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

Abstract

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 (A max ) and the steroid concentration required for half-maximal induction (EC 50 ) 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.Steroid hormone action has been studied intensively since the discovery of steroid hormone receptors almost 50 years ago (1) for three reasons. First, the receptors are obligatory mediators for the biological responses of the five classes of endogenous steroid hormones. Second, steroids and their receptors provide an ideal system in which to define the molecular mechanisms that regulate the controlled induction of gene transcription. Such studies have resulted in the identification of many species or factors that are required for transcription initiation, elongation, and termination (2-4). Third, the clinical benefits of steroid hormones are often severely limited by undesired side effects (5-8). A better molecular understanding of steroid action should lead to fewer side effects, which would significantly expand the clinical applications of steroids in treating human pathologies.The majority of factors identified to date for steroid-regulated gene induction participate in the early steps of transcription initiation (e.g. chromatin remodeling, assembly of the transcription preinitiation complex, and synthesis of the first oligonucleotides) (9). Two interesting classes of factors for steroid-regulated gene transcription are coactivators and corepressors (2-4). One of the earliest and most widely studied of these factors is TIF2/GRIP1, which is a member of the p160 family of coactivators with molecular masses of about 160 kDa (10, 11). Despite the detailed understanding of many of the factors involv...

An Approach to Greater Specificity for Glucocorticoids

“…This information is currently not available by any other method. It is therefore relevant that the sites of action of Cdk9, BRD4, NELF complex, NELF-E, and TIF2 are consistent with the biochemical data for these factors ( 41 ).…”

“…We define A max as the maximum amount of induced activity, which is determined from first-order Hill plot fits of the activity vs. steroid concentration with each combination of the factors. The fits of these plots are usually very good with ( R 2 ≥ 0.99) ( 41 , 44 ). One must then determine, usually by Western blots, whether the expression of each of the factors is, or is not, linear over the range of transfected cDNA used.…”

“…To make plots combining the results of several experiments, one can simply average the data (e.g., Figure 2 ). Alternatively, the data can be normalized, usually to the lowest amount of each of the two factors ( 41 ). In both situations, the average values ± error are then plotted.…”

“…With the identification and constancy of the CLS, it has been possible to combine the data of multiple GR transactivation experiments ( 34 , 41 , 43 ) to indicate how and where numerous factors exert their effects (Figure 3 ). With this approach, factors that previously were not thought to modify GR-regulated gene transcription have been readily identified and their site and mechanism of action determined.…”

“…With this approach, factors that previously were not thought to modify GR-regulated gene transcription have been readily identified and their site and mechanism of action determined. Examples of such novel modulators include PA1 (Pax2 transactivation domain interaction protein-associated protein 1) ( 43 ), each of the four components of the NELF complex ( 39 ), the kinase Cdk9 ( 44 ), and BRD4, which is a kinase involved in the initial steps of gene induction ( 41 ). This information is currently not available by any other method.…”