It is now well established that the parameters of GR-mediated gene induction (Amax, EC50, and PAA) can be modulated by changing the concentrations of involved transcriptional cofactors. Furthermore, we have recently reported that the different parameters can be controlled by different domains of the modulatory proteins. Thus, it is possible that only one or two, as opposed to all three, parameters may change under certain conditions (Awasthi and Simons, 2012, Mol Cell Endocrinol, 355, 121-134). Our earlier studies in human peripheral mononuclear cells (PBMCs) have confirmed that such changes with varying factor concentration affect the induction parameters of endogenous, as well as exogenous, GR-regulated genes (Luo and Simons Jr., 2009, Human Immunology, 70, 785-789). These results provide strong support for our hypothesis that the modulation of GR induction parameters is a relevant feature of human physiology. Current conventional assays determine the temporal ordering of cofactor association with DNA. However, almost all of the available mechanistic conclusions are phenomenological. In our continuing collaboration with Carson Chow, we have extended our experimentally supported mathematical theory of steroid hormone action to include a competition assay between any two cofactors that affect the Amax and/or EC50 of GR-regulated transactivation. This assay elucidates two previously unknown features of steroid receptor action: (1) the kinetically-defined mechanism of action of each cofactor and (2) the position in the cascade of reactions at which a cofactor acts relative both to a concentration limiting step (CLS) and to the other cofactor. The novel approaches of this assay have now uncovered new activities of components of the human negative elongation factor (NELF) complex, which participates in RNA polymerase II pausing shortly after transcription initiation, especially for synchronized gene expression. The NELF complex is comprised of four subunits: NELF-A, -B, -C/D, and -E. All four subunits attenuate GR-mediated gene induction, reduce the partial agonist activity of an antagonist, and increase the EC50 of an agonist during non-synchronized expression of exogenous and endogenous reporters. Stable knockdown of endogenous NELF-B has the opposite effects on an exogenous gene. The GR ligand-binding domain suffices for these biological responses. ChIP assays reveal that NELF-B diminishes GR recruitment to promoter regions of two endogenous genes. The competition assay reveals that NELF-A and NELF-B each act independently as competitive decelerators at two steps after the site of GR action and before or at the site of reporter gene action. A common motif in each NELF was identified that is required for full activity of both NELF-A and NELF-B. These studies allow us to position the actions of two new modulators of GR-regulated transactivation, NELF-A and NELF-B, relative to other factors in the overall gene induction sequence. Similar collaborative experiments are ongoing with Drs. Dinah Singer (NCI) and David Levens (NCI) to define the actions of other interesting cofactors. The application of the above competition assay will result in a rigorous kinetic, as opposed to a phenomenological, description of cofactor action while shedding new light on where important cofactors are acting, as opposed to simply binding. A long term application of this study concerns the widespread clinical use of steroid hormones to alter the induction (or repression) of numerous genes through binding to their cognate receptor proteins. However, steroid usage is limited by the current inability to control off-target, or non-specific, side-effects. Recent results from three separate areas of research with glucocorticoid and other steroid receptors (cofactor-induced changes in receptor structure, the ability of ligands to alter remote regions of receptor structure, and how cofactor concentration affects both ligand potency and efficacy) indicate that a key element of receptor activity is the intrinsically disordered amino-terminal domain. These results have been combined to construct a novel framework within which to logically pursue various approaches that could afford increased selectivity in steroid-based therapies. In summary, we are applying both novel and conventional methodologies to obtain previously unavailable molecular information both about the determinants of glucocorticoid steroid activity and about the modulation of the total activity (Amax) and dose-response curve (EC50) of agonists. The latter information stems from a rational approach to identify cofactors acting at specific steps in steroid hormone action. These modulatory factors permit a continuum of responses and constitute new therapeutic targets for differential control of gene expression by steroid hormones during development, differentiation, homeostasis, and endocrine therapies. These combined findings contribute to our long-term goal of defining the action of steroid hormones at a molecular level and of understanding their role in human physiology.
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