Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors.

We report here the identification of thyroid hormone response elements (TREs) that consist of a direct repeat, not a palindrome, of the half-sites. Unlike palindromic TREs, direct repeat TREs do not confer a retinoic acid response. The tandem TRE can be converted into a retinoic acid response element by increasing the spacing between the half-sites by 1 nucleotide, and the resulting retinoic acid response element is no longer a TRE. Decreasing the half-site spacing by 1 nucleotide converts the TRE to a vitamin D3 response element, while eliminating response to T3. These results correlate well with DNA-binding affinities of the thyroid hormone, retinoic acid, and vitamin D3 receptors. This study points to the general importance of tandem repeat hormone response elements and suggests a simple physiologic code exists in which half-site spacing plays a critical role in achieving selective hormonal response.     

Specificity of a retinoic acid response element in the phosphoenolpyruvate carboxykinase gene promoter: consequences of both retinoic acid and thyroid hormone receptor binding.

The ability of a retinoic acid (RA) response element (RARE) in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter to mediate effects of either RA or thyroid hormone (T3) on gene expression was studied. Fusion gene constructs consisting of PEPCK promoter sequences ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were used for this analysis. While T3 induced CAT expression to a small degree (about twofold) when such constructs were transiently transfected into H4IIE rat hepatoma cells, along with an expression vector encoding the alpha subtype of the T3 receptor (TR), this effect was mediated by promoter sequences distinct from the PEPCK RARE. Although TRs were capable of binding the PEPCK RARE in the form of putative monomers, dimers, and heterodimers with RA receptors (RARs), this element failed to mediate any positive effect of T3 on gene expression. In contrast, the PEPCK RARE mediated six- to eightfold induction of CAT expression by RA. When TRs were coexpressed along with RARs in transfected H4IIE cells, this RA induction was substantially blunted in a T3-independent manner. This inhibitory effect may be due to the binding of nonfunctional TRs or TR-RAR heterodimers to the PEPCK RARE. A model is proposed to explain the previously observed in vivo effects of T3 on PEPCK gene expression.     

RXR alpha, a promiscuous partner of retinoic acid and thyroid hormone receptors.

Retinoic acid receptor (RAR), thyroid hormone receptor (T3R) and vitamin D3 receptor (VD3R) differ from steroid hormone receptors in that they bind and transactivate through responsive elements organized as direct rather than inverted repeats. We now show that recombinant RAR and T3R are monomers in solution and cannot form stable homodimeric complexes on their responsive elements. Stable binding of the receptors to their responsive elements requires heterodimerization with a nuclear factor. This auxiliary factor is tightly associated with RAR and T3R in the absence of DNA and co-purifies with both receptors. As demonstrated by extensive purification, the same auxiliary factor is required for stable DNA binding of RAR as for that of T3R; the factor also facilitates the formation of a stable VD3R-DNA complex. The auxiliary factor is identical to the retinoid X receptor alpha (RXR alpha) by biochemical and functional criteria. The identification of RXR alpha as a dimerization partner for the RARs, T3Rs and VD3R has important implications as to the function of these receptors and their ligands in development, homeostasis and neoplasia.     

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.     

DNA binding of thyroid hormone receptors.

Thyroid hormone receptors, isolated from rat liver nuclei, bind to purified DNA. By contrast, free triiodothyronine and plasma proteins which bind thyroid hormone do not associate with DNA. Thus, the nuclear localization of thyroid hormone in target tissues may be explained by the association of its receptors with DNA.     

Capacity for cooperative binding of thyroid hormone (T3) receptor dimers defines wild type T3 response elements.

Thyroid hormone response elements (T3REs) have been identified in a variety of promoters including those directing expression of rat GH (rGH), alpha-myosin heavy chain (rMHC), and malic enzyme (rME). A detailed biochemical and genetic analysis of the rGH element has shown that it consists of three hexamers related to the consensus [(A/G)GGT(C/A)A]. We have extended this analysis to the rMHC and rME elements. Binding of highly purified thyroid hormone receptor (T3R) to T3REs was determined using the gel shift assay, and thyroid hormone (T3) induction was measured in transient tranfections. We show that the wild type version of each of the three elements binds T3R dimers cooperatively. Mutational analysis of the rMHC and rME elements identified domains important for binding T3R dimers and allowed a direct determination of the relationship between T3R binding and function. In each element two hexamers are required for dimer binding, and mutations that interfere with dimer formation significantly reduce T3 induction. Similar to the rGH element, the rMHC T3RE contains three hexameric domains arranged as a direct repeat followed by an inverted copy, although the third domain is weaker than in rGH. All three are required for full function and T3R binding. The rME T3RE is a two-hexamer direct repeat T3RE, which also binds T3R monomer and dimer. Across a series of mutant elements, there was a strong correlation between dimer binding in vitro and function in vivo for rMHC (r = 0.99, P less than 0.01) and rME (r = 0.67, P less than 0.05) T3REs. Our results demonstrate a similar pattern of T3R dimer binding to a diverse array of hexameric sequences and arrangements in three wild type T3REs. Addition of nuclear protein enhanced T3R binding but did not alter the specificity of binding to wild type or mutant elements. Binding of purified T3R to T3REs was highly correlated with function, both with and without the addition of nuclear protein. T3R dimer formation is the common feature which defines the capacity of these elements to confer T3 induction.     

