Differential capacity of wild type promoter elements for binding and trans-activation by retinoic acid and thyroid hormone receptors.

Retinoic acid receptor (RAR) and thyroid hormone receptor (T3R) are structurally similar and can bind as homodimers or T3R-RAR heterodimers to a single synthetic DNA response element. The interaction of these two types of receptors with wild type elements, however, has not been systematically investigated. Promoter elements from genes regulated by retinoic acid (RA) or thyroid hormone (T3) were tested for response to T3 and RA in transient transfections in both JEG and COS cells. The elements were classified as primarily responsive to RA or to T3 or responsive to both ligands. Binding of highly purified RAR alpha and T3R alpha to the various elements was assessed using the gel shift assay. Those elements predominantly responsive to one ligand showed preferential binding to the appropriate receptor. A series of point mutations were introduced into the rat GH T3 response element to further define sequence requirements for response to both RA and T3. Down-mutations in any of the three hexamers (previously demonstrated to be required for full response to T3 and full binding of T3R) also decreased RA induction and RAR binding. However, only one of two sets of up-mutations for T3 response also increased RA induction, demonstrating differences in hexamer preference between RAR and T3R. Variation in spacing of the three hexamers did not influence RA vs. T3 induction or RAR vs. T3R binding according to the predictions of a simple hexamer spacing model. There was a strong correlation between the extent of T3R dimer binding and strength of T3 induction for a subset of elements studied in JEG cells (r = 0.97, P < 0.01) and a weaker but significant correlation in COS cells (r = 0.65, P < 0.05)). In contrast, RAR dimer binding by the wild type elements did not quantitatively correlate with RA induction in either JEG (r = 0.13, P > 0.05) or COS cells (r = 0.21, P > 0.05). These results suggests that RAR interacts with a heterodimer partner(s) which influences binding site specificity, whereas T3R heterodimer partner(s) is less likely to alter binding site recognition. The observed difference in COS and JEG cells as well as the weak T3R binding-function relationship of the malic enzyme element, however, suggest that the influence of T3R heterodimer partner(s) on binding site specificity is likely to vary with cell type and the specific element tested.     

1α,25-Dihydroxyvitamin D3 receptor as a mediator of transrepression of retinoid signaling

The receptors for retinoic acid (RA) and for 1α,25-dihydroxyvitamin D₃ (VD), RAR, RXR, and VDR are ligand-inducible members of the nuclear receptor superfamily. These receptors mediate their regulatory effects by binding as dimeric complexes to response elements located in regulatory regions of hormone target genes. Sequence scanning of the tumor necrosis factor-α type I receptor (TNFαRI) gene identified a 3′ enhancer region composed of two directly repeated hexameric core motifs spaced by 2 nucleotides (DR2). On this novel DR2-type sequence, but not on a DR5-type RA response element, VD was shown to act through its receptor, the vitamin D receptor (VDR), as a repressor of retinoid signalling. The repression appears to be mediated by competitive protein–protein interactions between VDR, RAR, RXR, and possibly their cofactors. This VDR-mediated transrepression of retinoid signaling suggests a novel mechanism for the complex regulatory interaction between retinoids and VD. J. Cell. Biochem. 67:287–296, 1997. © 1997 Wiley-Liss, Inc.     

Dimerization interfaces formed between the DNA binding domains determine the cooperative binding of RXR/RAR and RXR/TR heterodimers to DR5 and DR4 elements.

We have previously reported that the binding site repertoires of heterodimers formed between retinoid X receptor (RXR) and either retinoic acid receptor (RAR) or thyroid hormone receptor (TR) bound to response elements consisting of directly repeated PuG(G/T)TCA motifs spaced by 1-5 bp [direct repeat (DR) elements 1-5] are highly similar to those of their corresponding DNA binding domains (DBDs). We have now mapped the dimerization surfaces located in the DBDs of RXR, RAR and TR, which are responsible for cooperative interaction on DR4 (RXR and TR) and DR5 (RXR and RAR). The D-box of the C-terminal CII finger of RXR provides one of the surfaces which is specifically required for the formation of the heterodimerization interfaces on both DR4 and DR5. Heterodimerization with the RXR DBD on DR5 specifically requires the tip of the RAR CI finger as the complementary surface, while a 7 amino acid sequence encompassing the 'prefinger region', but not the TR CI finger, is specifically required for efficient dimerization of TR and RXR DBDs on DR4. Importantly, DBD swapping experiments demonstrate not only that the binding site repertoires of the full-length receptors are dictated by those of their DBDs, but also that the formation of distinct dimerization interfaces between the DBDs are the critical determinants for cooperative DNA binding of these receptors to specific DRs.     

Half-site spacing and orientation determines whether thyroid hormone and retinoic acid receptors and related factors bind to DNA response elements as monomers, homodimers, or heterodimers.

The receptors for thyroid hormone (T3R) and retinoic acid (RAR) are members of a nuclear receptor subfamily that are capable of recognizing similar DNA sequences. Native response elements for T3R and RAR consist of two or more putative half-site binding motifs organized as imperfect direct or inverted repeats separated by different sized nucleotide gaps. To clarify how T3R, RAR, and related factors recognize DNA response elements, we analyzed the interaction of purified receptors with a series of inverted and direct repeats of an idealized AGGTCA half-site separated by different sized nucleotide gaps. Our results indicate that RAR and T3R can bind to half-sites as monomers and, depending on the orientation and distance between half-sites, also bind as homodimers or T3R-RAR heterodimers. T3R also binds to certain DNA elements as a heterodimer with one or more nuclear factors from eucaryotic cells. Thus, the orientation and spacing of half-sites play a central role in determining which configuration of receptors and nuclear factors will interact with a specific DNA element. This along with the ability of these factors to participate in reversible protein-protein interactions serve to broaden and diversify the responses mediated by T3R, RAR, and related members of this nuclear receptor subfamily.     

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 dimerization interfaces formed between the DNA binding domains of RXR, RAR and TR determine the binding specificity and polarity of the full-length receptors to direct repeats.

Heterodimers of retinoid X receptor (RXR) and retinoic acid receptor (RAR) bind preferentially to directly repeated elements with spacing of two (DR2) or five (DR5) base pairs, due to the specific heterocooperative interaction of their DNA binding domains (DBDs) on these elements. We have demonstrated in the accompanying paper that the heterodimeric DBD interface that is responsible for the cooperative binding to DR5 elements, specifically involves the D-box of the RXR CII finger and the tip of the RAR CI finger. We show here that a second type of dimerization interface, which specifically implicates the RAR T-box and the RXR CII finger to the exclusion of the D-box, determines the selective binding to DR2 elements. Interestingly, the same type of dimerization interface (RXR T-box and CII finger) is responsible for the cooperative binding of homodimers of the RXR DBD to DR1 elements. Based on the three-dimensional structure of the glucocorticoid receptor DBD, modeling of RXR/RAR, RXR/TR and RXR/RXR DBD cooperative interactions predicts that in all cases the DBD contributing the CII finger, i.e. that of RXR, has to be positioned 5' to its cooperatively bound partner. This binding polarity of the DBDs is conferred upon the full-length receptors, since crosslinking experiments indicate that RXR is always 5' to RAR in complexes between either DR5 or DR2 and RXR/RAR heterodimers. The possible significance of these observations for transactivation by retinoic acid receptors is discussed.