Kinetically Defined Mechanisms and Positions of Action of Two New Modulators of Glucocorticoid Receptor-regulated Gene Induction.

Most of the steps in, and many of the factors contributing to, glucocorticoid receptor (GR)-regulated gene induction are currently unknown. A competition assay, based on a validated chemical kinetic model of steroid hormone action, is now used to identify two new factors (BRD4 and negative elongation factor (NELF)-E) and to define their sites and mechanisms of action. BRD4 is a kinase involved in numerous initial steps of gene induction. Consistent with its complicated biochemistry, BRD4 is shown to alter both the maximal activity (Amax) and the steroid concentration required for half-maximal induction (EC50) of GR-mediated gene expression by acting at a minimum of three different kinetically defined steps. The action at two of these steps is dependent on BRD4 concentration, whereas the third step requires the association of BRD4 with P-TEFb. BRD4 is also found to bind to NELF-E, a component of the NELF complex. Unexpectedly, NELF-E modifies GR induction in a manner that is independent of the NELF complex. Several of the kinetically defined steps of BRD4 in this study are proposed to be related to its known biochemical actions. However, novel actions of BRD4 and of NELF-E in GR-controlled gene induction have been uncovered. The model-based competition assay is also unique in being able to order, for the first time, the sites of action of the various reaction components: GR < Cdk9 < BRD4 ≤ induced gene < NELF-E. This ability to order factor actions will assist efforts to reduce the side effects of steroid treatments.

Disruption of Ttll5/stamp gene (tubulin tyrosine ligase-like protein 5/SRC-1 and TIF2-associated modulatory protein gene) in male mice causes sperm malformation and infertility.

TTLL5/STAMP (tubulin tyrosine ligase-like family member 5) has multiple activities in cells. TTLL5 is one of 13 TTLLs, has polyglutamylation activity, augments the activity of p160 coactivators (SRC-1 and TIF2) in glucocorticoid receptor-regulated gene induction and repression, and displays steroid-independent growth activity with several cell types. To examine TTLL5/STAMP functions in whole animals, mice were prepared with an internal deletion that eliminated several activities of the Stamp gene. This mutation causes both reduced levels of STAMP mRNA and C-terminal truncation of STAMP protein. Homozygous targeted mutant (Stamp(tm/tm)) mice appear normal except for marked decreases in male fertility associated with defects in progressive sperm motility. Abnormal axonemal structures with loss of tubulin doublets occur in most Stamp(tm/tm) sperm tails in conjunction with substantial reduction in α-tubulin polyglutamylation, which closely correlates with the reduction in mutant STAMP mRNA. The axonemes in other structures appear unaffected. There is no obvious change in the organs for sperm development of WT versus Stamp(tm/tm) males despite the levels of WT STAMP mRNA in testes being 20-fold higher than in any other organ examined. This defect in male fertility is unrelated to other Ttll genes or 24 genes previously identified as important for sperm function. Thus, STAMP appears to participate in a unique, tissue-selective TTLL-mediated pathway for α-tubulin polyglutamylation that is required for sperm maturation and motility and may be relevant for male fertility.

Identification of location and kinetically defined mechanism of cofactors and reporter genes in the cascade of steroid-regulated transactivation.

A currently obscure area of steroid hormone action is where the component factors, including receptor and reporter gene, act. The DNA binding of factors can be precisely defined, but the location and timing of factor binding and action are usually not equivalent. These questions are addressed for several factors (e.g. glucocorticoid receptor (GR), reporter, TIF2, NCoR, NELF-A, sSMRT, and STAMP) using our recently developed competition assay. This assay reveals both the kinetically defined mechanism of factor action and where the above factors act relative to both each other and the equilibrium equivalent to the rate-limiting step, which we call the concentration limiting step (CLS). The utility of this competition assay would be greatly increased if the position of the CLS is invariant and if the factor acting at the CLS is known. Here we report that the exogenous GREtkLUC reporter acts at the CLS as an accelerator for gene induction by GRs in U2OS cells. This mechanism of reporter function at the CLS persists with different reporters, factors, receptors, and cell types. We, therefore, propose that the reporter gene always acts at the CLS during gene induction and constitutes a landmark around which one can order the actions of all other factors. Current data suggest that how and where GR and the short form of SMRT act is also constant. These results validate a novel and rational methodology for identifying distally acting factors that would be attractive targets for pharmaceutical intervention in the treatment of diseases involving GR-regulated genes.

