). Alternative splicing of the initial RNA transcript of the TR gene generates two mature mRNAs that each encode two proteins: TR-1 and c-erbA-2. In the rat, these proteins are identical from amino acid residues 1-370, but their respective sequences diverge markedly thereafter (Fig. 3). Consequently, c-erbA-2 cannot bind T₃ because it contains a 122-amino acid carboxy terminus that replaces a region in TR-1 that is critical for TH binding. Additionally, c-erbA-2 binds TREs weakly but cannot transactivate TH-responsive genes. Thus TR-1, but not c-erbA-2, is an authentic TR. Indeed, c-erbA-2 may act as an inhibitor of TH action possibly by competing for binding to TREs (253, 291). The TR-1 and c-erbA-2 system, then, represents one of the first examples in which multiple mRNAs generated by alternative splicing encode proteins that may be antagonistic to each other. Mitsuhashi et al. (330) also have described a second TR variant, c-erbA-2V, in which the first 39 amino acids of the divergent sequence are missing (330). Its function currently is unknown. Yet another interesting feature of the TR gene is the employment of the opposite strand to encode a gene product, rev-erbA. Rev-erbA mRNA contains a 269-nucleotide stretch which is complementary to the c-erbA-2 mRNA due to its transcription from the DNA strand opposite of that used to generate TR-1 and c-erbA-2 (293, 331). This protein also is a member of the nuclear hormone receptor superfamily. It is expressed in adipocytes and muscle cells, and can bind to TREs and retinoic acid response elements (RAREs) and repress gene transcription (190, 477, 597). However, rev-erbA should be considered an orphan receptor since its cognate ligand and function are not known. One potential role for rev-erbA may be to regulate the splicing that generates c-erbA-2 as increased levels of rev-erbA mRNA correlate with increased TR-1 mRNA relative to c-erbA-2 (80, 224, 290).

There also are two TRs derived from the TR gene (205, 284). This gene contains two promoter regions each of which is vital for the transcription of an mRNA coding for a distinctive protein. By the use of alternate promoter choice, one or both of the coding mRNAs are generated (566). The resultant TR isoforms are designated as TR-1 and TR-2. The amino acid sequences of the DNA binding, hinge region, and LBDs of these two TRs are identical, but the amino-terminal regions bear no homology (Fig. 3). Both are authentic receptors as they bind TREs and TH with high affinity and specificity and can mediate TH-dependent transcription. The expression of the two TR isoforms may be regulated by pituitary-specific transcription factors such as Pit-1 (566).

Both TR-1 and TR-1 mRNAs and proteins are ubiquitously expressed in rat tissues (204). However, TR-1 mRNA has highest expression in skeletal muscle and brown fat, whereas TR-1 mRNA has highest expression in brain, liver, and kidney. In contrast to the other TR isoforms, TR-2 mRNA and protein have tissue-specific expression in the anterior pituitary gland and specific areas of the hypothalamus as well as the developing brain and inner ear (47, 48, 98, 204, 590). In the chick, TR-2 mRNA also is expressed in the developing retina (473).

Careful low-stringency hybridization studies so far have not yielded any additional TR isoforms. Double TR and TR knockout mice are viable, and these mice did not have detectable [¹²⁵I]T₃ binding in nuclear extracts of several tissues (171, 316a). However, a number of short forms of TR and TR generated by alternative splicing of mRNA or by use of internal translational start sites have been found in embryonic stem cells and in fetal bone cells and may have biological significance (41, 75, 553, 567). The identification of a novel estrogen receptor isoform (ER) 10 years after the discovery of ER serves as a cautionary warning to remain open to the possibility of novel TR isoforms, particularly in restricted tissues or during transient periods in fetal development (265).

The regulation of the TR mRNAs is isoform and cell type dependent. In the intact rat pituitary, T₃ decreases TR-2 mRNA, modestly decreases TR-1 mRNA, and slightly increases rat TR-1 mRNA (204). Despite these opposing effects, the total T₃ binding decreases by 30% in the T₃-treated rat pituitary. Similar findings also were observed in GH₃ cells, a somatolactotropic rat cell line (205). In other tissues, T₃ slightly decreases TR-1 and c-erbA-2 mRNA except in the brain where c-erbA-2 levels are unaffected. TR-1 mRNA is minimally affected in nonpituitary tissues. Additionally, the hypothalamic tripeptide TRH decreases TR-2 mRNA, slightly decreases TR-1 mRNA, and minimally affects TR-1 mRNA in GH₃ cells (230). Retinoic acid blunts the negative regulation by T₃ in these cells (114, 231). Additionally, in patients with nonthyroidal illness in which their circulating free T₃ and T₄ levels were decreased, TR and TR mRNAs were increased in peripheral mononuclear cells and liver biopsy specimens (556). Thus induction of TR expression may compensate for decreased circulating TH levels in these patients.

