A functional glucocorticoid-responsive unit composed of two overlapping inactive receptor-binding sites: evidence for formation of a receptor tetramer.

Abstract

An unusual glucocorticoid-responsive element (called GRE A) was found to mediate the induction of the cytosolic aspartate aminotransferase gene by glucocorticoids and was bound by the glucocorticoid receptor in a DNase I footprinting assay. GRE A consists of two overlapping GREs, each comprising a conserved half-site and an imperfect half-site. The complete unit was able to confer glucocorticoid inducibility to a heterologous promoter (AMTV-CAT). Mutation of any of the half-sites, including the imperfect ones, abolished inducibility by the hormone, demonstrating that each of the isolated GREs was inactive. In electrophoretic mobility shift assays, purified rat liver glucocorticoid receptor (GR) formed a low-mobility complex with GRE A, presumably containing a GR tetramer. When purified bacterially expressed DBD was used, low-mobility complexes as well as dimer and monomer complexes were formed. In inactive mutated oligonucleotides, no GR tetramer formation was detected. Modification of the imperfect half-sites in order to increase their affinity for GR gave a DNA sequence that bound a GR tetramer in a highly cooperative manner. This activated unit consisting of two overlapping consensus GREs mediated glucocorticoid induction with a higher efficiency than consensus GRE.Steroid hormones regulate gene expression by interacting with intracellular receptors which, in turn, bind to specific DNA sequences and modulate the transcription of the target genes (6, 16). These receptors belong to a family of proteins that includes ligand-dependent transcriptional regulators as well as proteins for which no endogenous ligands have been identified (30). On the basis of the primary structure of these proteins and the sequence of the DNA-responsive elements, two classes of receptors have been identified: (i) receptors for estrogens, thyroid hormones, vitamin D, and retinoic acid as well as several orphan receptors (11) and (ii) receptors for glucocorticoids, mineralocorticoids, progestins, and androgens (6).During the last few years, a considerable amount of work has been devoted to identifying the DNA sequences recognized by these receptors. A consensus (half-site) sequence has been found for each class of receptor: TGACC for the estrogen receptor/thyroid hormone receptor subfamily and TGTTCT for the glucocorticoid receptor (GR) subfamily (2,4,39). It is believed that each of these sequence motifs binds one receptor molecule. Since receptors optimally bind to DNA as dimers, efficient hormone-responsive elements consist of two of these conserved sequence motifs (or half-sites) (36).The arrangement of these half-sites is at least as critical for efficient binding as the DNA sequence itself. In the estrogenresponsive element (ERE), half-sites are arranged as inverted repeats separated by 3 bp (25). The analysis of several promoters regulated by other receptors of this subfamily suggests that responsive elements are not frequently arranged as inverted repeats. In fact, several receptors of this subfamily, i.e., thyroid

Contribution of a Nuclear Factor 1 Binding Site to the Glucocorticoid Regulation of the Cytosolic Aspartate Aminotransferase Gene Promoter

“…The glucocorticoid effect is even more striking in the presence of cAMP, which is known to potentiate the induction of cAspAT by glucocorticoids (12). This fragment includes a glucocorticoid-responsive sequence (called GRE A) that we have previously shown to be functional when inserted into a heterologous promoter (13). The data suggest that the half-palindromic GRE present at Ϫ380 is not sufficient to confer glucocorticoid regulation.…”

“…Supershifted complexes are indicated by a star to the right. unique structure composed of two overlapping imperfect GREs; it binds two dimers of the glucocorticoid receptor in a highly cooperative manner (13).…”

“…Two regions of the cAspAT gene promoter contribute to the glucocorticoid regulation: a distal one (Ϫ1.983/Ϫ1.718 kilobases) which also carries the negative regulation by insulin, and a proximal one (Ϫ553/ Ϫ318 base pairs) (12). The latter one contains an unusual glucocorticoid-responsive sequence consisting of two overlapping GREs that can bind two receptor dimers in a highly cooperative manner (13). Because of this unusual structure and because of the complexity of the cAspAT gene promoter, it was of interest to search for other factors that can cooperate with the glucocorticoid receptor.…”

Induction of the Upstream Regulatory Region of Human Papillomavirus Type 31 by Dexamethasone Is Differentiation Dependent

“…A functional GRE is most commonly arranged as a 15-bp incomplete palindrome separated by a 3-bp spacer (Fig. 7A) (6,42 GREs (18), directly repeating half sites (13), and variable spacing of half sites (44). Furthermore, studies have shown that the rotational positioning of a GRE in a nucleosome is critical for its accessibility (28) and that GR binding to the GRE induces DNA bending (37).…”

Syndromes of glucocorticoid and mineralocorticoid resistance

“…Recent data suggest that GR and MR interact with hormone-responsive elements not only in the form of homodimers. Indeed, binding of GR tetramers has been demonstrated on the aspartate aminotransferase promoter (47), and there is evidence for heterodimerization of MR and GR leading to either synergy or inhibition of transactivation depending on the promoter and cellular context (20,48,49).…”