Incoherent feed-forward regulatory logic underpinning glucocorticoid receptor action

Abstract

The complexity and specificity of metazoan transcription are determined by combinatorial control of the composition and activity of regulatory complexes. To investigate the basis of this specificity, we focused on the glucocorticoid receptor (GR), a single regulatory factor that integrates multiple signals to give rise to many distinct patterns of expression. We measured the expression of a set of genes, each directly GR-regulated, but by different mechanisms in two cell lines. We varied ligand (dose, chemistry, and duration of treatment), GR (expression level and functionality), and a non-GR regulatory factor that commonly interacts with GR. Our study revealed distinct expression patterns within this set of genes, but all could be modeled by an incoherent feed-forward regulatory logic. Cellular signals, operating on GR and other factors within regulatory complexes, may define and modulate the kinetics and strength of the activating or inhibitory paths of the regulatory logic. Thus, characterizing systems behavior by perturbing single or multiple signals can reveal general principles of regulation, providing an approach to the dissection and deconvolution of combinatorial control.

Fig. 1.

Identification and characterization of DRGs. (A) Dexamethasone-stimulated changes in mRNA levels of DRGs. Cells were treated with vehicle [ethanol (EtOH)] or 10⁻⁶ M dex for 1.5, 3, or 24 h. Data are plotted in a log2 scale relative to EtOH. Data represent the mean ± SD, n = 3. (B) Immunoblots demonstrating the knockdown of GR. (C and D) Dexamethasone-stimulated changes in mRNA levels of DRGs after GR knockdown. Cells were transfected with either scrambled siRNA or siRNA specifically targeting GR transcripts, followed by treatment with EtOH or 10⁻⁶ M dex for 3 or 24 h. Transfection of two different scrambled siRNA sets showed similar results. Data are plotted in a log2 scale relative to EtOH. Data represent the mean ± SD, n = 3.

Fig. 2.

Dose–response curves and dose and temporal kinetics of DRGs in U2OS-hGR cells. (A–D) Dexamethasone-stimulated changes in mRNA levels of DRGs. Cells were treated with EtOH or dex (10⁻¹⁵ M to 10⁻⁵ M) for 3 h. Data are plotted in a log2 scale relative to EtOH. Data represent the mean ± SD, n = 3. (E–H) Dexamethasone-stimulated changes in mRNA levels of DRGs. Cells were treated with EtOH or dex (10 pM to 10 nM) for 0, 0.375, 0.75, 1.5, 3, 6, 12, or 24 h. Data are plotted in a log2 scale relative to EtOH. Data represent the mean, and Dataset S2 gives the mean and SD, n = 3.

Fig. 3.

Dose–response curves of DRGs induced by different ligands. (A–D) Dexamethasone-stimulated changes in mRNA levels of DRGs. Cells were treated with EtOH, RU486, prednisolone, cortisol, or corticosterone (10⁻¹¹ M to 10⁻⁵ M) for 3 h. Data are plotted in a log2 scale relative to EtOH (labeled CT). Data represent the mean, and Dataset S3 gives mean and SD, n = 3.

Fig. 4.

C-Jun interacts with glucocorticoid signaling in a context-dependent manner. (A) Immunoblots demonstrating the knockdown of c-Jun. The loading controls are the immunoblots of actin from the same samples. (B–I) Dexamethasone-stimulated changes in mRNA levels of DRGs after c-Jun knockdown. Cells were transfected with either scrambled siRNA (control) or siRNA specifically targeting c-Jun transcripts, followed by treatment with EtOH or dex (10⁻¹¹ M to 10⁻⁷ nM) for 3 h. Data are normalized with the level of RPL19 in the control and plotted in a log2 scale relative to EtOH (labeled CT). Data represent the mean ± SD, n = 3.

Fig. 5.

Regulation of ANKRD1 repression. (A) Immunoblots demonstrating the level of GR did not change in either wild type or A548T mutant. Cells were treated with EtOH or dex (10⁻¹¹ M to 10⁻⁵ M) for 1.5 h. Immunoblots at lower exposure showed similar relative GR intensities. (B) Dose–response curves of ANKRD1 in U2OS-hGR(A548T). U2OS-hGR(A548T) cells were treated with EtOH or dex (10⁻¹¹ M to 10⁻⁵ M) for 3 h. Data are plotted in a log2 scale relative to EtOH (labeled CT) and represent the mean ± SD, n = 3. (C) ChIP-seq data of GR occupancies at ANKRD1 gene (12). (D) ChIP showing GR occupancy at E1–E4 sites around ANKRD1. A549, U2OS-hGR, and U2OS-hGR(A548T) cells were treated with EtOH, 10⁻⁹ M, or 10⁻⁶ M dex for 45 min. Data are plotted in a log2 scale relative to EtOH and represent the mean ± SD, n = 3. The GR occupancies are significantly different between 1-nM and 1-μM dex treatment at all sites and in all three cell lines (P value < 0.05 by Student’s t test). (E) Model for the incoherent feed-forward loop control of ANKRD1 differential transcription.