Polarity and specific sequence requirements of peroxisome proliferator-activated receptor (PPAR)/retinoid X receptor heterodimer binding to DNA. A functional analysis of the malic enzyme gene PPAR response element

The malic enzyme (ME) gene is a target for both thyroid hormone receptors and peroxisome proliferator-activated receptors (PPAR). Within the ME promoter, two direct repeat (DR)-1-like elements, MEp and MEd, have been identified as putative PPAR response elements (PPRE). We demonstrate that only MEp and not MEd is able to bind PPAR/retinoid X receptor (RXR) heterodimers and mediate peroxisome proliferator signaling. Taking advantage of the close sequence resemblance of MEp and MEd, we have identified crucial determinants of a PPRE. Using reciprocal mutation analyses of these two elements, we show the preference for adenine as the spacing nucleotide between the two half-sites of the PPRE and demonstrate the importance of the two first bases flanking the core DR1 in 5'. This latter feature of the PPRE lead us to consider the polarity of the PPAR/RXR heterodimer bound to its cognate element. We demonstrate that, in contrast to the polarity of RXR/TR and RXR/RAR bound to DR4 and DR5 elements respectively, PPAR binds to the 5' extended half-site of the response element, while RXR occupies the 3' half-site. Consistent with this polarity is our finding that formation and binding of the PPAR/RXR heterodimer requires an intact hinge T region in RXR while its integrity is not required for binding of the RXR/TR heterodimer to a DR4.     

Heterodimerization preferences of thyroid hormone receptor alpha isoforms

Thyroid hormone receptors (TRs) are members of the steroid hormone receptor superfamily and are encoded by two different genes, alpha and beta. Three isoforms (alpha 1, alpha 2, and alpha 3) are created by alternative splicing of the TR alpha gene. In TR alpha 2 and alpha 3, the distal half of the putative dimerization domain is disrupted and the carboxy terminus of the protein is substituted with different amino acids. To evaluate the properties of these alterations in the dimerization region, DNA binding and dimerization of TR alpha isoforms were studied by electrophoretic mobility shift assays. TR alpha 1 formed a monomer or a homodimer on certain thyroid hormone responsive elements (TREs), whereas TR alpha 2 and alpha 3 did not bind effectively to any of the TREs studied. TR alpha 1 formed a heterodimer with 9-cis retinoic acid receptor alpha (RXR alpha) on all TREs studied. Although TR alpha 2 did not bind as a homodimer, it did bind as a heterodimer with RXR alpha to DR4 and MHC-TRE. TR alpha 3 bound as a heterodimer to a broader repertoire of TREs, including DR4, MHC, ME, and F2-TRE. These results indicate that the alterations in the dimerization region in TR alpha 2 and alpha 3 abrogated homodimer binding, but differentially affected heterodimerization with RXR alpha on various TREs.     

Cell type-dependent modulation of the dominant negative action of human mutant thyroid hormone beta 1 receptors

BACKGROUND: Mutations in the ligand-binding domain of the thyroid hormone receptor beta (TR beta) gene cause the syndrome of resistance to thyroid hormone (RTH). The clinical phenotype results from the antagonism of the normal TR alpha and the non-mutated TR beta alleles by the TR beta 1 mutants, via a dominant negative effect. There is, however, marked heterogeneity of organ resistance within and among kindreds with RTH. This study examines the potential role of cell type in modulating the dominant negative potency of human TR beta 1 (h-TR beta 1) mutants. MATERIALS AND METHODS: Transient transfections were performed in HeLa and NIH3T3 cells, using a wild type (WT) and three naturally occurring mutant h-TR beta 1 constructs, and three natural thyroid hormone response elements (TREs). Immunocytochemistry was performed to detect levels of TR beta 1 expression in these two cell types. In order to determine how TR beta 1 interacts with other cellular partners, gel-shift analyses using HeLa and NIH3T3 nuclear extracts were performed. RESULTS: Transfection studies using WT h-TR beta 1 in HeLa and NIH3T3 cells, showed that the 3,3',5-triiodothyronine (T3)-induced transactivation of the different TREs varied between cell types. Unlike the non-T3-binding h-TR beta 1 mutant, PV, mutants ED and OK displayed the expected T3-induced dose responsiveness in these two cell types. For each TRE examined, the magnitude of the dominant negative effect varied between the cell types. The levels of receptor expression in HeLa and NIH3T3 cells were identical, as determined by immunocytochemistry. Gel-shift analyses showed differences in the formation of hetero- and homodimers depending on both the cell type and TRE motif. CONCLUSIONS: The cell type in which a mutant receptor operates affects the relative amounts of hetero- and homodimers. Together with the nature of the mutation and the TRE-motif, this could modulate the dominant negative action of mutant receptors in different tissues, which, in turn, could contribute to the variable phenotypic characteristics of RTH.     

