Myocyte-specific enhancer factor 2 and thyroid hormone receptor associate and synergistically activate the alpha-cardiac myosin heavy-chain gene.

The muscle-specific regulatory region of the alpha-cardiac myosin heavy-chain (MHC) gene contains the thyroid hormone response element (TRE) and two A/T-rich DNA sequences, designated A/T1 and A/T2, the putative myocyte-specific enhancer factor 2 (MEF2) binding sites. We investigated the roles of the TRE and MEF2 binding sites and the potential interaction between thyroid hormone receptor (TR) and MEF2 proteins regulating the alpha-MHC promoter. Deletion mutation analysis indicated that both the A/T2 motif and TRE were required for muscle-specific expression of the alpha-MHC gene. The alpha-MHC enhancer containing both the A/T2 motif and TRE was synergistically activated by coexpression of MEF2 and TR in nonmuscle cells, whereas neither factor by itself activated the alpha-MHC reporters. The reporter construct containing the A/T2 sequence and the TRE linked to a heterologous promoter also showed synergistic activation by coexpression of MEF2 and TR in nonmuscle cells. Moreover, protein binding assays demonstrated that MEF2 and TR specifically bound to one another in vitro and in vivo. The MADS domain of MEF2 and the DNA-binding domain of TR were necessary and sufficient to mediate their physical interaction. Our results suggest that the members of the MADS family (MEF2) and steroid receptor superfamily (TR) interact with one another to synergistically activate the alpha-cardiac MHC gene expression.     

Specificity of a retinoic acid response element in the phosphoenolpyruvate carboxykinase gene promoter: consequences of both retinoic acid and thyroid hormone receptor binding.

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

Thyroid hormone receptors form distinct nuclear protein-dependent and independent complexes with a thyroid hormone response element

We have examined the binding of nuclear proteins and recombinant thyroid hormone receptors (TRs) to the palindromic thyroid hormone responsive element AGGTCATGACCT (TREp) using a gel electrophoretic mobility shift assay. Four specific protein-DNA complexes were detected after incubation of nuclear extracts (NE) from T3-responsive pituitary (GH3) cells with a TREp-containing DNA fragment. This was compared with the TREp binding of reticulocyte lysate-synthesized TRs. TR alpha 1 and TR beta 2 each formed a single major TR:TREp complex which comigrated with the least retarded complex formed by GH3 NE, while TR beta 1 formed multiple complexes suggesting that it can bind to TREp as an oligomer. Interestingly, coincubation of 35S-TR alpha 1, GH3 NE, and unlabeled TREp resulted in not only the 35S-TR:TREp complex, but in two additional more greatly retarded complexes containing 35S-TR alpha 1 and comigrating with those formed by GH3 extract alone. Incubation of each of the TRs with NE from COS-7 cells, which do not possess sufficient endogenous TRs to mediate T3-responses, resulted in formation of a new, more greatly shifted complex. A similar, heat labile activity which altered mobility of the TR:TRE complex was also present in NE from T3-unresponsive JEG-3 cells. At high concentration of NE, all of the TR bound to TREp was more greatly retarded than in the absence of NE. Truncation of TR alpha 1 at amino acid 210 prevented additional complex formation in the presence of NE without affecting DNA binding, suggesting that the carboxyl-terminus of the TRs is essential for interaction with nuclear proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thyroid hormone alters the DNA binding properties of chicken thyroid hormone receptors alpha and beta.

The effects of thyroid hormone agonists on thyroid hormone receptor (TR)/DNA complex formation was investigated to elucidate the mechanism by which TRs transactivate genes in response to ligand. The data, obtained from gel shift experiments, indicate that thyroid hormones alter the conformation of TRs bound to DNA, irrespective of if the element is occupied by monomeric TR, homodimeric TR/TR, or heterodimeric complexes with the retinoid receptors RAR or RXR. Furthermore, triiodo-thyronine (T3) prevents 2 TR molecules from binding to oligonucleotides containing direct repeats or inverted palindromes of the consensus AGGTCA motif, an effect that was not detected with palindromic elements. Heterodimers bound to direct repeats were less affected: RXR/TR were fully and RAR/TR complexes partially resistant to thyroid hormone. The data suggest that a ligand-induced conformational change in TR prevents double TR occupancy of a response element containing 2 direct repeats of the consensus binding motif, possibly by steric hindrance, whereas such an event does not prevent TR/RXR heterodimers from binding to DNA. Finally, our data show that a monomeric, liganded TR bound preferentially to the second half site in a AGGTCActcaAGGTCA element, and therefore indicate that nucleotides adjacent to the consensus half site contribute to binding specificity.     

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.