Each of the TR isoforms found in human, rat, and mouse are highly homologous with respect to their amino acid sequences (284). This conservation among species suggests that there may be important specialized functions for each isoform (109). However, the evidence for isoform-specific functions has been scant since most cotransfection experiments have failed to show important functional differences. Nonetheless, recent studies have suggested that TR-1 may exhibit isoform-specific regulation of the TRH and myelin basic protein genes, and TR-2 may play an important role in the regulation of the GH and TSH gene expression in the pituitary (4, 129, 208, 283, 305). Future studies with TR knockout mice, antisense oligonucleotides in tissue culture, and isoform-specific ligands, perhaps in conjunction with cDNA microarrays, should shed more light on the respective roles of TR isoforms in regulating specific target genes (24, 86, 136, 151).

	IV. THYROID HORMONE RECEPTOR FUNCTIONAL DOMAINS

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Mutational analyses of TRs and comparisons with other members of the nuclear hormone receptor superfamily have yielded much information on the structural features of TRs (284, 583). All TRs have a similar domain organization as that found in all nuclear hormone receptors: an amino-terminal A/B domain, a central DNA-binding domain containing two "zinc fingers" (DBD), a hinge region containing the nuclear localization signal, and a carboxy-terminal LBD (Fig. 4). It should be noted that each of these domains and regions may subserve multiple functions, and thus their names may only reflect the first function ascribed to them.

View larger version (11K): [in this window] [in a new window]   Fig. 4. General organization of major TR domains and functional subregions.

The DBD is located in the central portion of TR and has two zinc fingers, each composed of four cysteines coordinated with a zinc ion (Fig. 5). The integrity of each zinc finger is critical, as deletion of zinc fingers or amino acid substitution of these cysteine residues abrogates DNA-binding and transcriptional activity of steroid hormone receptors and TRs (173, 345, 460, 592). Within the first zinc finger, there is a "P box," comprised of amino acids located between and just distal to the third and fourth cysteines, which is similar to that of estrogen recetors (ERs), retinoic acid receptors (RARs), retinoid X receptors (RXRs), and vitamin D receptors (VDRs) (106, 317, 531). This critical region has been shown to be important in sequence-specific recognition of hormone response elements by different members of the nuclear hormone superfamily and contacts nucleic acids and phosphate groups within the major groove of the TRE (353, 413). Additionally, there are other important contact points within the minor groove of the TRE just downstream from the second zinc finger (A-box region). Also, as discussed below, TRs can heterodimerize with RXRs and can bind to TREs that are arranged as direct repeats separated by a four nucleotide gap. These TR/RXR heterodimers bind to TREs with a 5' to 3' polarity with TR in the downstream position (268, 391, 586). The ability to heterodimerize with RXR is critical for TR binding to the asymmetric TRE, as dimerization contacts stabilize the DNA binding and determine the spacing between half-sites. Within the DBD, there are dimerization interfaces in the TR just upstream of the first zinc finger, within the first zinc finger, and in a subregion distal to the second zinc finger (T box). The RXR dimerization surfaces are located in the second zinc finger including an arginine located in the D box, a region which previously has been shown to be important for distinguishing spacing between half-sites of hormone response elements (315, 531).

View larger version (12K): [in this window] [in a new window]   Fig. 5. DNA-binding domains of human TR. Schematic drawing of the two zinc fingers of human TR and the various subregions within the DNA-binding domains. Squares, TR/RXR heterodimerization contacts; ovals, direct base contacts; solid circles, direct phosphate contacts. [From Rastinejad et al. (413). Reprinted by permission from Nature, copyright 1995 Macmillan Magazines Ltd.]

The LBD not only is necessary for TH binding but also plays critical roles for dimerization, transactivation, and basal repression by unliganded TR. The recent solutions of the crystal structures of the liganded TR-1, unliganded RXRa, and RAR LBDs have greatly aided our understanding of its role on these functions and the attendant conformational changes that occur when T₃ binds to the receptor (Fig. 6) (46, 417, 544). Ligand is buried deep within a hydrophobic pocket in the LBD formed by discontinuous stretches that span almost the entire LBD. In particular, the most carboxy-terminal region (Helix 12) contributes its hydrophobic surface as part of the ligand-binding cavity. The hydrophobic residues face inward, whereas the conserved glutamate faces outward. The cavity also is bounded by hydrophobic surfaces from helices 3, 4, and 5. Although the crystal structure of unliganded TR has yet to be solved, the crystal structure of unliganded RXR shows that helix 12 projects into the solvent. Thus it is likely that helix 12 undergoes major conformational changes upon ligand binding, from a more open conformation to a closed one, which has been likened to a "mouse trap" mechanism. In an analogous manner, estrogen-bound LBD shows a similar structure as liganded-TR LBD with helix 12 facing inward (61). However, helix 12 of raloxifene-bound ER LBD is in a different position, lying in a groove between helices 3 and 5. Thus the relative positions of helix 12 and the boundary helices may determine whether coactivators can interact with TR. Indeed, studies using TR-LBD mutants based on the TR-LBD crystal structure have confirmed these regions for interacting with the coactivator GRIP-1 (107, 134).

View larger version (42K): [in this window] [in a new window]   Fig. 6. TR-1 ligand-binding domain crystal structure. Dark mass represents ligand. -Helices are indicated. [From Wagner et al. (544). Reprinted by permission from Nature, copyright 1995 Macmillan Magazines Ltd.]

