Glucocorticoid Modulatory Element Research Articles

Crystal structure and nuclear magnetic resonance analyses of the SAND domain from glucocorticoid modulatory element binding protein-1 reveals deoxyribonucleic acid and zinc binding regions.

The glucocorticoid-modulatory element-binding proteins, GMEB1 and GMEB2, are ubiquitous, multifunctional DNA-binding proteins with important roles in the modulation of transcription upon steroid hormone activation. The GMEB proteins have intrinsic transactivation ability, but also control the glucocorticoid response via direct binding to the glucocorticoid receptor. They are also mandatory host proteins for Parvovirus replication. Here we present the 1.55 A resolution crystal structure of a central portion of GMEB1, encompassing its SAND domain, which shares 80% sequence identity with the GMEB2 SAND domain. We demonstrate that this domain, also present in numerous proteins implicated in chromatin-associated transcriptional regulation, is necessary and sufficient to bind the glucocorticoid-modulatory element (GME) DNA target. We use nuclear magnetic resonance (NMR) and binding studies to map the DNA recognition surface to an alpha-helical region exposing the conserved KDWK motif. Using site-directed mutagenesis, key residues for DNA binding are identified. In contrast to the previously determined NMR structure of the Sp100b SAND domain, we find that the GMEB1 SAND domain also comprises a zinc-binding motif. Although the zinc ion is not necessary for DNA binding, it is found to determine the C-terminal conformation of the GMEB1 SAND domain. We also show that homologous zinc-binding motifs exist in a subset of SAND domain proteins and probe the roles of this novel motif.

New Glucocorticoid Receptor Partner?

Ubc9 is a human protein homolog of a yeast protein that is an E2 ubiquitin-conjugating enzyme. Ubc9 can promote conjugation of SUMO-1 (small ubiquitin-like modifier-1) to its various binding partners and thereby modifiy their function. Kaul et al. present evidence for a new role of Ubc9--regulation of transcriptional activation by the glucocorticoid receptor (GR). The authors found Ubc9 in a yeast two-hybrid screen for proteins that interact with the glucocorticoid modulatory element binding factors 1 and 2 (GMEB-1 and GMEB-2). GMEBs bind to DNA, glucocorticoid receptors, and coactivators and can inhibit transcriptional activity of the GR and decrease sensitivity of the receptor to agonists. Ubc9 also interacted in mammalian two-hybrid or pull-down assays with the GR itself. In transfected cells, Ubc9 increased transcriptional activity of GR and, when GR was expressed in large amounts, Ubc9 enhanced sensitivity of the GR to agonists and also modulated the response to GR antagonists. Unexpectedly, these effects did not require the sumoylation activity of Ubc9 and were even observed in the absence of glucocorticoid modulatory element. Assuming similar effects are mediated by endogenous Ubc9, the results indicate that Ubc9 may be a transcriptional adapter that helps control transcriptional activation by steroid receptors. S. Kaul, J. A. Blackford Jr., S. Cho, S. S. Simons Jr., Ubc9 is a novel modulator of the induction properties of glucocorticoid receptors. J. Biol. Chem. 277 , 12541-12549 (2002). [Abstract] [Full Text]

Ubc9 Is a Novel Modulator of the Induction Properties of Glucocorticoid Receptors

The EC(50) of agonists and the partial agonist activity of antagonists are crucial parameters for steroid hormone control of gene expression and endocrine therapies. These parameters have been shown to be modulated by a naturally occurring cis-acting element, called the glucocorticoid modulatory element (GME) that binds two proteins, GMEB-1 and -2. We now present evidence that the GMEBs contact Ubc9, which is the mammalian homolog of a yeast E2 ubiquitin-conjugating enzyme. Ubc9 also binds to glucocorticoid receptors (GRs). Ubc9 displays no intrinsic transactivation activity but modifies both the absolute amount of induced gene product and the fold induction by GR. With high concentrations of GR, added Ubc9 also reduces the EC(50) of agonists and increases the partial agonist activity of antagonists in a manner that is independent of the ability of Ubc9 to transfer SUMO-1 (small ubiquitin-like modifier-1) to proteins. This new activity of Ubc9 requires only the ligand binding domain of GR and part of the hinge region. Interestingly, Ubc9 modulation of full-length GR transcriptional properties can be seen in the absence of a GME. This, though, is consistent with the GME acting by increasing the local concentration of Ubc9, which then activates a previously unobserved target in the transcriptional machinery. With high concentrations of Ubc9 and GR, Ubc9 binding to GR appears to be sufficient to permit Ubc9 to act independently of the GME.

