A nuclear factor that enhances binding of thyroid hormone receptors to thyroid hormone response elements

Recent studies from this laboratory have demonstrated the presence of thyroid hormone response elements (TREs) in the 5'-flanking region of the rat alpha and TSH beta subunit genes. Using an avidin-biotin complex DNA binding assay, we have shown that these TREs bind the thyroid hormone (T3) receptor present in nuclear extracts of GH3 cells, as well as the in vitro synthesized Hc-erbA beta, which has been identified as a member of the family of T3 receptors. The binding of Hc-erbA beta to the alpha subunit TRE can be enhanced 3-4-fold by including GH3 nuclear extract in the binding assay. Binding to the TRE present in the TSH beta gene or the rat growth hormone gene was similarly enhanced, although to a lesser degree. The enhanced binding activity is trypsin-sensitive and heat labile, and is not reproduced by the addition of histones, bovine serum albumin, or cytosol instead of nuclear extract. Gel exclusion chromatography suggests a molecular size of approximately 65,000 Da. This protein, which is present in several different cell types, is also able to complement binding of the rat erbA alpha-1 and the pituitary-specific erbA beta-2 forms of the receptor. These data suggest that the binding of the T3 receptor to a TRE is augmented by another nuclear protein, which may be involved in the mechanism of action of thyroid hormone.     

The thyroid hormone receptors: molecular basis of thyroid hormone resistance.

Major progress has been achieved in the mechanism of action of thyroid hormones thanks to the identification of the T3 receptor as the product of the proto-oncogene c-erbA. Recognition of subsets of receptors with and without T3-binding properties and of the interaction of different receptors with each other leads to new insights in cell regulation and development. In thyroid hormone resistance, distinct mutations in the T3-binding domain of thyroid hormone receptor (TR)beta have been identified in unrelated families. No correlation between the type of mutation and tissue resistance has been established. Mutant TRs bind to thyroid hormone response elements (TREs) on both negative or positive T3-controlled genes. Subjects with heterozygous TR beta gene deletion are not affected, supporting the hypothesis that mutant TRs act through a dominant negative effect. In generalized thyroid hormone resistance, mutated TR beta may interfere through competition for TREs and/or formation of inactive dimers. Finally, deficiency in T3 receptor auxiliary protein or other accessory proteins or competition between mutant and normal TRs for these factors is not excluded.     

A homozygous deletion in the c-erbA beta thyroid hormone receptor gene in a patient with generalized thyroid hormone resistance: isolation and characterization of the mutant receptor.

Different point mutations have been identified in the T3-binding domain of the c-erbA beta thyroid hormone receptor gene that are associated with variant phenotypes of generalized thyroid hormone resistance (GTHR). In most cases of GTHR, heterozygotes are affected; a single mutant allele results in the inhibition of the function of normal thyroid hormone receptors. We report here a novel genetic abnormality, a 3-basepair (bp) deletion in the T3-binding domain of the beta-receptor in a kindred, S, with GTHR. One patient, S1, was the product of a consanguineous union of two heterozygotes and was homozygous for this defect. Heterozygotes from kindred S harbored a CAC deletion at nucleotides 1295-1297, which resulted in the deduced loss of amino acid residue threonine at codon 332, and they displayed elevated free T4 levels and inappropriately normal TSH levels characteristic of other kindreds with GTHR. However, patient S1, who had two mutant alleles, had markedly elevated TSH and free T4 levels and displayed profound abnormalities in brain development and linear growth. A fibroblast c-erbA beta cDNA extending from codon 175 to stop codon 457 was cloned from patient S1, sequenced, and used to create a full-length mutant cDNA. The kindred S mutant receptor was synthesized in vitro and did not bind T3. This mutant receptor did bind with similar avidity as the wild-type human beta-receptor to thyroid hormone response elements of the human TSH beta (-12 to 43 bp) and rat GH (-188 to -160 bp) genes. Kindred S showed the effect in man of heterozygous and homozygous expression of a dominant negative form of c-erbA beta.     

3,5,3'-triiodothyronine receptor auxiliary protein (TRAP) enhances receptor binding by interactions within the thyroid hormone response element.