Effects of purified bovine whey factors on cellular immune functions in ruminants

Retinoid X receptors (RXRs) form heterodimers with thyroid hormone receptors (TRs). RXRs increase DNA binding affinity of TRs and T3-mediated transactivation on positive T3 response elements (TREs). However, the role of RXRs on negative TREs, and the relation of RXRs to the dominant negative effect of mutant TRs, are not defined. To clarify the function of RXRs on negative TREs, we performed transient cotransfection studies using the rat glycoprotein hormone alpha promoter fused to luciferase gene (alphaLuc), and human TRH promoter fused to luciferase gene (TRH-Luc) as reporters. We found that the JEG-3 cell-alphaLuc system was very sensitive to TR regulation. Using TRbeta1 wild-type (WT) expression vector, 6.2 ng/well (170 ng/10 cm dish), and 0.2 ng/well (11 ng/10 cm dish) caused maximal, and half maximal, inhibition of Luc activities in the presence of 1 nM T3. A T3 dose dependent inhibition study was also performed. From these studies, we determined that the appropriate conditions in which to study alphaLuc transactivation, in a linear portion of the dose response curve, was using 0.8 ng/well TRbeta1 expression vector and 0.1 nM T3. Under these conditions, TRbeta1 mutant R316H (GH), but not G345R (Mf), showed a weak dominant negative effect at a 1:1 ratio in the presence of 0.1 nM T3 although neither mutant had detectable T3 binding affinity. Moreover this dominant negative effect of R316H on the alphaLuc reporter was enhanced in the presence of RXRgamma. Mutant G345R showed a stronger dominant negative effect than did R316H when using a double palindromic TRE fused to herpes simplex thymidine kinase-Luc reporter as a positive TRE. These results conform to the clinical features of R316H which is associated with apparent pituitary resistance of thyroid hormone (PRTH). Mutant R316H also showed a weak dominant negative effect with TRH-Luc at a 1:1 ratio in the absence or presence of RXRgamma. However RXRgamma did not enhance the dominant negative effect as it did using alphaLuc reporter gene. Electrophoretic gel mobility shift assay (EMSA) showed that RXR alpha augmented the DNA binding affinity of wild type and R316H TRs as heterodimers on the previously reported negative TREs of glycoprotein hormone alpha promoter, suggesting that RXR does not produce its response by removing TRs from these TREs. RXR alpha augmented DNA binding affinity of TRbeta1WT, and R316H showed a weaker heterodimer band than did the wild type in EMSA. Using the TRH-Luc reporter, basal activity was increased by wild type TRbeta1. However a TRbeta1 DNA binding domain mutant, (C127S) which can not bind to DNA, did not increase the basal activity. This indicates that DNA binding of the TR is required for increasing basal activity of TRH promoter. These results indicate that (1) RXR-TR heterodimers play a role in basal transactivation and T3 suppression of negatively regulated genes, and (2) RXRs increase the dominant negative effect of some mutant TRs on specific negative TREs. (3) This effect occurs without removing TRs from the TRE. (4) The differential dominant negative effect of mutant R316H (negative TRE > positive TRE) may explain, at least in part, the presentation of R316H as PRTH. (5) Augmentation of basal activity by wild type TRs on a negative TRE requires DNA binding.     

