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. 2015 Mar 12;29(5):716–729. doi: 10.1210/me.2014-1403

Figure 6.

Figure 6.

Regulation of GRα occupancy by Hic-5 on GBRs near block genes. A, Extent to which Hic-5 restricts Dex induction and repression of block genes. U2OS(GRα) cells were transfected with nonspecific siRNA or siHic-5 and treated with ethanol (ETOH) or Dex for 24 hours, and global mRNA levels were determined by microarray analysis (see Supplemental Dataset 4); the Hic-5–blocked gene set was identified as described for Figure 3A. The log2 fold change in the mRNA levels of all Hic-5–blocked genes caused by 24-hour Dex treatment (from microarray data) is shown (y-axis) with results for each gene in Hic-5–depleted cells shown in pink and results for the same gene in Hic-5–positive cells shown in blue. B, Hic-5–blocked, shared, and Hic-5–dependent GRα peaks found near Hic-5–blocked genes. Hic-5–blocked gene sets determined from gene expression microarray analyses of cells treated with Dex for 4 or 24 hours were divided into genes that were induced or repressed by Dex, and the number of GRα binding events observed within ± 50 kb of the TSSs are divided into 3 categories: Hic-5–blocked peaks, shared peaks, and Hic-5–dependent peaks. C, GR binding sites at the GRAMD4 and GAS1 loci are shown in the Integrative Genomics Viewer. The red and black peaks indicate GR binding events in Hic-5–depleted and Hic-5–positive conditions, respectively. Blue bars under the peaks were identified as significant by the peak-calling algorithm. Input tracks for both conditions are shown as controls. The genomic coordinates of GRAMD4 peaks are as follows: GBR1, chr22:47,000,100–47,001200; GBR2, chr22:47,027,100–47028200; GBR3, chr22:47,030,800–47031900, and GAS1-GBR, chr9:89,598,400–89599300. D, RT-qPCR validation of the Dex-regulated expression of Hic-5–blocked genes shown in C. mRNA expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels for each sample and represent means ± SEM for 3 biological replicates. E, Validation of GR occupancy by ChIP-qPCR. GRα occupancy was analyzed at the sites shown in C by quantitative ChIP (normalized to input chromatin) in U2OS(GRα) cells transfected with siNS or siHic-5 and then treated with ethanol or 100 nM Dex for 1 hour. For GRAMD4, GBRs 1, 2, and 3 are shown left to right in C. Error bars represent means and ranges of variation for 2 technical replicates from one experiment, and results shown are representative of 3 independent experiments.