Evidence for a Common Step in Three Different Processes for Modulating the Kinetic Properties of Glucocorticoid Receptor-induced Gene Transcription

The dose-response curve of steroid hormones and the associated EC(50) value are critical parameters both in the development of new pharmacologically active compounds and in the endocrine therapy of various disease states. We have recently described three different variables that can reposition the dose-response curve of agonist-bound glucocorticoid receptors (GRs): a 21-base pair sequence of the rat tyrosine aminotransferase gene called a glucocorticoid modulatory element (GME), GR concentration, and coactivator concentration. At the same time, each of these three components was found to influence the partial agonist activity of antiglucocorticoids. In an effort to determine whether these three processes proceed via independent pathways or a common intermediate, we have examined several mechanistic details. The effects of increasing concentrations of both GR and the coactivator TIF2 are found to be saturable. Furthermore, saturating levels of either GR or TIF2 inhibit the ability of each protein, and the GME, to affect further changes in the dose-response curve or partial agonist activity of antisteroids. This competitive inhibition suggests that all three modulators proceed through a common step involving a titratable factor. Support for this hypothesis comes from the observation that a fragment of the coactivator TIF2 retaining intrinsic transactivation activity is a dominant negative inhibitor of each component (GME, GR, and coactivator). This inhibition was not due to nonspecific effects on the general transcription machinery as the VP16 transactivation domain was inactive. The viral protein E1A also prevented the action of each of the three components in a manner that was independent of E1A's ability to block the histone acetyltransferase activity of CBP. Collectively, these results suggest that three different inputs (GME, GR, and coactivator) for perturbing the dose-response curve, and partial agonist activity, of GR-steroid complexes act by converging at a single step that involves a limiting factor prior to transcription initiation.

Properties of the glucocorticoid modulatory element binding proteins GMEB-1 and -2: potential new modifiers of glucocorticoid receptor transactivation and members of the family of KDWK proteins.

An important component of glucocorticoid steroid induction of tyrosine aminotransferase (TAT) gene expression is the glucocorticoid modulatory element (GME), which is located at -3.6 kb of the rat TAT gene. The GME both mediates a greater sensitivity to hormone, due to a left shift in the dose-response curve of agonists, and increases the partial agonist activity of antiglucocorticoids. These properties of the GME are intimately related to the binding of a heteromeric complex of two proteins (GMEB-1 and -2). We previously cloned the rat GMEB-2 as a 67-kDa protein. We now report the cloning of the other member of the GME binding complex, the 88-kDa human GMEB-1, and various properties of both proteins. GMEB-1 and -2 each possess an intrinsic transactivation activity in mammalian one-hybrid assays, consistent with our proposed model in which they modify glucocorticoid receptor (GR)-regulated gene induction. This hypothesis is supported by interactions between GR and both GMEB-1 and -2 in mammalian two-hybrid and in pull-down assays. Furthermore, overexpression of GMEB-1 and -2, either alone or in combination, results in a reversible right shift in the dose-response curve, and decreased agonist activity of antisteroids, as expected from the squelching of other limiting factors. Additional mechanistic details that are compatible with the model of GME action are suggested by the interactions in a two-hybrid assay of both GMEBs with CREB-binding protein (CBP) and the absence of histone acetyl transferase (HAT) activity in both proteins. GMEB-1 and -2 share a sequence of 90 amino acids that is 80% identical. This region also displays homology to several other proteins containing a core sequence of KDWK. Thus, the GMEBs may be members of a new family of factors with interesting transcriptional properties.