We have previously demonstrated that binding of in vitro synthesized thyroid hormone receptor (TR) to thyroid hormone response elements (TREs) is enhanced by the addition of nuclear extracts from several different cell types, suggesting that binding of TR is partially dependent on a T3 receptor auxiliary protein (TRAP). We have used the avidin-biotin complex DNA-binding assay to discriminate between regions of TREs that bind TR alone and sites that are influenced by interactions with TRAP. Mutations in the TREs from rat GH and glycoprotein hormone alpha-subunit genes show that a specific DNA sequence is required for TRAP-mediated enhancement of TR binding. Mutations in the B half-site of the rat GH TRE or in similar sequences [(T/A)GGGA] in the alpha-subunit TRE ablate the enhancement of TR binding by TRAP. Furthermore, binding of TR to a natural half-site in the TSH beta-subunit gene (bases -16 to 6), which lacks an additional AGGGA-like sequence, is not enhanced by the addition of TRAP. Binding of TR to TREs was also tested at physiological salt concentrations in the avidin-biotin complex DNA-binding assay. Binding of human TR beta to TREs decreases dramatically at 140 mM KCl compared to binding at 50 mM KCl; however, the addition of TRAP enhances the binding to almost 4-fold of basal binding, suggesting that TRAP may be important for stabilization of TR binding to TREs in the cell.     

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.     

Novel mode of deoxyribonucleic acid recognition by thyroid hormone receptors: thyroid hormone receptor beta-isoforms can bind as trimers to natural response elements comprised of reiterated half-sites

Thyroid hormone receptors (TRs) regulate gene expression by binding to specific DNA sequences, denoted thyroid hormone response elements (TREs). The accepted paradigm for TRs proposes that they bind as homo- or heterodimers to TREs comprised of two AGGTCA half-site sequences. In the prototypic TRE, these half-sites are arranged as direct repeats separated by a four-base spacer. This dimeric model of TR binding, derived from analysis of artificial DNA sequences, fails to explain why many natural TREs contain more than two half-sites. Therefore, we investigated the ability of different TR isoforms to bind to TREs possessing three or more half-sites. We report that the TRbeta isoforms (TRbeta0, TRbeta1, TRbeta2), but not TRalpha1, can bind to reiterated DNA elements, such as the rat GH-TRE, as complexes trimeric or greater in size. The TRbeta0 isoform, in particular, formed homo- and heterotrimers (with the retinoid X receptor) with high efficiency and cooperativity, and TRbeta0 preferentially used reporters containing these reiterated elements to drive gene expression in vivo. Our data demonstrate that TRbeta isoforms can form multimeric receptor complexes on appropriately reiterated DNA response elements, providing a functional distinction between the TR isoforms and an explanation for TREs possessing three or more half-sites.     

The T3R alpha gene encoding a thyroid hormone receptor is essential for post-natal development and thyroid hormone production.

The diverse functions of thyroid hormones are thought to be mediated by two nuclear receptors, T3R alpha1 and T3R beta, encoded by the genes T3R alpha and T3R beta respectively. The T3R alpha gene also produces a non-ligand-binding protein T3R alpha2. The in vivo functions of these receptors are still unclear. We describe here the homozygous inactivation of the T3R alpha gene which abrogates the production of both T3R alpha1 and T3R alpha2 isoforms and that leads to death in mice within 5 weeks after birth. After 2 weeks of life, the homozygous mice become progressively hypothyroidic and exhibit a growth arrest. Small intestine and bones showed a strongly delayed maturation. In contrast to the negative regulatory function of the T3R beta gene on thyroid hormone production, our data show that the T3R alpha gene products are involved in up-regulation of thyroid hormone production at weaning time. Thus, thyroid hormone production might be balanced through a positive T3R alpha and a negative T3R beta pathway. The abnormal phenotypes observed on the homozygous mutant mice strongly suggest that the T3R alpha gene is essential for the transformation of a mother-dependent pup to an 'adult' mouse. These data define crucial in vivo functions for thyroid hormones through a T3R alpha pathway during post-natal development.