TR and TR-1 isoforms can bind T₃ and various TH analogs with subtle differences in affinity. TR binds T₃ with slightly higher affinity than TR-1 (449). Triac (3,5,3'-triiodothyroacetic acid) binds TR-1 with similar affinity as T₃ and binds TR-1 with two- to threefold higher affinity than T₃. Several novel thyromimetics have been designed which bind TR-1 (GC-1 and CGS 23425) with 10- to 50-fold higher affinity (86, 512). The transcriptional activities of these isoform-specific compounds parallel their binding affinities and may offer novel therapeutic treatments of diseases such as hypercholesterolemia while sparing the heart (which contains mostly TR) from side effects. The crystal structures of hTR and hTR LBDs have been solved and may provide important information for designing even more selective thyromimetics in the future (419).

The LBD also is involved in several other important receptor functions. Scattered throughout the LBD are discontinuous heptad repeats that have been proposed to form hydrophobic interfaces for TR homo- and heterodimerization (145). Mutations in the ninth heptad repeat region have selectively decreased TR homo- and heterodimer formation, suggesting that there may be different subregions of the LBD that are important for TR dimerization (21, 140, 344, 592). Indeed, the TR-1 LBD crystal structure demonstrates that there is a hydrophobic surface in the ninth heptad repeat region that could serve as a potential dimerization interface (544). A natural TR mutation from a patient with resistance to TH at amino acid 316 also displayed decreased homodimer formation, suggesting that additional regions of the LBD may be important for dimerization (189, 360, 592). The relative contributions to dimerization by the LBD and DBD interfaces may depend on the receptor. A recent study suggests that a region that contains the ninth heptad region called the "I box" may be important for RAR heterodimerization with RXR in solution and for binding to direct repeats of variable spacing (392). On the other hand, the DBD dimerization interface may be important for dictating binding to direct repeats of a specific spacing (in this case, a 5-nucleotide gap). Recent studies suggest that the ninth heptad region may be more important for heterodimerization of TR-1, whereas the DBD may play the dominant role for c-erbA-2 because it lacks a complete ninth heptad region due to alternative splicing (416, 568).

Baniahmad et al. (27) used a GAL4-fusion system to identify at least three transcriptional activation regions in the LBD and designated them as 2, 3, and 4 (27). Uppalari and Towle (534) also have used a yeast transfection system to describe several activation regions in TR-1 LBD as well as in the hinge region (534). In particular, 4 located near the carboxy terminus has high homology with LBD sequences found in other nuclear hormone receptors previously designated as the activation function-2 (AF-2) domain (Fig. 7). This sequence located within helix 12 has been shown to be important for ligand-dependent transcriptional activation by other nuclear hormone receptors (31, 105, 278). Recently, Chatterjee and co-workers (521) have made point mutations in this region and have observed normal T₃ binding and DNA binding, but no transcriptional activation, using a GAL4/TR LBD fusion protein system (521). As discussed earlier, helix 12 likely undergoes major conformational changes upon ligand binding (419). Studies with steroid hormone receptors and TRs have demonstrated that the AF-2 domain is important for interactions with coactivators such as SRC-1 and related family members (419). Interestingly, mutations in the AF-2 region of the TR LBD had modest effects on T₃-dependent interaction of the coactivator TRAM-1 (SRC-3), whereas a mutation in helix 3 of the TR LBD severely impaired T₃-dependent interaction with TRAM-1 but had little effect on interaction with SRC-1 (503). Ligand-dependent GRIP-1 (SRC-2) interactions with TR involve helices 3, 5, 6, and 12 (134). These findings suggest that different subregions of TR may differentially contribute to interaction with specific coactivators. Additionally, several groups reported that corepressors may interact with sequences on helices 3, 5, and 6 that overlap sequences involved in interacting with coactivators (214, 348, 390). Several mutations from patients with resistance to TH and artificial mutations in helices 3 and 5 do not interact with coactivators or corepressors (97, 348, 390).

View larger version (22K): [in this window] [in a new window]   Fig. 7. Comparison of AF-2 regions of nuclear hormone receptors. Conserved xE sequences are underlined.

The hinge region between the DBD and T₃-binding domain likely contains an amino acid sequence that is associated with nuclear localization (126). This lysine-rich sequence is highly conserved among nuclear hormone receptors and bears homology with the simian virus 40 T antigen nuclear localization sequence. TRs are likely imported into the nucleus shortly after synthesis as they are predominantly found in the nucleus and can bind DNA, even in the absence of hormone. Furthermore, unlike some steroid hormone receptors, TRs do not associate with cytoplasmic heat shock proteins (103). Recent studies using green fluorescent fusion proteins of wild-type TR and TR hinge region mutants demonstrated this region may be important for T₃-mediated translocation of TR into the nucleus (605).