Ligand-dependent conformational changes in thyroid hormone and retinoic acid receptors are potentially enhanced by heterodimerization with retinoic X receptor

Recently, many lines of evidence have been accumulated indicating that thyroid hormone receptor (TR) and retinoic acid receptor (RAR) undergo a ligand-dependent conformation change. Since most of these results were obtained by either gel-shift assay or circular dichroism spectroscopic studies, it was not clear which part of the receptor bore the major conformational change. Moreover, it is not clear whether the formation of heterodimer between TR or RAR and retinoic X receptor (RXR) has any effects on this structural change. Utilizing partial proteolytic analysis, we demonstrated that thyroid hormone and retinoic acid induce a specific protease-resistant conformation to their cognate receptors. Studies of various deletion mutants reveal that the entire ligand binding domain of these receptors is involved in this change, and suggest that ligand may induce a more compact structure in its binding domain. Evidence from native gel electrophoresis supports this notion. This conformational change occurs in the absence of DNA and occurs indenpendently of other domains in the receptor. Heterodimerization between TR or RAR and the RXR has little effect on receptor conformation in the absence of hormone but does enhance the ligand-dependent structural change. Interestingly, dual hormone treatment, i.e. thyroid hormone and 9-cis RA, intensifies this enhancement. We suggest that the observed protease-resistant conformation may introduce a different configuration to the receptor and therefore may effect the receptor in various ways, but most likely is involved in converting the receptor from a negative regulator to a positive activator.     

Functional characterization of the rat growth hormone promoter elements required for induction by thyroid hormone with and without a co-transfected beta type thyroid hormone receptor

We have extensively characterized the sequences of the rat growth hormone (rGH) promoter required for induction by T3 (thyroid hormone, 3,5,3'-L-triiodothyronine) in a transient transfection system. Oligonucleotides containing portions of the rGH promoter sequence with various deletions and point mutations were placed upstream of the first 137 base pairs of the rGH promoter or the heterologous herpes virus thymidine kinase promoter in chloramphenicol acetyltransferase expression vectors. The rGH137 and thymidine kinase promoters show no or minimal response to T3 in the basal state. The constructs were tested in GH4C1 rat pituitary cells and COS cells (functionally deficient in thyroid hormone receptor) with and without a co-transfected plasmid expressing a beta type c-erbA gene coding for a functional T3 receptor. Oligonucleotides containing the T3 receptor binding site confer hormone-dependent induction in a manner that is independent of either orientation or variation in position on the helix relative to the promoter. Point mutations in the sequence -189 to -173 result in loss of T3 induction, and bases between -173 and -167 were also required for a full T3 response. The minimal length to confer T3 induction to the rGH promoter was 23 base pairs (-190 to -167). Point mutations creating a perfect duplication of 7 base pairs within the receptor binding site conferred 12-fold T3 response to the rGH137 promoter, 3-fold greater than the wild type rGH237 construct. T3 inductibility was also transferred to the thymidine kinase promoter by an oligonucleotide containing the sequence -200 to -157, demonstrating that cell type specific elements located 3' to 157 of the rGH promoter are not required for thyroid hormone responsiveness.     

An assay for thyroid hormone binding to chromatin receptors

I have measured the interaction of T₃ with highly soluble, expanded, rat liver chromatin using a new assay for the study of hormone binding to nucleoprotein. Bound hormone and free hormone were rapidly and quantitatively separated by the adsorption of the hormone-nucleoprotein complex onto hydroxylapatite. This procedure satisfies several criteria for a successful binding assay: (1) The binding capacity is stable throughout the time required to reach equilibrium, (2) the ratio of specific to nonspecific binding (signal/noise) is at least 20:1, (3) large numbers of samples can be handled easily, (4) the amount of bound hormone is directly proportional to the quantity of chromatin employed, (5) the hormone and its analogs display a range of affinities for the binding site, and (6) the binding occurs to a limited number of sites, over a free hormone concentration range which is similar to the hormone concentrations found in vivo.     

The binding of thyroid hormone receptors to DNA

The behaviour of tri-iodothyronine (T3)- and thyroxine (T4)-receptor complexes when bound to native DNA-cellulose is reported. Equal and large proportions of both T3- and T4-receptor complexes bind to DNA but although T3-receptor complexes are 99% recoverable by 0.5 M NaCl buffer elution, only 60-70% of the T4-receptor complexes are regained. The balance appears as free T4, apparently released as the T4-receptor complexes bind to the DNA whilst the corresponding receptor remains bound. This effect is independent of T4-receptor complex/DNA ratio up to ca. 4 fmol/micrograms DNA, of the presence of an equal amount of unoccupied receptor and of an eight-fold concentration range of both T4-receptor complex and DNA at a fixed ratio, in the cellulose matrix. Pre-formed receptor-DNA material, likewise, only accepts some 60% of the expected quantity of T4 whereas the capacity for T3 appears to be similar to that of free receptors.