Kinetically-defined component actions in gene repression

Abstract

Gene repression by transcription factors, and glucocorticoid receptors (GR) in particular, is a critical, but poorly understood, physiological response. Among the many unresolved questions is the difference between GR regulated induction and repression, and whether transcription cofactor action is the same in both. Because activity classifications based on changes in gene product level are mechanistically uninformative, we present a theory for gene repression in which the mechanisms of factor action are defined kinetically and are consistent for both gene repression and induction. The theory is generally applicable and amenable to predictions if the dose-response curve for gene repression is non-cooperative with a unit Hill coefficient, which is observed for GR-regulated repression of AP1LUC reporter induction by phorbol myristate acetate. The theory predicts the mechanism of GR and cofactors, and where they act with respect to each other, based on how each cofactor alters the plots of various kinetic parameters vs. cofactor. We show that the kinetically-defined mechanism of action of each of four factors (reporter gene, p160 coactivator TIF2, and two pharmaceuticals [NU6027 and phenanthroline]) is the same in GR-regulated repression and induction. What differs is the position of GR action. This insight should simplify clinical efforts to differentially modulate factor actions in gene induction vs. gene repression.

Fig 1. Reaction diagram for a single step in the gene induction sequence.

The product from the previous step Y _i-1 combines with the accelerator X _i to form a new product $Y_i^{*}$ with equilibrium or affinity constant q _i. A decelerator D _i inhibits the reaction by binding to X _i to form $X_i^{\text{'}}$ with affinity constant ${\gamma q}_i^{\text{'}}$ and/or by binding to $Y_i^{*}$ to form $Y_i^{\text{'}}$. The two products $Y_i^{\text{'}}$ and $Y_i^{*}$ enter the following reaction in the combination $\left[Y_i\right]=\left[Y_i^{*}\right]+\beta \left[Y_i^{\text{'}}\right]$. The decelerator is called competitive when α = 0, uncompetitive when γ = 0, noncompetitive when α = γ, linear when β = 0, and partial when β>0.

Fig 2. Dose-response curve for GR repression (for eight doses of Dex) is linear-fractional plot.

U2OS.rGR cells were transiently transfected with 35ng of AP1LUC plasmid and (A) 0ng or (B) 10ng of TIF2 plasmid and then treated with 15ng/ml of PMA and the indicated concentrations of Dex. Luciferase activity was determined and the data plotted and fit to Equation (1) as described in Materials and Methods (error bars are ± S.D. of triplicates).

Fig 3. Plots of dose-response parameters for varying concentrations of AP1LUC and TIF2.

Experiments were conducted with triplicate samples of U2OS.rGR cells that were transiently transfected with the indicated concentrations of AP1LUC and TIF2 plasmids and treated with 25ng/ml of PMA for four concentrations of Dex. Average plots vs. TIF2 for (A) A_max, (B) A_min, (C) IC₅₀, and (D) A_max×IC₅₀/A_min and vs. AP1LUC for (E) A_max and (F) IC₅₀ were obtained by first normalizing the data to the value for the lowest amount of AP1LUC and factor and then averaging and plotting the values (n = 4–12, ± S.E.M.).

Fig 4. Dose-response parameters for varying concentrations of AP1LUC and NU6027.

Experimental assays were conducted as in Fig. 3 with 10ng/ml of PMA and four concentrations of Dex. Average plots of (A) A_max, (B) A_min, (C) A_max×IC₅₀/A_min, and (D) IC₅₀ vs. NU6027 were obtained by first normalizing the data to the value for the lowest amount of AP1LUC and factor and then averaging and plotting the values (n = 5, ± S.E.M.).

Fig 5. Dose-response parameters for varying concentrations of AP1LUC and phenanthroline.

Experimental assays were conducted as in Fig. 4. Average plots of (A) A_max, (B) A_min, (C) A_max×IC₅₀/A_min, and (D) IC₅₀ vs. NU6027 were obtained by first normalizing the data to the value for the lowest amount of AP1LUC and factor and then averaging and plotting the values (n = 5, ± S.E.M.).

Fig 6. Predicted reaction scheme of PMA induction of Luciferase activity from synthetic reporter (AP1LUC) by AP1 that is repressed by steroid-bound receptor (GR).

The position of the CLS, and positions of action of TIF2, NU6027, phenanthroline, and GR, as determined by the data of Figs. 3–5, are indicated. A’ and A” represent unknown, post-CLS steps, each of which can lead to Luciferase activity but the efficiency from A” is much less than A’.