The hinge region also has additional properties. The laboratories of Evans (83) and Rosenfeld and co-workers (211) identified corepressor proteins that can interact with unliganded TRs, RARs, and v-erbA and mediate repression of basal transcription by these receptors and v-erbA (see sect. VIII). Mutations in the TR-1 hinge region abrogate the basal repression by corepressor. Additionally, a v-erbA mutation in the hinge region that abrogates its oncogenic potential also failed to interact with a corepressor, silencing mediator for RAR and TR (SMRT) (83). These findings suggest that the hinge region of unliganded TRs located on helix 1 may serve as a contact surface with corepressors or have allosteric effects on their interaction. Recent work by several groups also suggest that sequences within helices 3, 5, and 6 of the LBD contribute to corepressor binding (214, 348, 390).

The amino-terminal regions have variable lengths and divergent sequences among the TR isoforms. Even among different species, this region is less well conserved for a given TR isoform, because the rat and human TRs are 97 and 99% identical in their DBDs and LBDs, respectively, but only 85% identical in their amino-terminal domains (254). The role(s) of the amino-terminal domain is poorly understood. Studies of the glucocorticoid receptor have suggested that there is a major activation function domain, 1, which has structural similarities with viral acidic activator proteins such as VP16 (209). Previous work by Tora and co-workers (524, 525) with progesterone and truncated estrogen receptors also have suggested that the amino-terminal domain may modulate cell-specific and promoter-specific transcription. Cotransfection studies generally have demonstrated only a few examples of isoform-specific transcriptional activation by TRs. Farsetti et al. (129) have shown that TR-1 has higher transcriptional activity than TR-1 via a myelin basic protein TRE reporter in the context of the native promoter, but not with the viral thymidine kinase promoter (129). Jeannin et al. (224) observed similar TR-1-specific effects on myelin basic protein, but not on malic enzyme, gene expression suggesting that isoform-specific gene regulation may occur in a subset of target genes (224). Similar TR-1-specific effects on the regulation of the TRH promoter also have been observed (181, 305). TR-2 may have a more potent role in the negative regulation of glycoprotein -subunit and TSH than other TR isoforms (208, 275). In particular, TR-2 mediates strong ligand-independent activation of negatively regulated target genes such as glycoprotein hormone -subunit and TSH (275). Indeed, recent studies of TR knockout mice have implicated TR-2 as the major TR isoform in negative regulation of TSH (3).

The role of the amino-terminal domain in transcriptional activation is still controversial. Some studies have shown that deletion of the amino-terminal domain of TR-1 had no effect on T₃-dependent transcriptional activation by TR-1 (516, 592), suggesting that it does not contain a major activation function domain like the glucocorticoid receptor. On the other hand, studies of TR-1 and TR-1 from several species have shown that the amino-terminal domain may be important for transcriptional activation and interactions with the general transcription factor TFIIB (25, 183, 520). Additionally, it has been shown that the chick TR-2 amino-terminal region may have two activation domains (472). A minimal subregion of amino acids 21-50 allows human TR-2 to interact with coactivators in the absence of ligand and may account for the ligand-independent activation of some positively regulated target genes (358). However, one study of the amino-terminal region suggested that rat TR-2 does not appear to have a strong activation domain in the amino-terminal region (520), whereas other studies have shown that TR-2 transactivates similar to TR-1 (205, 478). It is possible that differences in species, cell types, TREs, and minimal promoters may account for these different observations (129, 491, 580). The amino-terminal domain also may modulate ligand-independent repression via positively regulated TREs because one study showed that TR-1 is more potent than TR-1 or TR-2 in mediating basal repression in the absence of T₃ (208). Recent studies showed that the amino terminus of TR-2 may interact with the silencing domain of the corepressor, SMRT, and thereby block the recruitment of other components of the corepressor complex (575). Finally, the amino-terminal domain of TR also may influence the conformation of the DBD and the repertoire of TREs to which it can bind (184, 232).

	V. THYROID HORMONE RESPONSE ELEMENTS

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Steroid hormone receptors bind as homodimers to conserved palindromic hormone response elements that mediate hormone regulation of target genes (160). In contrast, TRs can bind to TREs as monomers, homodimers, and heterodimers in vitro. In general, most of these TREs are located upstream from the minimal promoter, but in certain cases, also can be located in 3'-flanking sequences downstream from the coding region (40, 600). Mutational analyses of the rat growth hormone gene TRE, and sequence comparison among known TREs from other T₃-responsive genes, have suggested a putative consensus hexamer half-site sequence of (G/A)GGT(C/G)A (84, 555). However, there can be considerable variation found in primary nucleotide sequences of TREs as well as the number, spacing, and orientation of their half-sites (555). In particular, TRs can bind to TREs in which half-sites are arranged as palindromes (TRE_pal), direct repeats (DRs), and inverted palindromes (IPs). The optimal spacing for these half-site arrangements are zero, four, and six nucleotides, respectively (TRE_pal0, DR4, and IP6) (Fig. 8). Almost all positively regulated target genes contain two or more half-sites; however, TRs can activate transcription via an artificial single octamer half-site, perhaps even as monomers (240).

View larger version (13K): [in this window] [in a new window]   Fig. 8. Half-site orientation and optimal nucleotide spacing between half-sites. N refers to nucleotides, and arrows show direction of half-sites on the sense strand. TRE, thyroid hormone response element.