Properties of the Glucocorticoid Modulatory Element Binding Proteins GMEB-1 and -2: Potential New Modifiers of Glucocorticoid Receptor Transactivation and Members of the Family of KDWK Proteins

An important component of glucocorticoid steroid induction of tyrosine aminotransferase (TAT) gene expression is the glucocorticoid modulatory element (GME), which is located at −3.6 kb of the rat TAT gene. The GME both mediates a greater sensitivity to hormone, due to a left shift in the dose-response curve of agonists, and increases the partial agonist activity of antiglucocorticoids. These properties of the GME are intimately related to the binding of a heteromeric complex of two proteins (GMEB-1 and -2). We previously cloned the rat GMEB-2 as a 67-kDa protein. We now report the cloning of the other member of the GME binding complex, the 88-kDa human GMEB-1, and various properties of both proteins. GMEB-1 and -2 each possess an intrinsic transactivation activity in mammalian one-hybrid assays, consistent with our proposed model in which they modify glucocorticoid receptor (GR)-regulated gene induction. This hypothesis is supported by interactions between GR and both GMEB-1 and -2 in mammalian two-hybrid and in pull-down assays. Furthermore, overexpression of GMEB-1 and -2, either alone or in combination, results in a reversible right shift in the dose-response curve, and decreased agonist activity of antisteroids, as expected from the squelching of other limiting factors. Additional mechanistic details that are compatible with the model of GME action are suggested by the interactions in a two-hybrid assay of both GMEBs with CREB-binding protein (CBP) and the absence of histone acetyl transferase (HAT) activity in both proteins. GMEB-1 and -2 share a sequence of 90 amino acids that is 80% identical. This region also displays homology to several other proteins containing a core sequence of KDWK. Thus, the GMEBs may be members of a new family of factors with interesting transcriptional properties.

Ability of the glucocorticoid modulatory element to modify glucocorticoid receptor transactivation indicates parallel pathways for the expression of glucocorticoid modulatory element and glucocorticoid response element activities

The glucocorticoid modulatory element (GME) of the rat tyrosine aminotransferase gene is located at −3.6 kb and 1 kb upstream of the glucocorticoid response elements (GREs). The GME has the unique transcriptional properties of modulating both the dose–response curve of agonists bound to the glucocorticoid receptor (GR) and the residual agonist activity of GR-bound antisteroids. The expression of GME activity involves the binding of two novel proteins (GMEB-1 and GMEB-2) that we have recently cloned. However, the mechanistic details are limited. The DNA sequence requirements for GME activity (CGTC) also remain poorly defined, which restricts efforts to identify other GME modulated genes. To help understand the mechanism for the unusual activities of the GME and to identify permissive gene environments for GME activity, we compared the changes in GME activity and GRE action (i.e. the fold induction by GR) caused by modifying several parameters. Phasing between the GME and downstream tandem GREs was unimportant, in contrast to other cis-acting elements like the GRE, while GME activity decreased rapidly when placed at increasingly larger distances 3′ to a tandem GRE. A minimal promoter was less effective in supporting GME than GRE activity. Although CREB binds to the GME, overexpression of CREB reduced GRE, but not GME, activity and a CRE was inactive when substituted for the GME. No effect of the GME was observed on the binding of GRs to a single GRE. However, the GME upstream of a single GRE was also unable to produce a left shift in the Dex dose–response curve under conditions where the GME was active with two GREs. In the absence of any GREs, the GME displayed intrinsic activity by elevating basal level expression. Collectively, these results indicate that an optimal position for a functional GME is within 250 bp upstream of a tandem GRE driving a complex promoter. Furthermore, as the changes in GME activity did not correlate with those for fold induction from the GRE, the mechanisms for expression of GME and GRE activities appear to utilize parallel, as opposed to common pathways.

Cloning and characterization of hGMEB1, a novel glucocorticoid modulatory element binding protein

A 21-bp element called glucocorticoid modulatory element (GME) modulates the glucocorticoid receptor-mediated responses via the binding of an as yet poorly characterized trans-acting complex of proteins containing the 88-kDa GMEB1 and the 67-kDa GMEB2. Using heat shock protein 27 (HSP27) as bait in the yeast two-hybrid assay, we cloned a 1.83-kb cDNA encoding a novel 573-amino acid protein called human GMEB1 (hGMEB1). hGMEB1 possesses a KDWK domain, contains sequences almost identical (36/38) to three tryptic peptides of rat GMEB1 and shares 38% identity with rat GMEB2. hGMEB1 is ubiquitously expressed as a 85-kDa protein in all cell lines and tissues examined. In vitro translated hGMEB1 bound specifically to GME oligonucleotides yielding a complex of similar size to the complex obtained using rat liver nuclear extracts. Both complexes were supershifted with an antibody specific to hGMEB1. Co-immunoprecipitation experiments confirmed the in vivo interaction of HSP27 with hGMEB1.

