Chromatin immunoprecipitation (ChIP) scanning identifies primary glucocorticoid receptor target genes

Abstract

The global physiological effects of glucocorticoids are well established, and the framework of transcriptional regulation by the glucocorticoid receptor (GR) has been described. However, the genes directly under GR control that trigger these physiological effects are largely unknown. To address this issue in a single cell type, we identified glucocorticoid-responsive genes in A549 human lung adenocarcinoma cells by microarray analysis and quantitative real-time PCR. Reduction of GR expression by RNA interference diminished the effects of dexamethasone on all tested target genes, thus confirming the essential role of GR in glucocorticoid-regulated gene expression. To identify primary GR target genes, in which GR is a component of the transcriptional regulatory complex, we developed a strategy that uses chromatin immunoprecipitation to scan putative regulatory regions of target genes for sites occupied by specifically bound GR. We screened 11 glucocorticoid-regulated genes, and we identified GR-binding regions for eight of them (five induced and three repressed). Thus, our approach provides a means for rapid identification of primary GR target genes and glucocorticoid-response elements, which will facilitate analyses of transcriptional regulatory mechanisms and determination of hormone-regulated gene networks.

Fig. 1.

The effect of GR RNAi on the expression of GR target genes. (A) GR RNAi reduced GR expression in A549 cells. A549 cells were transfected with GR siRNA (1.5 μg) for 48 h. Cells were then collected and lysed. GR and actin were detected by immunoblotting with N499 and anti-actin antibody (Sigma). (B) GR RNAi reduced the induction of EKI2 and GILZ gene expression by dexamethasone. (C) GR RNAi compromised the repression of GEM and SNK gene expression by dexamethasone. Cells were transfected as described for A and treated with dexamethasone or ethanol for 4–5 h. RNA was isolated and the expression of GILZ, EKI2, SNK, and GEM was determined by qPCR. Data show a representative result from at least two independent RNAi experiments.

Fig. 2.

Identifying primary GR-regulated genes and mapping their GR-binding regions. A549 cells were treated with ethanol or dexamethasone for 90 min, and ChIP-scanning experiments were performed on GILZ (A), THBD (B), SDPR (C), SLC19A2 (D), PPG (E), BHLHB2 (F), GEM (G), and SNK (H). The PCR primers for ChIP scanning that correspond to distinct regions in each promoter are indicated. The fold enrichment values for the experimental regions were determined by normalizing to the internal control hsp70 gene value. Hatched bars indicate the scanning regions, which were enriched >2-fold. Data represent the SEM of the fold enrichment (dexamethasone-treated cells divided by ethanol-treated cells) from at least three experiments.

Fig. 3.

Analysis of glucocorticoid responses of distinct GR-binding sites of glucocorticoid-induced genes. Reporter plasmids (75 ng) were cotransfected with pcDNA3-hGR (150 ng) and RSV-βGal (100 ng) into A549 cells in a 24-well plate. After 4 h, cells were washed with PBS and treated with 0.1 μM dexamethasone for an additional 16–20 h. Cells were then lysed and assayed for luciferase and β-Gal activities. Data represent the SEM of the fold induction (dexamethasone-treated cells divided by ethanol-treated cells) of luciferase activity from at least three experiments.

Fig. 4.

Mutational analysis of potential GREs in GILZ GR-binding sites. (A) Within a 1-kb fragment encompassing GR-binding and GR-responsive regions (pGILZ1, Figs. 2 A and 3), four potential GREs were revealed by sequence analysis. These GREs were mutated as shown (mutated residues are underlined). (B) The effect of single or combinatorial mutations of potential GREs on glucocorticoid responses. Reporter plasmids (75 ng) were cotransfected with pcDNA3-hGR (150 ng) and RSV-βGal (100 ng) into A549 cells in a 24-well plate. After 24 h, cells were washed with PBS and treated with 0.1 μM dexamethasone for an additional 16–20 h. Cells were then lysed and assayed for luciferase and β-Gal activities. Data represent the SEM of the fold induction (dexamethasone-treated cells divided by ethanol-treated cells) of luciferase activity from at least three experiments.

Fig. 5.

Analysis of glucocorticoid responses of distinct GR-binding sites of glucocorticoid-repressed genes. Reporter plasmids (75 ng) were cotransfected with pcDNA3-hGR (150 ng) and RSV-βGal (100 ng) into A549 cells in a 24-well plate. After 4 h, cells were washed with PBS and treated with 0.1 μM dexamethasone for an additional 16–20 h. Cells were then lysed and assayed for luciferase and β-Gal activities. Data represent the SEM of the percentage of full luciferase expression (dexamethasone-treated cells divided by ethanol-treated cells) of luciferase activity from at least three experiments.