Approximately 30 natural TREs have been described so far with DRs, followed by IPs, as the most common motifs (555). Several groups also have observed that TR homo- and heterodimers bind to TREs arranged as IPs and DRs better than palindromes (17, 268, 333, 588). In the case of DRs, it has been shown that VDRs preferentially transactivate via reporter vectors containing DRs with a three-nucleotide gap (DR3), TRs via a four-nucleotide gap (DR4), and RARs via a gap of five nucleotides (DR5), according to a "3-4-5" rule (160, 532). Heterodimerization with RXR and specific DNA contact points in the DBD may play important roles in determining heterodimer spacing on direct repeats (153, 160, 413). However, the DNA binding and transcriptional activation via these elements do not appear to be absolutely receptor specific because TRs can bind and transactivate weakly on DR5 and DR6, and RARs can bind and transactivate on DR4 (557, 588). VDRs can bind to DR4 and DR5 (587) but cannot transactivate via these HREs. However, VDR has dominant negative activity on T₃ and RA-mediated transcription via these elements. Additionally, the primary sequence of the half-site may be important in maintaining receptor-specific binding to DRs as mutation of the third nucleotide of the half-site hexamer from a G to a T enabled VDR binding and ligand-mediated transactivation via a DR4 (418). It also has been shown that flanking and spacing sequences in TREs can affect DNA-binding and transcriptional activation, either by making contacts with TR or local DNA bending (216, 240, 242, 244).

TRs can form heterodimers with RXRs, which also are members of the nuclear hormone receptor superfamily (see below). This enables TR complexes to bind to TREs in a specific orientation relative to the minimal promoter. Palindromic and inverted palindromic TRE half-site sequences are symmetric and thus do not dictate a particular heterodimer orientation on the TRE. On the other hand, TRE half-sites in DR4 have a 5' to 3' polarity so the direct repeat motif could specify the heterodimer orientation on the TRE. Several approaches have been used to study this issue. Mutant TRs or RXRs containing amino acid substitutions in the P box of the first zinc finger of the DNA-binding domain that allow preferential binding to a glucocorticoid hormone response element (GRE) half-site have been used to study TR/RXR heterodimer binding to hybrid response elements containing TRE and GRE half-sites (268, 391, 586). These results showed that TR binds to the downstream half-site and RXR to the upstream half-site when TR/RXR heterodimer binds to DR4. Methylation interference studies also showed that TR-1 and TR-1/RXR preferentially bound to the downstream half-sites of DR4 and the IP F2 (216). The apparent polarity of TR/RXR complexes on F2 may be due to degeneracy of one of the half-site sequences or possibly contributions by the flanking and spacing sequences. Cotransfection studies in which the orientation of these TREs were reversed also decreased T₃-mediated transcriptional activity (216). Taken together, these findings suggest that TR/RXR heterodimers bind with a specific polarity which, in turn, can modulate transcriptional activity. This "shape" of the TR complex may be important in protein-protein interactions with coactivators and corepressors that link the liganded TR/RXR heterodimer with the transcriptional machinery. In this connection, Kurokawa et al. (267) demonstrated that RAR/RXR heterodimers bound to DR1 and DR5 with different polarities. In the former case, they remained bound to a corepressor and mediated constitutive basal repression, and in the latter case, they dissociated from corepressor in the presence of all-trans-retinoic acid and mediated transcriptional activation. Similarly, it has been shown that the TRE sequence can affect corepressor release from TR in the presence of ligand (368).

	VI. THYROID HORMONE RECEPTOR COMPLEXES

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Early studies of TR binding to specific DNA sequences utilized methods such as the avidin-biotin complex/DNA (ABCD) assay that did not allow direct visualization of TR complexes bound to DNA (63, 161, 585). However, successful employment of electrophoretic mobility shift assays (EMSAs) demonstrated that TRs could bind to synthetic and natural TREs as monomers, homodimers, and heterodimers in vitro (144, 288, 557, 584). TR-1 may have a greater tendency than TR-1 to form homodimers on several different TREs, suggesting that these two TR isoforms may have different dimerization potentials (109, 607). Domain swap experiments have suggested that the amino-terminal region of TR-1 may inhibit homodimer formation via an allosteric mechanism (208).

Initially, TRs were thought to mediate their effects on transcription as homodimers, similar to steroid hormone receptors. However, two groups observed that TRs surprisingly heterodimerized with proteins from pituitary and liver nuclear extracts (63, 341). These proteins were called TR auxiliary proteins (TRAPs) and enhanced TR binding to TREs (63, 341). Because these proteins were expressed in nuclear extracts from many different tissues and species (63, 287, 341, 494), TR/TRAP heterodimers potentially could be formed in all cells that contain TRs. Because these heterodimers appeared to bind better to TREs than TR homodimers, it was speculated that they played a role in T₃-regulated transcription.

