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. 1996;6(3):169-84.

Cross-talk between thyroid hormone and specific retinoid X receptor subtypes in yeast selectively regulates cognate ligand actions

P G Walfish  1 , Y F Yang, T Ypganathan, L A Chang, T R Butt
Affiliations

Cross-talk between thyroid hormone and specific retinoid X receptor subtypes in yeast selectively regulates cognate ligand actions

P G Walfish et al. Gene Expr. 1996.

Abstract

Thyroid (T3) hormone beta1 (TR) and 9-cis retinoic acid (9c-RA) retinoid X receptors (RXR) can form heterodimer complexes that bind to hormone response elements (HREs) in target genes to either activate or repress transcription. However, the action of each cognate ligand and the accessory cellular factors that can differentially regulate the transcriptional responses of a heterodimer-DNA complex are not well understood. Studies in most mammalian cell lines have demonstrated that 9c-RA cannot bind or transactivate TR/RXR-T3 response element (TRE) complexes. In contrast, when identical heterodimer complexes were coexpressed in the yeast (Saccharomyces cerevisiae) with single copy typical TREs [i.e., DR+4 (direct repeat), F2 (everted repeat), or PAL (inverted repeat) DNA response elements] we observed that i) unliganded TRbeta1 homodimers had constitutive action on F2 and PAL but not DR4 TREs; ii) TRbeta1 homodimer responsivity to T3 ligand was relatively weak (less than twofold) and was only demonstrable on F2 but not PAL or DR4-TREs, whereas TRbeta1 heterodimers responded to T3 when RXRgamma but not RXR alpha was the heterodimeric partner; iii) RXR responsivity to 9c-RA (three- to sixfold) could be demonstrated only on palindromic TREs that could be enhanced by TRbeta1 on all TREs; iv) T3 + 9c-RA ligands increased (additively or synergistically) transactivation when RXRgamma but not alpha heterodimerized with TRbeta1 on both typical as well as atypical (DR1, DR3, DR5, and F2M) TREs. Substitutions for wild-type TRbeta1 of C-terminus mutants deficient in dimerization with RXRs abrogated the anticipated single and dual cognate ligand-induced effects on TRbeta1/RXRgamma transactivation of DR4 TREs, whereas mutants with preserved dimerization function but impaired T3 transactivation regions could maintain an enhanced 9c-RA response but were devoid of the anticipated T3 and dual (T3 + 9c-RA) cognate ligand-induced effects. Thus, the ligand-inducible response of TR and RXR homodimers expressed in yeast are relatively weak but can be further enhanced by TRbeta1 cross-talk with specific RXR subtypes in the presence of both cognate ligands.

