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.     

Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently.

We have purified and cloned a HeLa cell nuclear protein that strongly stimulates binding of retinoic acid and thyroid hormone receptors (RARs and TRs) to response elements. The purified protein is a human retinoid X receptor beta (hRXR beta). Three murine members of the RXR family (mRXR alpha, beta, and gamma) have also been cloned, and their interactions with RARs and TRs have been investigated. Under conditions where RAR, RXR, and TR bound poorly as homodimers to various response elements, strongly cooperative RAR-RXR and TR-RXR binding was observed. The binding efficiency was dependent on the sequence, relative orientation, and spacing of the repeated motifs of response elements. We show also that unstable RAR-RXR heterodimers were formed in solution, and that C-terminal sequences and the DNA-binding domains of both receptors were required for efficient formation of stable heterodimers on response elements. These findings suggest a convergence of the signaling pathways of some members of the nuclear receptor superfamily.     

The patterns of binding of RAR, RXR and TR homo- and heterodimers to direct repeats are dictated by the binding specificites of the DNA binding domains.

We show here that, in addition to generating an increase in DNA binding efficiency, heterodimerization of retinoid X receptor (RXR) with either retinoic acid receptor (RAR) or thyroid hormone receptor (TR) alters the binding site repertoires of RAR, RXR and TR homodimers. The binding site specificities of both homo- and heterodimers appear to be largely determined by their DNA binding domains (DBDs), and are dictated by (i) homocooperative DNA binding of the RXR DBD, (ii) heterocooperative DNA binding of RXR/RAR and RXR/TR DBDs, and (iii) steric hindrance. No homodimerization domain exists in the DBDs of TR and RAR. The dimerization function which is located in the ligand binding domain further stabilizes, but in general does not change, the repertoire dictated by the corresponding DBD(s). The binding repertoire can be further modified by the actual sequence of the binding site. We also provide evidence supporting the view that the cooperative binding of the RXR/RAR and RXR/TR DBDs to directly repeated elements is anisotropic, with interactions between the dimerization interfaces occurring only with RXR bound to the 5' located motif. This polarity, which appears to be maintained in the full-length receptor heterodimers, may constitute a novel parameter in promoter-specific transactivation.