Several groups showed that TRs heterodimerize with RXRs, members of the nuclear hormone receptor superfamily which have high homology with RARs (250, 300, 321, 589, 594, 602). RXRs bind their cognate ligand, 9-cis-retinoic acid, with high affinity (202, 304). They can form homodimers as well as heterodimers with RARs, VDRs, and peroxisome proliferator activated receptors (PPARs) (160). Several lines of evidence suggest that RXRs are the major TRAPs and thus play a critical role in T₃-mediated transcription. First, they were observed to enhance TR binding to TREs. Second, studies using anti-RXR antibodies showed that the major endogenous TRAPs are RXRs or related proteins (287, 420, 494). Third, TR/TRAP and TR/RXR heterodimer complexes both remained bound to TREs in the presence of T₃ (17, 333, 422, 584, 589) (see below). Fourth, heterodimer-selective, but not homodimer-selective, mutants were able to mediate transcriptional activation of TRE-containing reporters (21, 140, 344, 592). Mutant TRs containing deletions or amino acid substitutions of amino acids at positions 290-310 or in the ninth heptad region of the LBD concomitantly decreased heterodimerization and transactivation (21, 140, 344, 360, 592). Fifth, RXR enhanced T₃-mediated transcription in yeast cells that do not contain endogenous TRAPs (65, 187). And last, RXR enhanced T₃-mediated transcription in a reconstitutable in vitro transcription system (294).

TR/RXR heterodimer formation increases the repertoire of target genes that can be regulated by T₃ as heterodimers bind to TREs with variable sequence and orientation of half-sites (160). Moreover, there are at least three members of the RXR subfamily, so it is possible that different RXR isoforms may form TR/RXR heterodimers that have different TRE-binding specificities and/or abilities to transactivate target genes. Additionally, it is possible that an endogenous ligand like 9-cis-retinoic acid can bind and activate the heterodimer partner of the TR complex. Of note, addition of both 9-cis-retinoic acid and T₃ synergistically activated transcription on two different TRE-containing reporters (227, 429). However, in other cases, TR can block 9-cis-binding to RXR and thereby abrogate retinoid stimulation of target genes (91, 147, 266). Finally, TRs can heterodimerize with other members of the nuclear receptor family including RAR, PPAR, chicken ovalbumin upstream promoter transcription factor (COUP-TF), and VDRs (45, 99, 314, 448, 519, 589). The functional significance of these heterodimers, most of which have been demonstrated in vitro by EMSA, is not known. However, they do not appear to be major endogenous TRAPs and may have restricted roles in particular target genes or tissues (287, 494). If they do have physiological roles, though, these heterodimers increase further the diversity of TR complexes and the potential of receptor cross-talk on target genes.

Although TRs can form monomers, homodimers, and heterodimers on TREs in EMSA (284, 583), the role(s) of TR monomers and homodimers on transcription is not well understood. In contrast to steroid hormone receptors in which ligand enhances receptor binding to HREs, T₃ decreases TR-1 and TR-1 homodimer as well as TR-1/TR-1 dimer binding to several TREs arranged as DRs and IPs (17, 333, 422, 584). Unliganded TR homodimers also interact better with corepressors than unliganded TR/RXR heterodimers in vitro (207, 311). Thus it is possible that unliganded homodimers may form a complex with corepressors that are involved in basal repression of target genes in the absence of T₃, which then dissociates from the TRE in the presence of T₃.

Cross-linking and coimmunoprecipitation studies suggest that TR monomers and TR/RXR heterodimers exist in solution, whereas TR homodimers form weakly in solution (268, 288, 581). T₃ and 9-cis-retinoic acid promote dimerization in solution, and thus may preform a transcriptionally active complex before DNA binding (95, 234). It appears that dimerization promotes DNA binding (392), and DNA binding enhances dimerization (56, 366, 581).

	VII. PHOSPHORYLATION OF THYROID HORMONE RECEPTORS

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Recently several groups also have observed that increasing the phosphorylation state of cells can enhance T₃-mediated transcriptional activation of target genes (229, 308, 496). The mechanisms for this enhanced transcriptional activation are not known but may involve phosphorylation of TR, RXR, or coactivators. In support of the potential role of TR phosphorylation in transcriptional activation, it recently has been demonstrated that TR can be phosphorylated in vitro and in vivo (165, 167, 308, 492). Chick TR-1 has at least two serine phosphorylation sites in the amino-terminal A/B domain, but the functional role(s) is not known (167). Additionally, these sites do not appear to be conserved across species. The human TR-1 can be phosphorylated in vivo and in vitro (308), although the phosphorylation sites have not been determined. Two groups have used HeLa cytosol extract to in vitro phosphorylate Escherichia coli-expressed TR-1 (39, 492). Sugawara et al. (492) examined the binding of phosphorylated TR-1 to several TREs and found that phosphorylation selectively enhanced TR homodimer, but not TR/RXR heterodimer, binding to several different TREs. Bhat et al. (39) showed that phosphorylation enhanced DNA binding by both TR complexes. Interestingly, phosphorylation by protein kinase A can decrease v-erbA and chick TR-1 monomer binding to TREs (530). These results suggest that phosphorylation may be another mechanism, in addition to T₃ binding, that can modulate TR complex binding to TREs. Additionally, T₃ itself can modulate the phosphorylation state of TR (518).

