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.     

DNA binding and dimerization determinants for thyroid hormone receptor alpha and its interaction with a nuclear protein.

The gel retardation assay was used to analyze the role of the thyroid hormone receptor alpha (TR alpha) ligand-binding domain (LBD) in controlling receptor interaction with a thyroid hormone responsive element (TRE). While wild type receptor TR alpha binds to the TRE mainly as monomer, deletion of 85 amino acids from its C-terminus results in a mutant receptor with enhanced DNA binding that forms several slow mobility complexes as revealed by gel retardation assay. Receptor deletion mutants that lack most of the LBD show significantly elevated DNA binding and are still able to bind to DNA as two complexes. Thus, the C-terminal end of TR alpha appears to interfere with the dimerization/oligomerization function and DNA binding of TR alpha. All C-terminal deletion mutants have lost their T3-responsive activator function, but some show constitutive activity. Nuclear factor from several cell lines, including CV-1, F9, and GC cells, interacts with TR alpha receptor to form a larger molecular weight complex as determined by gel retardation assay. This factor could not be detected in HeLatk- cells, where TR alpha does not activate a TRE-containing reporter gene. The nuclear factor is heat sensitive and does not bind to TRE itself but can interact with TR alpha in the absence of DNA. Deletion analysis demonstrates that the leucine zipper-like sequence located in the LBD of TR alpha is involved in this interaction. Together, our data suggest that TR alpha contains a dimerization function outside the LBD which is inhibited by the carboxy-terminal region, while the leucine zipper-like sequence in the LBD is required for interaction with a nuclear factor.     

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.