Modulation of TAT gene induction by glucocorticoids involves a neutralizing sequence

Recent studies have indicated that two elements in addition to the glucocorticoid response element (GRE) are involved in the induction of the endogenous TAT gene in Fu5-5 rat hepatoma cells. The first is the 21 bp glucocorticoid modulatory element (GME) at −3648 bp, which causes reporter constructs to display both a left shift in the dose–response curve for glucocorticoids and increased percentages of agonist activity for antiglucocorticoids. The second is a negative element at −3340 to −3050 that blocks the action of the GME. This last observation raised the question of how GME activity can be expressed in Fu5-5 cells in the intact TAT gene that contains both the GME and the negative element. The present study identifies a third element, a “neutralizing” sequence, that restores the activity of the GME even when otherwise inactivated by the negative element. This neutralizing sequence was located within the region surrounding the GREs of the TAT gene but is separate from the GREs. The activity of the individual GME and negative elements was found to depend upon spacing. However, in combination with the natural GRE, the native TAT gene spacing of the GME and negative elements was able to reproduce the activity of the intact gene. Thus, a total of three additional elements (an activator, a negative element, and a neutralizer) appear to cooperate with the GREs in glucocorticoid induction of the TAT gene in Fu5-5 cells. While such a grouping of elements may be novel among steroid regulated genes, it is a not uncommon occurrence for the transcriptional control of other genes.

Cloning and Characterization of a Novel Binding Factor (GMEB-2) of the Glucocorticoid Modulatory Element

The 21-base pair glucocorticoid modulatory element (GME) of the rat tyrosine aminotransferase gene is the only cis-acting element known to modulate the transcriptional activity of receptors bound to glucocorticoid response elements. Specifically, the GME increases the activity of complexes bound both by physiological concentrations of glucocorticoids, due to a left shift in the dose-response curve, and by saturating concentrations of anti-glucocorticoids. For this reason, the nuclear protein(s) that has been demonstrated to bind to the GME is of major interest as a possible transcription factor with hitherto undescribed properties. Subsequent studies indicated that not one but two proteins of 88 and 67 kDa (= GMEB-1 and -2, respectively) formed a heteromeric complex with double-stranded GME oligonucleotides in gel shift assays and participated in the expression of GME activity (Oshima, H., Szapary, D., and Simons, S. S., Jr. (1995) J. Biol. Chem. 270, 21893-21910). Here, we report the use of polymerase chain reaction of degenerate oligonucleotides and 5'- and 3'-rapid amplification of cDNA ends to clone two cDNAs of 2. 0 and 1.9 kilobase pairs that probably result from alternative splicing. Both cDNAs encoded open reading frames containing all four previously sequenced peptides. The longer 2.0-kilobase pair cDNA encoded an open reading frame for an acidic, 529-amino acid protein and afforded a major 67-kDa and a minor 58-kDa protein after in vitro transcription/translation. Both proteins were recognized by a mono-epitopic antibody raised against a peptide of GMEB-2. The in vitro translated protein bound to GME DNA in gel shift assays. However, the binding to GME DNA increased markedly after mixing with authentic GMEB-1 to give a gel-shifted complex that was similar to that derived from HTC cell cytosol. GMEB-2 shares a unique domain (KDWKR) with proteins derived from diverse organisms as follows: Drosophila (DEAF-I), rat (Suppressin), and Caenorhabditis elegans (three unknown open reading frames). Collectively, these data suggest that the 67-kDa GMEB-2 not only is an important factor for the modulation of glucocorticoid receptor bound to glucocorticoid response elements but also may belong to a novel family of transcription factors.

A negative tyrosine aminotransferase gene element that blocks glucocorticoid modulatory element-regulated modulation of glucocorticoid-induced gene expression.