Recently, Davis et al. (113) have shown that TR-1 associates with mitogen-activated protein (MAP) kinase in coimmunoprecipitation studies and that ligand binding may stimulate TR phosphorylation by MAP kinase. Interestingly, previous studies have shown that MAP kinase can modulate transcriptional activity of ER and PPAR by phosphorylation of the receptor (238, 406). Moreover, MAP kinase phosphorylation of the steroidogenic factor-1 (SF-1) and ER AF-1 regions leads to enhanced coactivator recruitment and transcriptional activation (188, 528). Stimulation of the protein kinase A pathway also potentiated T₃-mediated transcription in a cell-specific manner (301), suggesting multiple kinase pathways may modulate transcriptional activity of TR. Power et al. (405) have demonstrated that some nuclear hormone receptors can be activated by dopamine stimulation in the absence of ligand, mostly likely via receptor phosphorylation. It is possible that some ligand-independent effects by TR may be due to receptor phosphorylation by cell-specific kinases or phosphatases, or due to cell-specific expression of certain membrane-signaling receptors that can activate transcription by unliganded TR. In this connection, cell-type specific phosphorylation may stabilize TR-1 protein (517). Additionally, it has been shown that DNA binding by the alternative splice variant of TR-1, c-erbA-2, is regulated by casein kinase II phoshorylation of serines in its carboxy terminus (239). This phosphorylation is critical for determining the dominant negative activity (ability to block wild-type TR action) by c-erbA-2. These findings suggest that phosphorylation potentially may regulate diverse and important TR functions, although the precise location of phosphorylation sites, their regulation, and their functional roles remain to be elucidated.

	VIII. MOLECULAR MECHANISMS OF THYROID HORMONE RECEPTOR ACTION

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In contrast to steroid hormone receptors that are transcriptionally inactive in the absence of ligand, unliganded TRs bind to TREs and may modulate transcription of target genes (Fig. 9). Several laboratories showed that unliganded TRs can repress basal transcription of positively regulated TREs in cotransfection studies (26, 53, 603). It was not known initially whether these observations were physiologically relevant or peculiar to cotransfection systems. Early observations showing that T₃ decreased TR homodimer binding to TREs led to the hypothesis that unliganded TR homo- and/or heterodimers might mediate basal repression (584). This notion was further supported by the demonstration that TR binding to TREs was important for mediating basal repression, as mutations in TR-1 DBD or the TRE primary sequence abrogated basal repression (26, 592). Unliganded TRs have been shown to interact directly with TFIIB, a key component of the basal transcription machinery (25, 183, 394, 520, 523), and potentially can interfere with the assembly of a functional preinitiation complex at the promoter. Studies of TR action in in vitro transcription systems (142) suggested that direct interaction between TRs and the basal transcriptional machinery could help mediate ligand-dependent basal repression. On the other hand, several studies also suggested that soluble corepressors may be critical for mediating basal repression (407, 522).

View larger version (18K): [in this window] [in a new window]   Fig. 9. Model for repression, derepression, and transcriptional activation by TR in the absence or presence of T3.

The cloning and functional characterization of several corepressors have greatly enhanced our understanding of basal repression and shed light on these previous issues (Fig. 10). Several laboratories used the yeast two-hybrid system or biochemical purification to clone proteins that exhibited decreased interaction with TR and RAR in the presence of their cognate ligands (83, 211, 295, 442). One of them was a 270-kDa protein called nuclear receptor corepressor (NCoR), which also was isolated as an RXR-interacting protein, RIP 13 (211, 459). It contains three transferable repression domains and two carboxy-terminal -helical interaction domains. NCoR was able to mediate basal repression by TR and RAR, as well as orphan members of the nuclear hormone receptor family such as rev-erbA and COUP-TF. It had little or no interaction with steroid hormone receptors and did not mediate basal repression by these receptors. NCoR also has been shown to interact with TFIIB, TAFII32, and TAFII70, so part of its ability to repress transcription may be due to its ability to interact with the basal transcripitonal machinery. Recently, a truncated version of NCoR, NCoRi, which is missing the repressor region, has been identified, which may represent an alternative-splice variant of NCoR (207). This protein blocks basal repression by NCoR and potentially may serve as a natural antagonist for NCoR if it is expressed in signficant amounts in tissues. Another corepressor, SMRT, has been identified and has homology with NCoR (83, 295, 442). The original sequence for SMRT was derived from a partial clone, but it is now appreciated that full-length SMRT is similar in size and has similar repression and nuclear receptor interaction domains as NCoR. SMRT also is able to mediate basal repression of TR and RAR in cotransfection studies. Another protein, small ubiquitious nuclear corepressor (SUN-CoR), was isolated and enhanced basal repression by TR and rev-erbA (596). This 16-kDa protein may form part of a corepressor complex as it interacts with NCoR.

View larger version (10K): [in this window] [in a new window]   Fig. 10. Comparison of the organization and structure of putative nuclear hormone receptor corepressors. NCoR, nuclear receptor corepressor.