A theoretical framework for gene induction and experimental comparisons.

Ligand-mediated gene induction by steroid receptors is a multistep process characterized by a dose-response curve for gene product that follows a first-order Hill equation. This behavior has classically been explained by steroid binding to receptor being the rate-limiting step. However, this predicts a constant potency of gene induction (EC(50)) for a given receptor-steroid complex, which is challenged by the findings that various cofactors/reagents can alter this parameter in a gene-specific manner. These properties put strong constraints on the mechanisms of gene induction and raise two questions: How can a first-order Hill dose-response curve (FHDC) arise from a multistep reaction sequence, and how do cofactors modify potency? Here we introduce a theoretical framework in which a sequence of steps yields an FHDC for the final product as a function of the initial agonist concentration. An exact determination of all constants is not required to describe the final FHDC. The theory predicts mechanisms for cofactor/reagent effects on gene-induction potency and maximal activity and it assigns a relative order to cofactors in the sequence of steps. The theory is supported by several observations from glucocorticoid receptor-mediated gene induction. It identifies the mechanism and matches the measured dose-response curves for different concentrations of the combination of cofactor Ubc9 and receptor. It also predicts that an FHDC cannot involve the DNA binding of preformed receptor dimers, which is validated experimentally. The theory is general and can be applied to any biochemical reaction that shows an FHDC.

STAMP alters the growth of transformed and ovarian cancer cells.

BACKGROUND: Steroid receptors play major roles in the development, differentiation, and homeostasis of normal and malignant tissue. STAMP is a novel coregulator that not only enhances the ability of p160 coactivator family members TIF2 and SRC-1 to increase gene induction by many of the classical steroid receptors but also modulates the potency (or EC50) of agonists and the partial agonist activity of antisteroids. These modulatory activities of STAMP are not limited to gene induction but are also observed for receptor-mediated gene repression. However, a physiological role for STAMP remains unclear. METHODS: The growth rate of HEK293 cells stably transfected with STAMP plasmid and overexpressing STAMP protein is found to be decreased. We therefore asked whether different STAMP levels might also contribute to the abnormal growth rates of cancer cells. Panels of different stage human cancers were screened for altered levels of STAMP mRNA. Those cancers with the greatest apparent changes in STAMP mRNA were pursued in cultured cancer cell lines. RESULTS: Higher levels of STAMP are shown to have the physiologically relevant function of reducing the growth of HEK293 cells but, unexpectedly, in a steroid-independent manner. STAMP expression was examined in eight human cancer panels. More extensive studies of ovarian cancers suggested the presence of higher levels of STAMP mRNA. Lowering STAMP mRNA levels with siRNAs alters the proliferation of several ovarian cancer tissue culture lines in a cell line-specific manner. This cell line-specific effect of STAMP is not unique and is also seen for the conventional effects of STAMP on glucocorticoid receptor-regulated gene transactivation. CONCLUSIONS: This study indicates that a physiological function of STAMP in several settings is to modify cell growth rates in a manner that can be independent of steroid hormones. Studies with eleven tissue culture cell lines of ovarian cancer revealed a cell line-dependent effect of reduced STAMP mRNA on cell growth rates. This cell-line dependency is also seen for STAMP effects on glucocorticoid receptor-mediated transactivation. These preliminary findings suggest that further studies of STAMP in ovarian cancer may yield insight into ovarian cancer proliferation and may be useful in the development of biomarker panels.

Glucocorticoid receptor ligand binding domain is sufficient for the modulation of glucocorticoid induction properties by homologous receptors, coactivator transcription intermediary factor 2, and Ubc9.

