Identification of Location and Kinetically Defined Mechanism of Cofactors and Reporter Genes in the Cascade of Steroid-regulated Transactivation

Abstract

Background: Current descriptions of steroid hormone action are largely phenomenological rather than mechanistic. Results: Methodology is described for determining kinetically defined mechanisms and relative sites of action of any two cofactors with steroid receptors. Conclusion: Position and mode of reporter gene action are constant. Significance: Location and mechanistic action of cofactors, relative to each other and reporter, is assignable in sequence for receptor-regulated gene transactivation.

PA1 Protein, a New Competitive Decelerator Acting at More than One Step to Impede Glucocorticoid Receptor-mediated Transactivation

“…With an exogenous reporter gene under similar conditions, PA1 competitively inhibits the kinetically defined actions of the coactivator TIF2. Using a newly developed competition assay (27,28), we further show that PA1 does not interact directly with TIF2 but operates at two sites upstream from where TIF2 functions. The fact that PA1, a novel competitor of GR transactivation, can act at two different sites in the same transactivation sequence suggests that other cofactors may also affect more than one step at a molecular level in steroid-regulated gene transcription.…”

“…As shown below, much more information can be gleaned from these graphs using our recently developed assay (27,28). The assay is based on a previously validated theory of gene induction that models the entire process as a sequence of enzymatic reactions (35).…”

“…These actions of activation and inhibition do not refer to the effect on the final output, as is the case for coactivators and corepressors, but they only apply to the individual reaction on which they act. To avoid confusion, we have adopted the terms accelerator and decelerator to refer to the enzymatic equivalent action of the cofactor at its site of action (28). Thus, as in enzyme kinetics, it is very possible that a decelerator could decrease the output of a given step to cause an increase in the final product, in which case it would be called a coactivator.…”

“…Although our experiments are for a closed system without fluxes, we can still identify an equilibrium analog of the rate-limiting step that we call a concentration limiting step (CLS). The competition assay assigns the site of factor action relative not only to the other factor but also relative to the CLS, the position of which we recently found is constant under a wide variety of conditions and is defined by the site of action of the reporter gene (28).…”

“…Of the infinite number of possible shapes, the theory predicts that the graphical behavior of the curves in each plot can only be one of just 35 types, each of which is associated with unique kinetic actions (e.g. accelerator or competitive decelerator) (28) and positions of factor action (27). In the case of an accelerator, the identification of the relevant graphical behavior requires determining the level of endogenous accelerator relative to added accelerator, which is accomplished by Western blot analysis.…”