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Figures

FIG. 1
FIG. 1
Transactivational responses in yeast of TRβ 1 and RXR receptors when coexpressed with consensus and natural T3 response elements. (A) Ligand-dependent transactivation of TREs by TR and RXRs expressed as homo- or heterodimers. Yeast triple transformants expressing TRβ 1, RXRs, alone or in combination, with either DR4, F2, or PAL lacZ reporter were grown in the presence of no ligand (open bar), 1 μM T3 (stripped bar), 1 μM 9c-RA (hatched bar), or 1 μM T3 + 9c-RA (solid bar). β-Galactosidase (β-GAL) assay was performed with yeast extract and the activity was expressed as Miller units/mg protein. The mean and SEs of three different experiments are shown. “Null” indicates the basal reporter activity using expression plasmids devoid of receptor. (B) Ligand-dependent transactivation of atypical TREs by TR and RXRs expressed as homo- or heterodimers. Reporter plasmids containing either DR1, DR3, F2M, or DR5 elements were cotransfected with TRβ 1 and/or RXRs in the presence of no ligand, T3, 9c-RA, or T3 + 9c-RA and similarly analyzed as in (A).
FIG. 2
FIG. 2
Electrophoretic mobility shift assays (EMSA) to determine DNA binding properties of recombinant TR and RXRs produced in yeast cells. (A) EMSA of radiolabeled TRE probes with TRβ 1 and RXRs. EMSA were performed using a labeled F2, F2M, or PAL (sequences given in Fig. 1B), DR1, DR3, DR4, and DR5 (sequences given in the Materials and Methods section) in the presence of TRβ 1 or RXR(α or γ), alone or in combination as indicated. “Null” represents the basal DNA binding activity of vector devoid of a nuclear receptor. (B) Binding specificity of DNA-receptor complex. EMSA was performed with a labeled DR4 probe and yeast extracts containing TRβ 1/RXRs heterodimers. The panel shows specific and supershifted complexes of TRβ 1/RXRα heterodimer with polyclonal antibodies of TRβ (Ab-TRβ) or RXRα (Ab-RXRα) as indicated. The supershifted complex is indicated by an arrow. “Control” indicates no antibody was added to the binding reaction, and “preimmune” represents serum collected prior to immunization. (C) Western blot analysis of recombinant receptors. Yeast extracts prepared in the presence or absence of cognate ligands containing homo- or heterodimeric receptors were blotted where indicated with anti-TR or RXRs antibody as described in the Materials and Methods section.
FIG. 3
FIG. 3
Allosteric regulation of dual cognate transcriptional enhancement by RXR subtype-specific heterodimers. Ligand-dependent transactivation of TRβ 1/RXR(α or γ) heterodimers on a DR4 TRE reporter plasmid. TRβ 1 coexpressed with RXRα or γ were analyzed for transcriptional responses in the presence of varying concentrations of T3 and/or 9c-RA. The left panels for each figure refer to TRβ 1/RXRα and the right panels to TRβ 1/RXRγ heterodimer responses. The upper panels refer to the transactivation responses to serial concentrations of 9c-RA in the absence (▪) or presence (▴) of a fixed saturating concentration (10–6 M) of T3, whereas the lower panels show the transactivation responses to serial concentrations of T3 in the absence (▾) or presence (♦) of a fixed saturating concentration (10–6 M) of 9c-RA.
FIG. 4
FIG. 4
Structural functional characteristics of C-terminus mutants of TRβ 1 with various LBD C-terminus mutants. (A) Transactivation properties of TRβ 1 and C-terminus mutants compared to wild-type when coexpressed with a F2 reporter plasmid in the absence of ligand (open bar) or presence of T3 (striped bar). “Null” indicates the basal reporter activity using expression plasmids devoid of receptors. (B) DNA binding properties of the four C-terminus mutants of TRβ 1 mutants when coexpressed with RXRs. EMSA was performed using a labeled DR4 or F2 probe with LBD mutants coexpressed with RXRs as indicated.
FIG. 5
FIG. 5
Substitution of cis terminal TRβ 1 for wild-type inhibits single and dual cognate ligand action on heterodimers. (A) The transactivation properties of wild-type TRβ 1 compared to TRβ 1 LBD C-terminus mutants RXR heterodimers RXRs in the presence of DR4 and F2 TREs. LBD TRβ 1 mutants coexpressed with RXRα or RXRγ were analyzed in the presence of no ligand, T3, 9c-RA, or T3 + 9c-RA on β-GAL reporter plasmids. (B) Transactivation properties of wild-type compared to mutant TRβ 1/RXRγ heterodimer in the presence of β-GAL reporter plasmids containing either DR1, DR5, DR3, or F2M HREs in the presence of no ligand, T3, 9c-RA, or T3 + 9c-RA. (C) Effects of C-terminus TRβ 1 mutant D300A compared to T455 on dual cognate-dependent transcriptional enhancement when coexpressed with a β-GAL DR4 reporter. The transactivational responses for 9c-RA alone (▪) and in the presence of fixed concentration (10–6 M) of T3 (▴) are shown for the D300A/ RXRγ heterodimer (upper panels) and for T455/RXRγ heterodimers (lower panels).
FIG. 5
FIG. 5
Substitution of cis terminal TRβ 1 for wild-type inhibits single and dual cognate ligand action on heterodimers. (A) The transactivation properties of wild-type TRβ 1 compared to TRβ 1 LBD C-terminus mutants RXR heterodimers RXRs in the presence of DR4 and F2 TREs. LBD TRβ 1 mutants coexpressed with RXRα or RXRγ were analyzed in the presence of no ligand, T3, 9c-RA, or T3 + 9c-RA on β-GAL reporter plasmids. (B) Transactivation properties of wild-type compared to mutant TRβ 1/RXRγ heterodimer in the presence of β-GAL reporter plasmids containing either DR1, DR5, DR3, or F2M HREs in the presence of no ligand, T3, 9c-RA, or T3 + 9c-RA. (C) Effects of C-terminus TRβ 1 mutant D300A compared to T455 on dual cognate-dependent transcriptional enhancement when coexpressed with a β-GAL DR4 reporter. The transactivational responses for 9c-RA alone (▪) and in the presence of fixed concentration (10–6 M) of T3 (▴) are shown for the D300A/ RXRγ heterodimer (upper panels) and for T455/RXRγ heterodimers (lower panels).
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