Several factors modulate the position of the dose-response curve of steroid receptor-agonist complexes and the partial agonist activity of antagonist complexes, thereby causing differential gene activation by circulating hormones and unequal gene repression during endocrine therapies with antisteroids. We now ask whether the modulatory activity of three factors (homologous receptor, coactivator transcription intermediary factor 2, and Ubc9) requires the same or different domains of glucocorticoid receptors (GRs). In all cases, we find that neither the amino terminal half of the receptor, which contains the activation function-1 activation domain, nor the DNA binding domain is required. This contrasts with the major role of activation function-1 in determining the amount of gene expression and partial agonist activity of antisteroids with most steroid receptors. However, the situation is more complicated with Ubc9, where GR N-terminal sequences prevent the actions of Ubc9, but not added GR or transcription intermediary factor 2, at low GR concentrations. Inhibition is relieved by deletion of these sequences or by replacement with the comparable region of progesterone receptors but not by overexpression of the repressive sequences. These results plus the binding of C-terminal GR sequences to the suppressive N-terminal domain implicate an intramolecular mechanism for the inhibition of Ubc9 actions at low GR concentrations. A shift from noncooperative to cooperative steroid binding at high GR concentrations suggests that conformational changes reposition the inhibitory N-terminal sequence to allow Ubc9 interaction with elements of the ligand binding domain. Collectively, these results indicate a dominant role of GR C-terminal sequences in the modulation of the dose-response curve and partial agonist activity of GR complexes. They also reveal mechanistic differences both among individual modulators and between the ability of the same factors to regulate the total amount of gene expression.

Equilibrium interactions of corepressors and coactivators with agonist and antagonist complexes of glucocorticoid receptors.

Corepressors and coactivators can modulate the dose-response curve and partial agonist activity of glucocorticoid receptors (GRs) complexed with agonist and antagonist steroids, respectively, in intact cells. We recently reported that GR-antagonist complexes bind to the coactivator TIF2, (transcriptional intermediary factor 2), which is consistent with the whole-cell effects of coactivators being mediated by direct interactions with GR complexes. We now ask whether the whole-cell modulatory activity of corepressors also entails binding to both GR-agonist and -antagonist complexes and whether the association of corepressors and coactivators with GR complexes involves competitive equilibrium reactions. In mammalian two-hybrid assays with two different cell lines and in cell-free pull-down and whole-cell immunoprecipitation assays, the corepressors NCoR (nuclear receptor corepressor) and SMRT (silencing mediator of retinoid and thyroid hormone receptor) associate with agonist and antagonist complexes of GRs. Both N- and C-terminal regions of GR are needed for corepressor binding, which requires the CoRNR box motifs that mediate corepressor binding to other nuclear/steroid receptors. Importantly, whole-cell GR interactions with corepressors are competitively inhibited by excess coactivator and vice versa. However, the regions of the coactivator TIF2 that compete for GR binding to corepressor and coactivator are not the same, implying a molecular difference in GR association with coactivators and corepressors. Finally, when the whole-cell ratio of coactivators to corepressors is altered by selective cofactor binding to exogenous thyroid receptor beta +/- thyroid hormone, the GR dose-response-curve and partial agonist activity are appropriately modified. Such modifications are independent of histone acetylation. We conclude that mutually antagonistic equilibrium interactions of corepressors and coactivators modulate the dose-response curve and partial agonist activity of GR complexes in a manner that is responsive to the intracellular ratio of these two classes of cofactors. This modulation provides an attractive mechanism for differential control of gene expression during development, differentiation, homeostasis, and endocrine therapies.

Research resource: modulators of glucocorticoid receptor activity identified by a new high-throughput screening assay.

