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. 2009 Dec;19(12):2163-71.
doi: 10.1101/gr.097022.109. Epub 2009 Oct 2.

Genomic determination of the glucocorticoid response reveals unexpected mechanisms of gene regulation

Timothy E Reddy  1 , Florencia Pauli, Rebekka O Sprouse, Norma F Neff, Kimberly M Newberry, Michael J Garabedian, Richard M Myers
Affiliations

Genomic determination of the glucocorticoid response reveals unexpected mechanisms of gene regulation

Timothy E Reddy et al. Genome Res. 2009 Dec.

Abstract

The glucocorticoid steroid hormone cortisol is released by the adrenal glands in response to stress and serves as a messenger in circadian rhythms. Transcriptional responses to this hormonal signal are mediated by the glucocorticoid receptor (GR). We determined GR binding throughout the human genome by using chromatin immunoprecipitation followed by next-generation DNA sequencing, and measured related changes in gene expression with mRNA sequencing in response to the glucocorticoid dexamethasone (DEX). We identified 4392 genomic positions occupied by the GR and 234 genes with significant changes in expression in response to DEX. This genomic census revealed striking differences between gene activation and repression by the GR. While genes activated with DEX treatment have GR bound within a median distance of 11 kb from the transcriptional start site (TSS), the nearest GR binding for genes repressed with DEX treatment is a median of 146 kb from the TSS, suggesting that DEX-mediated repression occurs independently of promoter-proximal GR binding. In addition to the dramatic differences in proximity of GR binding, we found differences in the kinetics of gene expression response for induced and repressed genes, with repression occurring substantially after induction. We also found that the GR can respond to different levels of corticosteroids in a gene-specific manner. For example, low doses of DEX selectively induced PER1, a transcription factor involved in regulating circadian rhythms. Overall, the genome-wide determination and analysis of GR:DNA binding and transcriptional response to hormone reveals new insights into the complexities of gene regulatory activities managed by GR.

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Figures

Figure 1.
Figure 1.
Genomic distribution of glucocorticoid receptor binding sites and response elements. (A) Enrichment of GR binding sites in genomic features. The percentage of all identified GR binding sites in each type of region, indicated in parentheses, was divided by the relative amount of the human genome each type of region occupies and plotted on a log2 scale. (B) The revised glucocorticoid response element (GRE) identified in the most significant GR binding sites identified by ChIP-seq. (C) Distribution of the number of GREs in identified high-confidence GR binding sites across the human genome. (D,E) Example ChIP-seq data showing GR and Pol II (RNA Pol2) binding at GREs near the NFKBIA and PER1 genes. ChIP-seq data are plotted as the density of 25-bp tags mapping to the region. The density is separated to show reads mapping to the positive (orange) and negative (blue) strand of the reference genome. For RNA-seq data, darker indicates more expressed.
Figure 2.
Figure 2.
Gene expression response to dexamethasone and overlap with glucocorticoid receptor binding. (A) Histogram showing the distribution of expression change for the DEX-responsive genes, as measured with RNA-seq. DEX-induced genes are shown in orange, while DEX-repressed genes are shown in blue. (B) Comparison of DEX response in genes that have GR binding within 10 kb of their transcription start site (purple) and genes without GR binding in the same region (white). (C) Percentage of DEX-responsive genes (y-axis) that have GR binding within a given distance from the transcription start site (x-axis). Orange line, up-regulated genes; blue line, down-regulated genes. Vertical lines indicate median distance to nearest binding site. Dashed lines indicate binding sites called with a lower confidence threshold of FDR < 5%.
Figure 3.
Figure 3.
Dose response to dexamethasone. (A) Gene expression response, measured with RT-QPCR, of DEX-responsive genes in response to treatment with increasing concentrations of DEX. The up-regulated genes (orange line) and down-regulated genes (blue line) showed similar response, with EC50 ∼ nM (dashed line). Error bars, SD. (B) PER1 (blue line) is particularly responsive to low concentrations of DEX, compared with all other genes measured (gray lines). Error bars, maximum and minimum over three biological replicates. (C) PER1 dose response (green lines) verified with primers designed against various exons and exon–exon junctions, as indicated. For comparison, the overall response of activated (orange line) and repressed genes (blue line) is reproduced. Error bars, SD.
Figure 4.
Figure 4.
Time-course response to dexamethasone. Gene expression response, measured with RT-QPCR, of DEX-responsive genes over increasing duration of exposure to 100 nM DEX treatment. (A) Average gene expression response (y-axis) across the time-course (x-axis). Error bars, SD. Fifty percent response time for up- and down-regulated genes are indicated with dashed and dotted lines, respectively. (B) Time of exposure to 100 nM DEX required to achieve 20%, 50%, and 80% response for up-regulated (orange bars) and down-regulated (blue bars) genes. Error bars, SE in each parameter from curve fitting.
Figure 5.
Figure 5.
Effect of glucocorticoid receptor knockdown on dexamethasone response. (A) Western blot showing the effect of siRNA-mediated knockdown on GR protein levels. (B) Gene expression response of 46 DEX-responsive genes to 5-h 100 nM DEX treatment in cells transfected either with control (white bars) or GR-targeting (black bars) siRNA pools. Three independent knockdown experiments were performed for each of two siRNA pools. Error bars, SEM across all six measurements. Measurements for each siRNA pool are available in Supplemental Figures S6 and S7. (C) Box plot showing overall effect of siRNA-mediated knockdown of the GR on the gene expression response for DEX-induced and DEX-repressed genes. Whiskers indicate 10%-90% range.
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