DNA binding specificity of mutant glucocorticoid receptor DNA-binding domains

Mutation of a small number of amino acids in the DNA-binding domain of the estrogen receptor to the corresponding sequence of the glucocorticoid receptor switches the specificity of the receptor in transactivation assays (Mader, S., Kumar, V., de Verneuil, H., and Chambon, P. (1989) Nature 338, 271-274). We have made the corresponding reciprocal mutations in the context of the glucocorticoid receptor DNA-binding domain and studied the binding of wild type and mutant purified proteins to palindromic glucocorticoid and estrogen response elements as well as to elements of intermediate sequence, using gel mobility shift assays. We show here that a protein with two altered amino acids binds glucocorticoid and estrogen response elements with a low but equal affinity, whereas a protein with an additional changed residue has a high affinity for estrogen response elements but still retains a considerable affinity for glucocorticoid response elements. Using binding sites of intermediate sequence we have further characterized the interaction with DNA. The in vitro DNA binding results are confirmed by in vivo transactivation assays in yeast. Finally we suggest a testable model for amino acid/base pair interactions involved in recognition by the glucocorticoid receptor DNA-binding domain of its target sequence.     

Determinants of target gene specificity for ROR alpha 1: monomeric DNA binding by an orphan nuclear receptor.

The ROR alpha isoforms are orphan members of the steroid/thyroid/retinoid receptor superfamily. Previous DNA-binding studies indicated that ROR alpha isoforms bind to response elements consisting of a single copy of the core recognition sequence AGGTCA preceded by a 6-bp A/T-rich sequence and that the distinct amino-terminal domains of each isoform influence DNA-binding specificity. In this report, we have investigated in detail the protein determinants of target gene specificity for the ROR alpha 1 isoform and have now identified the minimal sequence both in its amino- and carboxy-terminal domains required for high-affinity DNA binding. High-resolution methylation and ethylation interference analyses and mixing of truncated proteins in a DNA-binding assay show that ROR alpha 1 presumably binds along one face of the DNA helix as a monomer. By analogy to previous studies of the orphan receptors NGFI-B and FTZ-F1, extensive mutational analysis of the ROR alpha 1 protein shows that a domain extending from the carboxy-terminal end of the second conserved zinc-binding motif is required for specific DNA recognition. However, point mutations and domain swap experiments between ROR alpha 1 and NGFI-B demonstrated that sequence-specific recognition dictated by the carboxy-terminal extension is determined by distinct subdomains in the two receptors. These results demonstrate that monomeric nuclear receptors utilize diverse mechanisms to achieve high-affinity and specific DNA binding and that ROR alpha 1 represents the prototype for a distinct subfamily of monomeric orphan nuclear receptors.     

DNA binding properties of the vitamin D3 receptor zinc finger region.

The DNA binding domains of the nuclear receptor superfamily are highly conserved and consist of residues that fold into two zinc finger-like motifs, suggesting that the structures of this region among the members of the superfamily are likely to be very similar. Furthermore, the response elements that these receptors bind to are similar in sequence and organization. Nevertheless, these receptors selectively recognize target response elements and differentially regulate linked genes. In order to study the details of receptor:DNA binding, we have overexpressed and purified the vitamin D3 receptor DNA binding domain (VDRF) and have begun characterizing its DNA binding properties. We find that the VDRF protein binds strongly and specifically to direct repeats constituting a vitamin D response element from the mouse osteopontin (Spp-1) promoter region but weakly to the human osteocalcin vitamin D response element. Unlike receptors that recognize hormone response elements oriented as inverted repeats, such as the glucocorticoid receptor (GR) and estrogen receptor, VDRF appears to bind half-sites noncooperatively, without the free energy contribution of dimerization seen when the glucocorticoid receptor DNA binding domain associates with a glucocorticoid response element. By comparing and contrasting the DNA binding properties of the vitamin D and glucocorticoid receptors, we suggest a model for how receptors that prefer direct repeats differ in their binding strategy from those that recognize inverted repeats.     

Determinants for DNA-binding site recognition by the glucocorticoid receptor.

The glucocorticoid receptor binds with high specificity to glucocorticoid response elements, discriminating them from other closely related binding sites. Three amino acids in the recognition alpha-helix of the DNA-binding domain of the receptor are primarily responsible for this specific DNA binding activity. In this study we analyze in detail how these residues determine the specific DNA binding by studying a series of mutant glucocorticoid receptor DNA-binding domains containing all combinations of glucocorticoid and estrogen receptor-specific residues at these positions. Statistical analysis of the results enables us to create models describing the association between amino acids and base pairs. Several strategies appear to be used in accomplishing discrimination between the glucocorticoid and estrogen response elements. Single residues (i.e., Val-443 in the glucocorticoid receptor and Glu-439 in the estrogen receptor) appear to form both positive contacts with specific base pairs in the cognate binding site and negative contacts in the non-cognate site. In the glucocorticoid receptor Ser-440 is pleiotropically negative for all sites tested but the negative effect is stronger for the estrogen response element thus contributing to binding site discrimination. Furthermore, combinations of amino acids appear to act synergistically, most often causing a reduction in binding to non-cognate sites.     