Glucocorticoid steroids affect almost every type of tissue and thus are widely used to treat a variety of human pathological conditions. However, the severity of numerous side effects limits the frequency and duration of glucocorticoid treatments. Of the numerous approaches to control off-target responses to glucocorticoids, small molecules and pharmaceuticals offer several advantages. Here we describe a new, extended high-throughput screen in intact cells to identify small molecule modulators of dexamethasone-induced glucocorticoid receptor (GR) transcriptional activity. The novelty of this assay is that it monitors changes in both GR maximal activity (A(max)) and EC(50) (the position of the dexamethasone dose-response curve). Upon screening 1280 chemicals, 10 with the greatest changes in the absolute value of A(max) or EC(50) were selected for further examination. Qualitatively identical behaviors for 60% to 90% of the chemicals were observed in a completely different system, suggesting that other systems will be similarly affected by these chemicals. Additional analysis of the 10 chemicals in a recently described competition assay determined their kinetically defined mechanism and site of action. Some chemicals had similar mechanisms of action despite divergent effects on the level of the GR-induced product. These combined assays offer a straightforward method of identifying numerous new pharmaceuticals that can alter GR transactivation in ways that could be clinically useful.

Role of activation function domain-1, DNA binding, and coactivator GRIP1 in the expression of partial agonist activity of glucocorticoid receptor-antagonist complexes.

The determinants of the partial agonist activity of most antisteroids complexed with steroid receptors are not well understood. We now examine the role of the N-terminal half of the glucocorticoid receptor (GR) including the activation domain (AF-1), the DNA binding site sequence, receptor contact with DNA, and coactivator binding on the expression of partial agonist activity in two cell lines for GRs bound by five antiglucocorticoids: dexamethasone mesylate (Dex-Mes), dexamethasone oxetanone (Dex-Ox), progesterone (Prog), deoxycorticosterone (DOC), and RU486. Using truncated GRs, we find that the N-terminal half of GR and the AF-1 domain are dispensable for the partial agonist activity of antiglucocorticoids. This contrasts with the AF-1 domain being required for the partial agonist activity of antisteroids with most steroid receptors. DNA sequence (MMTV vs a simple GRE enhancer) and cell-specific factors (CV-1 vs Cos-7) exert minor effects on the level of partial agonist activity. Small activity differences for some complexes of GAL4/GR chimeras with GR- vs GAL-responsive reporters suggest a contribution of DNA-induced conformational changes. A role for steroid-regulated coactivator binding to GRs is compatible with the progressively smaller increase in partial agonist activity of Dex-Mes > Prog > RU486 with added GRIP1 in CV-1 cells. This hypothesis is consistent with titration experiments, where low concentrations of GRIP1 more effectively increase the partial agonist activity of Dex-Mes than Prog complexes. Furthermore, ligand-dependent GRIP1 binding to DNA-bound GR complexes decreases in the order of Dex > Dex-Mes > Prog > RU486. Thus, the partial agonist activity of a given GR-steroid complex in CV-1 cells correlates with its cell-free binding of GRIP1. The ability to modify the levels of partial agonist activity through changes in steroid structure, DNA sequence, specific DNA-induced conformational changes, and coactivator binding suggests that useful variations in endocrine therapies may be possible by the judicious selection of these parameters to afford gene and tissue selective results.

PCR expression mutagenesis: a high-throughput mutation assay applied to the glucocorticoid receptor ligand-binding domain.

Glucocorticoid receptors (GRs) are extensively studied members of the steroid hormone receptor superfamily that regulate the transcription rates of numerous genes. Notwithstanding, the role of each GR amino acid in the various steps of transactivation is still unknown. A recent report shows that linear DNA has the same capacity as super-helical plasmid DNA for gene expression in transient transfection assays. Based on this observation, we describe a high-throughput assay to analyze a large set of alanine point mutations that are introduced by two rounds of PCR. The PCR products are then directly transfected into cells. This PCR expression mutagenesis (PEM) technique is used to identify several new residues of the GR ligand binding domain that influence ligand binding and/or transactivation. PEM thus provides a quick method for screening large quantities of mutant proteins. In combination with automation, PEM provides a more rapid and efficient tool for probing the role of each amino acid in the biological functions of a given protein.

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.