Studies of TR and v-erbA have defined the importance of the hinge region for interactions with NCoR and SMRT, because mutations in this region abrogate basal repression without affecting transcriptional activation (83, 211, 324). Interestingly, rev-erbA contains two amino-terminal regions which interact with NCoR and are required for basal repression, suggesting that nuclear hormone receptors may have different interaction sites with corepressors (597). Within the interaction domains of NCoR and SMRT are consensus LXXI/HIXXXI/L sequences that resemble the LXXLL sequences that enable coactivators to interact with nuclear hormone receptors (214, 348, 390). Interestingly, these motifs allow both corepressors and coactivators to interact with similar amino acid residues on helices 3, 5, and 6, which are part of the ligand pocket of TR. Differences in the length and specific sequences of the corepressor and coactivator interaction sites coupled with the conformational changes in the AF-2 region upon ligand binding may determine whether corepressor or coactivator binds to TR (390). Additionally, corepressors can bind to the TR heterodimer partner RXR. It appears that helix 12 of RXR masks a corepressor binding site in RXR, which is unmasked upon heterodimerization with TR (599).

Recently, several groups have shown that corepressors can complex with other repressors, Sin 3 and histone deacetylase 1 (HDAC1), that are mammalian homologs of well-characterized yeast transcriptional repressors RPD1 and RPD3 (13, 200, 271, 347, 352). Thus local histone deacetylation may play a critical role in basal repression by nuclear hormone receptors. Moreover, this mechanism of basal repression may be employed by other transcription factors such as Mad/Max and Myc/Mxi heterodimers (13, 200, 271). Anti-NCoR antibodies have been shown to coimmunoprecipitate HDAC activity (200). Additionally, microinjection of specific antibodies generated against mSin3 and RPD3 were able to block basal repression by NCoR (200). Recent studies by Lazar (286) and Wong (J. M. Wong, personal communication) suggest that HDAC4 may interact directly with NCoR at a different repressor site than Sin3 and HDAC1. Recent coimmunoprecipitation studies also suggest that HDAC3 may be the major HDAC associated with SMRT (286). Another component of the corepressor may be the protooncogene c-Ski which has been shown to be involved in transcriptional represson by Mad and TR (354). It is likely that histone deacetylation by unliganded TR/corepressor complex may help maintain local chromatin structure in a state that shuts down basal transcription. In this connection, studies examining TRA promoter in a Xenopus oocyte system showed that simultaneous chromatin assembly and TR/RXR binding were required for basal repression of transcription (562, 563). This repression was relieved by addition of T₃ and was accompanied by chromatin remodeling. These data demonstrate that histone acetylation and deacetylation, and the consequent changes in chromatin stucture and nucleosome positioning, may be important determinants of gene transcription (Fig. 11). Additionally, DNA methylation may play a role in basal repression as methyl-CpG-binding proteins can associate with a corepressor complex containing Sin3 and HDACs (351, 543). This repression was relieved by the deacetylase inhibitor trichostatin A. These findings suggest that two repression processes, DNA methylation and histone deacetylation, may be linked via methyl-CpG-binding proteins.

View larger version (32K): [in this window] [in a new window]   Fig. 11. Molecular model for basal repression in the absence of T3 and transcriptional activation in the presence of T3. X refers to potential unidentified cofactors. See text for details.

TR also can activate transcription in the absence of ligand. Samuels and co-workers (201) showed that unliganded TR surprisingly could transactivate via rGH and PRL TREs in pituitary cell lines (89). Additionally, several groups have reported ligand-independent gene transcription in neuroblastoma and Xenopus cells (346, 385). It is not known whether cell-specific activators or inhibitors or other mechanisms account for these observations. Last, TRs can activate transcription of negatively regulated genes such as RSV LTR, glycoprotein hormone -subunit, TSH, and TRH in the absence of ligand (88, 206, 305, 342, 433). Brent et al. (57) showed that a negative TRE (nTRE) from the glycoprotein hormone -subnunit gene could be placed in different positions relative to the rat growth hormone minimal promoter (even downstream), and still mediate negative regulation. They showed that the nTRE sequence may play a more important role than its position relative to the minimal promoter. Additionally, several groups have found nTREs located in the 3'-untranslated region of target genes (40, 600). Recent studies have suggested that ligand-independent activation may be mediated by TR recruitment of corepressors, and this may be mediated by protein-protein interactions of DNA (500, 501). Interestingly, these effects could be blocked by pharmacological inhibition of HDAC activity. These findings suggest that histone deacetylation may promote ligand-independent activation of a negatively regulated target gene. Another study showed that T₃-mediated negative regulation may also require HDAC activity (445). On the other hand, NCoRi (which does not contain the repression domains) enhances ligand-independent regulation in a reporter containing the TRH promoter (207). NCoRi activation function is stronger than NCoR, so the ligand-independent activation function may map to the carboxy terminus of NCoR (93). These studies suggest a role for corepressors in ligand-independent activation of some negatively regulated target genes, but the precise mechanism needs to be further defined.

The physiological role of corepressors is only partially understood. Corepressors have been implicated in leukemias that have RAR fusions and acute myolegenous leukemia/eight-twenty-one (ETO) fusions from chromosomal translocations (158, 176). Additionally, the TR/TR double-knockout mice have a milder phenotype than congenitally hypothyroid mice, suggesting that basal repression by unliganded receptor may have more deleterious effects on transcription in target tissues. Lazar (286) recently foun