Crystal Structure of the Mineralocorticoid Receptor DNA Binding Domain in Complex with DNA

The steroid hormone receptors regulate important physiological functions such as reproduction, metabolism, immunity, and electrolyte balance. Mutations within steroid receptors result in endocrine disorders and can often drive cancer formation and progression. Despite the conserved three-dimensional structure shared among members of the steroid receptor family and their overlapping DNA binding preference, activation of individual steroid receptors drive unique effects on gene expression. Here, we present the first structure of the human mineralocorticoid receptor DNA binding domain, in complex with a canonical DNA response element. The overall structure is similar to the glucocorticoid receptor DNA binding domain, but small changes in the mode of DNA binding and lever arm conformation may begin to explain the differential effects on gene regulation by the mineralocorticoid and glucocorticoid receptors. In addition, we explore the structural effects of mineralocorticoid receptor DNA binding domain mutations found in type I pseudohypoaldosteronism and multiple types of cancer.     

Identification of androgen-selective androgen-response elements in the human aquaporin-5 and Rad9 genes

The AR (androgen receptor) is known to influence the expression of its target genes by binding to different sets of AREs (androgen-response elements) in the DNA. One set consists of the classical steroid-response elements which are partial palindromic repeats of the 5'-TGTTCT-3' steroid-receptor monomer-binding element. The second set contains motifs that are AR-specific and that are proposed to be partial direct repeats of the same motif. On the basis of this assumption, we used an in silico approach to identify new androgen-selective AREs in the regulatory regions of known androgen-responsive genes. We have used an extension of the NUBIScan algorithm to screen a collection of 85 known human androgen-responsive genes compiled from literature and database searches. We report the evaluation of the most promising hits resulting from this computational search by in vitro DNA-binding assays using full-size ARs and GRs (glucocorticoid receptors) as well as their isolated DBDs (DNA-binding domains). We also describe the ability of some of these motifs to confer androgen-, but not glucocorticoid-, responsiveness to reporter-gene expression. The elements found in the aquaporin-5 and the Rad9 (radiation-sensitive 9) genes showed selective AR versus GR binding in band-shift assays and a strong activity and selectivity in functional assays, both as isolated elements and in their original contexts. Our data indicate the validity of the hypothesis that selective AREs are recognizable as direct 5'-TGTTCT-3' repeats, and extend the list of currently known selective elements.     

The stringency and magnitude of androgen-specific gene activation are combinatorial functions of receptor and nonreceptor binding site sequences.

The mechanism by which specific hormonal regulation of gene expression is attained in vivo is a paradox in that several of the steroid receptors recognize the same DNA element in vitro. We have characterized a complex enhancer of the mouse sex-limited protein (Slp) gene that is activated exclusively by androgens but not by glucocorticoids in transfection. Potent androgen induction requires both the consensus hormone response element (HRE) and auxiliary elements residing within the 120-bp DNA fragment C' delta 9. Multiple nonreceptor factors are involved in androgen specificity, with respect to both the elevation of androgen receptor activity and the inactivity of glucocorticoid receptor (GR), since clustered base changes at any of several sites reduce or abolish androgen induction and do not increase glucocorticoid response. However, moving the HRE as little as 10 bases away from the rest of the enhancer allows GR to function, suggesting that GR is repressed by juxtaposition to particular factors within the androgen-specific complex. Surprisingly, some sequence variations of the HRE itself, within the context of C' delta 9, alter the stringency of specificity, as well as the magnitude, of hormonal response. These HRE sequence effects on expression correspond in a qualitative manner with receptor binding, i.e., GR shows a threefold difference in affinities for HREs amongst which androgen receptor does not discriminate. Altering the HRE orientation within the enhancer also affects hormonal stringency, increasing glucocorticoid but not androgen response. The effect of these subtle variations suggests that they alter receptor position with respect to other factors. Thus, protein-protein interactions that elicit specific gene regulation are established by the array of DNA elements in a complex enhancer and can be modulated by sequence variations within these elements that may influence selection of precise protein contacts.     

Creation of "super" glucocorticoid receptors by point mutations in the steroid binding domain

Almost all modifications of the steroid binding domain of glucocorticoid receptors are known to cause a reduction or loss of steroid binding activity. Nonetheless, we now report that mutations of cysteine 656 of the rat receptor, which was previously suspected to be a crucial amino acid for the binding process, have produced "super" receptors. These receptors displayed an increased affinity for glucocorticoid steroids and a decreased relative affinity for cross-reacting steroids such as progesterone and aldosterone. The increased in vitro affinity of the super receptors was maintained in a whole cell bioassay. These results indicate that additional modifications of the glucocorticoid receptor, and probably the other steroid receptors, may further increase the binding affinity and/or specificity.