T3 receptor suppression of Sp1-dependent transcription from the epidermal growth factor receptor promoter via overlapping DNA-binding sites

Expression of the human epidermal growth factor receptor (EGFR) gene is inhibited by ligand-activated thyroid hormone receptor (T3R). Binding sites for Sp1 and for the T3R.retinoid X receptor (RXR) complex overlap in a functional core of the EGFR promoter. Sp1 inhibited binding of the T3R complex to this 36-base pair (bp) EGFR element in vitro but did not affect binding of the T3R complex to a positive thyroid hormone response element (TRE). In Drosophila SL2 cells, which lack Sp1 and T3R, function of the EGFR promoter was strongly dependent on Sp1. Sp1-dependent promoter function was inhibited by ligand-activated T3R but not by mutant T3R defective in DNA or T3 binding. RXR increased the extent of inhibition. Sp1 enhanced activity of the 36-bp element placed 5' to a minimal TATA promoter and this enhancement was also repressed by T3R. Mutations in the 36-bp element were unable to separate Sp1 and T3R functions. However, addition of a second half-site 5' to the existing site in an inverted repeat configuration created a positive TRE. In the absence of ligand, T3R inhibited Sp1 stimulation from this altered element; addition of T3 reversed the inhibition. When a dimeric TRE is separated from Sp1-binding sites strong synergism was observed. The nature and location of the TRE thus strongly influence biological responses. A TRE site in the EGFR promoter that overlaps an Sp1-binding site inhibits Sp1 function but is unable to direct positive function.     

Thyroid hormone receptor binds with unique properties to response elements that contain hexamer domains in an inverted palindrome arrangement

Thyroid hormone receptor (T3R)-accessory protein heterodimers preferentially bind to thyroid hormone response elements (TREs), which contain hexamer domains arranged as a direct repeat separated by a 4-basepair gap (DR + 4). T3R homodimers, however, preferentially bind to elements that consist of an inverted palindrome. We now report on unique T3R binding patterns and functional characteristics of two such elements that mediate T3 regulation. We performed mutational analysis of the chicken lysozyme silencer F2 (LysF2) TRE and demonstrated that the two functional binding domains are arranged as an inverted palindrome separated by 6 basepairs. Both the LysF2 TRE and the similarly arranged myelin basic protein TRE bind T3R dimers at very low T3R concentrations. Despite the high relative affinity for T3R dimer binding, the T3 induction conferred by these elements is low compared to that of previously characterized TREs with a DR + 4 arrangement. The laminin-B1 gene element, previously shown to bind retinoic acid receptor, contains at least four hexameric binding domains. All of the domains can bind T3R simultaneously and are involved in conferring T3 induction, but bind with a different pattern than that reported for retinoic acid receptor. T3R homodimer binding to a series of mutant laminin-B1 elements and T3 induction were significantly correlated (r = 0.82; P < 0.05). T3R homodimer binding to LysF2 element mutants was not correlated with T3 induction (r = 0.32; P > 0.05); however, T3R-nuclear protein heterodimer binding was significantly correlated (r = 0.67; P < 0.05). T3R-nuclear protein heterodimers, but not homodimers, bound consistently to mutations of the LysF2 element that altered the gap between hexamers. The overall discordance between strong T3R binding to these elements and weak T3 induction indicates that the unusual hexamer arrangement places the T3R complex in an unfavorable configuration for maximal T3-dependent transactivation. The differential T3 sensitivity of generalized resistance to thyroid hormone-associated T3R mutants to the LysF2 element compared with the DR + 4 arrangement suggests that these unique features may have physiological significance.     

Absence of thyroid hormone receptor beta-retinoid X receptor interactions in auditory function and in the pituitary-thyroid axis

THYROID hormone receptor beta-deficient (TRbeta-/-) mice have defective auditory-evoked brain stem responses (ABR). Since in vitro, TRbeta binds to DNA as homodimers or as heterodimers with retinoid X receptors (RXRs), we investigated whether the TRbeta-/- phenotype may reflect loss of RXR-TRbeta heterodimer or TRbeta homodimer function. Normal ABR thresholds were recorded in RXRbeta-/-, RXRgamma-/-, RXRalpha-/+ and RXR compound mutant mice. When RXR mutations were introduced onto TRbeta-/+ or TRbeta-/- backgrounds, thresholds were dictated solely by TRbeta and not RXR genotype. TRbeta-/-mice also over-produce thyroid hormones and thyroid stimulating hormone; however, levels of these hormones were unaltered by RXR mutations. This suggests that, contrary to in vitro models, RXRs may be dispensable and that TRbeta may function in vivo by an RXR-independent mechanism in the auditory system and pituitary-thyroid axis.