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 identify new cofactors affecting GR-controlled gene induction with previously unrecognized activities. The human negative elongation factor (NELF) complex, which participates in RNA polymerase II pausing shortly after transcription initiation, 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. In view of the known role of the NELF complex in RNA polymerase II pausing and the fact that one site of action of NELF-A and -B in GR transactivation could involve polymerase pausing, we asked if cofactors (Cdk9 and ELL) best known to affect paused polymerase could reverse the effects of NELF-A and -B on GR. Unexpectedly, Cdk9 and ELL augmented, rather than prevented, the effects of NELF-A and -B. Furthermore, Cdk9 actions are not blocked either by Ckd9 inhibitors (DRB or flavopiridol) or by two Cdk9 mutants defective in kinase activity. The mode and site of action of NELF-A and -B mutants with an altered NELF domain are similarly affected by wild-type and kinase-dead Cdk9. We conclude that Cdk9 is a new modulator of GR action, that Ckd9 and ELL have novel activities in GR-regulated gene expression, that NELF-A and -B can act separately from the NELF complex, and that Cdk9 possesses activities that are independent of Cdk9 kinase activity. Finally, the competition assay has succeeded in ordering the site of action of several cofactors of GR transactivation. The above studies clearly demonstrate the ability of our approach to give novel information regarding more selective modulation of GR actions at specific organ/gene targets. This new information is a result not only of examining dose-response curves but also of quantitating the overall response as opposed to the conventional approach of focusing on interactions of ligands and factors with just the ligand binding domain of receptors. This standard approach overlooks the activity of the unstructured AF1 domain in the amino terminus of steroid receptors. We have drawn attention to recent studies showing that highly flexible intrinsically disordered regions of transcription factors, including that of the N-terminal domain AF1 of steroid receptors, not only are critical for several aspects of steroid receptor action but also can be exploited as drug targets, thereby opening unique opportunities for endocrine-based therapies. The above approach has also been used by the HESI RISK21 project, which formed the Dose-Response/Mode-of-Action Subteam to develop strategies for using all available data (in vitro, in vivo, and in silico) to advance the next-generation of chemical risk assessments. A goal of the Subteam is to enhance the existing Mode of Action/Human Relevance Framework and Key Events/Dose Response Framework (KEDRF) to make the best use of quantitative dose-response and timing information for Key Events (KEs). The resulting Quantitative Key Events/Dose-Response Framework (Q-KEDRF) provides a structured quantitative approach for systematic examination of the dose-response and timing of KEs resulting from a dose of a bioactive agent that causes a potential adverse outcome. Two concepts are described as aids to increasing the understanding of mode of actionAssociative Events and Modulating Factors. These concepts are illustrated in estrogen-induced uterotrophic responses in rodents, which demonstrate how quantitative dose-response modeling for KE, the understanding of temporal relationships between KEs, and a counterfactual examination of hypothesized KEs can determine whether they are Associative Events or true KEs. 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. More importantly, the identification of factors influencing steroid hormone action at sites well downstream of steroid binding, and close to the final, desired effect, offer the prospect of modifiable targets that should evoke fewer undesirable side effects due to their proximity to the final response. 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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