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 assays determine the temporal ordering of cofactor binding to DNA. However, almost all of the available mechanistic conclusions are phenomenological. In our continuing collaboration with Carson Chow, we now 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. This assay was used to further define the actions of PTIP associated protein 1 (PA1) (Cho et al., 2007, J Biol Chem, 282, 20395-20406), which we find is a new corepressor of GR transactivation. PA1 suppresses Amax, increases the EC50, and reduces the PAA of an exogenous reporter gene in a manner that is independent of associated PTIP. PA1 is fully active with, and strongly binds to, the C-terminal half of GR. PA1 reverses the effects of the coactivator TIF2 on GR-mediated gene induction but is unable to augment the actions of the corepressor SMRT. Analysis of competition assays between PA1 and TIF2 with an exogenous reporter indicates that the kinetic definition of PA1 action is a competitive decelerator at two sites upstream of where TIF2 acts. With the endogenous genes IGFBP1 and IP6K3, PA1 also reduces GR induction, increases the EC50, and decreases the PAA. ChIP and re-ChIP experiments indicate that PA1 accomplishes this inhibition of the two genes via different mechanisms: PA1 appears to increase GR dissociation from, and reduce GR transactivation at, the IGFBP1 promoter regions but blocks GR binding to IP6K3 promoter. We conclude that PA1 is a new competitive decelerator of GR transactivation and can act at more than one molecularly defined step in a manner that depends upon the specific gene. 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. More conventional approaches are also being used to understand that molecular details of cofactor action. Previously, we reported that the hitherto relatively inactive amino terminus of the coactivator TIF2 bound to the amino terminal regions of both GR and the progesterone receptor (PR) (Wang et al., 2007, Biochemistry, 48, 8036-8049). For both GR and PR, it is the amino terminal region that contains the sequence that has the most activity for the induction of gene transcription. In collaboration with Dr. Raj Kumar (The Commonwealth Medical College, PA), we have obtained biophysical support for the binding of the amino terminal fragments of GR and TIF2. These results suggest that amino terminal interactions of GR and TIF2 are as important as, or even more so than, the well-known binding of the central domain of TIF2 to the C-terminal region of GR for understanding TIF2-augmented GR transactivation. 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.

Project Start
Project End
Budget Start
Budget End
Support Year
21
Fiscal Year
2012
Total Cost
$384,930
Indirect Cost
City
State
Country
Zip Code
Chow, Carson C; Ong, Karen M; Kagan, Benjamin et al. (2015) Theory of partial agonist activity of steroid hormones. AIMS Mol Sci 2:101-123
Zhu, Rong; Lu, Xinping; Pradhan, Madhumita et al. (2014) A kinase-independent activity of Cdk9 modulates glucocorticoid receptor-mediated gene induction. Biochemistry 53:1753-67
Simons Jr, S Stoney; Edwards, Dean P; Kumar, Raj (2014) Minireview: dynamic structures of nuclear hormone receptors: new promises and challenges. Mol Endocrinol 28:173-82
Luo, Min; Lu, Xinping; Zhu, Rong et al. (2013) A conserved protein motif is required for full modulatory activity of negative elongation factor subunits NELF-A and NELF-B in modifying glucocorticoid receptor-regulated gene induction properties. J Biol Chem 288:34055-72
Simons Jr, S Stoney; Kumar, Raj (2013) Variable steroid receptor responses: Intrinsically disordered AF1 is the key. Mol Cell Endocrinol 376:81-4
Zhang, Zhenhuan; Sun, Yunguang; Cho, Young-Wook et al. (2013) PA1 protein, a new competitive decelerator acting at more than one step to impede glucocorticoid receptor-mediated transactivation. J Biol Chem 288:42-58
Blackford Jr, John A; Guo, Chunhua; Zhu, Rong et al. (2012) Identification of location and kinetically defined mechanism of cofactors and reporter genes in the cascade of steroid-regulated transactivation. J Biol Chem 287:40982-95
Dougherty, Edward J; Guo, Chunhua; Simons Jr, S Stoney et al. (2012) Deducing the temporal order of cofactor function in ligand-regulated gene transcription: theory and experimental verification. PLoS One 7:e30225
Simons Jr, S Stoney; Chow, Carson C (2012) The road less traveled: new views of steroid receptor action from the path of dose-response curves. Mol Cell Endocrinol 348:373-82
Awasthi, Smita; Simons Jr, S Stoney (2012) Separate regions of glucocorticoid receptor, coactivator TIF2, and comodulator STAMP modify different parameters of glucocorticoid-mediated gene induction. Mol Cell Endocrinol 355:121-34

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