Tyrosine aminotransferase (TAT) is the prototypic steroid-inducible gene. Recently, we have found that the modulation of TAT induction properties is reproduced by a novel cis-acting TAT gene element, the glucocorticoid modulatory element (GME). This GME lies about 1 kb upstream of the glucocorticoid response elements (GREs) of the TAT gene and binds a heterooligomer of two recently defined proteins. We now report the existence of an additional TAT gene element between the GME and the GREs that blocks the action of the GME and thus prevents the left shift in the glucocorticoid dose-response curve caused by the GME. This negative element has the properties of a silencer because its activity is relatively position- and orientation-independent. The interaction appears to be stoichiometric in that the effects of a single negative element can be overcome by a second GME. This negative element also has an intrinsic inhibitory activity in the absence of the GME. The majority of the negative element activity could be elicited by a 56-bp sequence between -3105 and -3050 bp of the TAT gene. Multiple, clustered mutations of this sequence reduced, but did not eliminate, the negative activity. Further efforts to restrict the negative element were unsuccessful, suggesting that multiple sequences are required for full activity. High affinity, sequence-specific binding of a trans-acting factor(s) was observed in gel shift assays. This binding was half-maximally competed by a 4.4-fold excess of nonradioactive probe and was very stable once formed (delta H [symbol: see text] dissoc. = 32 kcal/mol), suggesting that low concentrations of a high affinity binding protein(s) exist in nuclear extracts. Further support for this conclusion came from the observation that cotransfection of a plasmid containing multiple copies of the 56-bp negative element was able to relieve the negation of GME activity in a GME-56-bp-GRE reporter construct. These data directly support the role of a trans-acting factor(s) in binding to the 56-bp negative element and blocking GME activity. Collectively, these data suggest that glucocorticoid induction of TAT gene expression is subject to multiple levels of control by several new cis-acting elements and thus is much more complex than previously appreciated.

Sequence-selective interactions of transcription factor elements with tandem glucocorticoid-responsive elements at physiological steroid concentrations.

Synergism in transcription is said to occur when the combined response from two DNA elements for the binding of trans-acting factors is greater than the sum of the responses from each element in isolation. The synergism of steroid receptors with themselves or with other trans-acting factors at saturating concentrations of steroid has proved to be an important component of steroid-regulated gene transcription. We have recently described a glucocorticoid modulatory element (GME) of the rat tyrosine aminotransferase gene that, in conjunction with a trans-acting factor, modulates the transcriptional activity of receptor-glucocorticoid and -antiglucocorticoid complexes with homologous and heterologous genes and promoters (Oshima, H., and Simons, S. S., Jr. (1992) Mol. Endocrinol. 6, 416-428). We now report that, under certain circumstances, the GME displays synergistic activity with a glucocorticoid-responsive element (GRE). However, several properties of GME action are different from those previously observed for synergism. The effects of the GME were marked at subsaturating or physiological concentrations of glucocorticoids but insignificant at saturating concentrations, which are the established conditions for synergism. The GME was found to increase the agonist activity of partial antiglucocorticoids, while synergism involving antisteroids has yet to be reported. Furthermore, the GRE was active in conjunction with two tandem repeats of a GRE, which was a combination that did not support conventional synergism. Most importantly, the effects of the GME were greater than with any other trans-acting factor binding element tested, indicative of a sequence-selective activity. The efficacy of the GME was also insensitive to the spacing between elements. Thus, the GME provides a mechanism for selective transcriptional modulation by physiological concentrations of steroid, and by antisteroids, of a common class of genes that are under the control of two or more GREs.

Modulation of transcription factor activity by a distant steroid modulatory element.

Variations in the biological activity of antisteroids, as determined by their percent agonist activity, is a well known but poorly understood phenomenon. For example, in tyrosine aminotransferase (TAT) induction by the antiglucocorticoid dexamethasone 21-mesylate in rat hepatoma tissue culture cells, the percent agonist activity varies with the density of cultured cells. A 21-basepair sequence of the rat TAT gene has now been isolated which confers all of the induction properties of the endogenous TAT gene to homologous and heterologous promoters and genes. We call this 21-basepair sequence, which acts in concert with a trans-acting factor identified by gel shift experiments, a glucocorticoid modulatory element. The changes in induction properties were found to be independent of the fold induction by dexamethasone, thus arguing that the GME does not synergize with the glucocorticoid response element. A model incorporating this new element is advanced which can explain the observed variations of TAT induction and may be generally applicable for the mechanism of action of other steroid hormones.