Cluster analysis of genes regulated upon TA and/or TNF treatment. (A) Comparison of the genes regulated by TA, TNF, and TA + TNF. Venn diagram represents the overlap between the genes regulated upon each treatment. (B) Cluster analysis of the regulated genes. Using k-means clustering, the 1932 collectively regulated genes were clustered into six distinct clusters based on their changes in RNAPII occupancy upon treatment with TA, TNF, and TA + TNF, relative to DMSO, as depicted by the heatmap. The number of genes in each cluster is indicated. The cluster profiles are as follows: (Cluster 1) largely unaffected by TA and down-regulated by TNF and to a higher extent by TA + TNF; (Cluster 2) up-regulated by TA and TA + TNF, and down-regulated by TNF; (Cluster 3) up-regulated by TNF and down-regulated by TA and TA + TNF; (Cluster 4) largely unaffected by TA and strongly up-regulated in the presence of TNF (a subset of these genes is down-regulated by TA + TNF); (Cluster 5) mildly up-regulated by TA or TNF and strongly up-regulated by TA + TNF; (Cluster 6) up-regulated by TA and down-regulated by TNF and to a lesser extent by TA + TNF. The main characteristics of genes in clusters 2, 4, 5, and 6 are indicated at right.

Analysis of genome-wide GR- and p65-binding sites upon TA and/or TNF treatment. (A) The number of GR- and p65-binding sites identified upon TA, TNF, and TA + TNF treatment using the respective DMSO ChIP-seq data as control. (B) Median distance (in Kb) of GR- and p65-binding sites to the TSS of the nearest gene regulated by TA, TNF, and TA + TNF, as indicated. (C) Response elements for GR, NFKB, and AP1 identified as the predominant motifs in the GR and p65 binding sites. Motifs were identified by a de novo motif search and visualized using WebLogo. (D) Motif occurrence within the GR and p65 binding sites. The bar graph shows the percentage of GR and p65 sites containing the indicated motifs.

Gained p65-binding sites and their correlation to gene expression profile. (A) Profile of p65 binding sites. Venn diagram of the overlap of p65 sites detected upon treatment with TNF or TA + TNF. (B) Tag density maps depicting the pattern of GR occupancy around (peak mode ±2.5 kb) gained p65 sites. Color density indicates the level of GR occupancy (square root of tag density; see scale below) in a 250-bp window. The position of the example presented in E (ZFAND5) is indicated. (C) Motif occurrence within gained p65 sites co-occupied by GR. The bar graph shows the percentage of sites containing the indicated motifs. (D) Boxplots of GR and p65 tag counts distributed under peak locations (log₂ scale) of gained p65 sites co-occupied by GR, upon TA + TNF treatment, in WT cells, and the respective tag distributions under the same locations in GR_KD cells. (E) p65 and GR ChIP-seq data illustrate binding of p65 and GR at gained p65 sites co-occupied by GR, detected upon the indicated treatments, in WT and p65_KD cells. Data were viewed in the UCSC Genome Browser. The maximum number of overlapping tags, representing peak height, is indicated on the y-axis. (F) Model of GR and p65 interaction at gained p65 sites co-occupied by GR. (G) Enrichment of gained p65 sites co-occupied by GR that are assigned to the genes of each cluster. Peak enrichment fold ratio is as described in Figure 5G.

Genes with altered RNAPII occupancy upon TA and/or TNF treatment. (A) The number of genes with altered RNAPII occupancy (increased and decreased) in response to TA, TNF, and TA + TNF treatment, as indicated. (B) RNAPII occupancy vs mRNA levels. Scatter plots depicting the Pearson correlation (r) between changes in mRNA levels (y-axis) and in RNAPII occupancy (x-axis) for randomly selected regulated genes. Changes in mRNA and RNAPII occupancy were expressed as fold induction of the indicated treatment over DMSO (in log₂ scale).

Genome-wide GR- and p65-binding sites upon TA or TNF treatment. (A,E) RNAPII and GR (A) or p65 ChIP-seq data (E) illustrate RNAPII occupancy and GR and p65 binding in the promoter and/or within the gene body of target genes. Strong binding sites were detected upon TA (green) and TNF treatment (pink), whereas residual binding was observed in the absence of ligand (gray). Data were viewed in the University of California at Santa Cruz (UCSC) Genome Browser. The maximum number of overlapping tags, representing peak height, is indicated on the y-axis. (B,F) Validation of GR-binding sites (B) and p65-binding sites (F). Strip plots of GR ChIP–qPCR data for 25 randomly selected GR sites from WT and GR_KD cells (B) and p65 ChIP–qPCR data for 20 randomly selected p65 sites from WT and p65_KD cells (F) treated as indicated. Results are expressed as a percentage of input chromatin. Binding sites with fold induction (relative to DMSO) >2 are regarded as validated. Establishment of GR_KD and p65_KD cells are described in Supplemental Figure 2. (C,G) Genomic location analysis of GR-binding sites (C) and p65-binding sites (G). GR sites identified upon TA treatment (C) and p65 sites identified upon TNF treatment (G) were divided into the following categories based on their position relative to the nearest gene: distant (>25 Kb), within 5 Kb upstream, within 5 kb downstream, within 5–25 kb upstream, within 5–25 kb downstream and intragenic (exon, intron, and within 0–5 kb up- and downstream). The Pink Thing (http://pinkthing.cmbi.ru.nl) was used for the analysis. (D) Comparison of genome-wide GR profiling data. Venn diagram of the overlap of GR sites identified in the present study (8306 sites, ChIP-seq using HeLa B2 cells treated with TA for 4 h) with those identified in Reddy et al. (2009) (4392 sites, ChIP-seq using A549 cells treated with Dex for 1 h). (H) Comparison of genome-wide p65 profiling data. Venn diagram of the overlap of p65 sites identified in the present study (12,552 sites, ChIP-seq using HeLa B2 cells treated with TNF for 1 h) with those identified in Lim et al. (2007) (5858 sites, ChIP-PET using THP-1 cells treated with LPS for 1 h).

Gained GR-binding sites and their correlation to gene expression profile. (A) Profile of GR-binding sites. Venn diagram of the overlap of GR sites detected upon treatment with TA or TA + TNF. (B) Tag density maps depicting the pattern of p65 occupancy around (peak mode ±2.5 kb) gained GR sites. Color density indicates the level of p65 occupancy (square root of tag density; see scale below) in a 250-bp window. The position of the example presented in E (ZBTB20) is indicated. (C) Motif occurrence within gained GR sites co-occupied by p65. The bar graph shows the percentage of sites containing the indicated motifs. (D) Boxplots of GR and p65 tag counts distributed under peak locations (log₂ scale) of gained GR sites co-occupied by p65, upon TA + TNF treatment, in WT cells and the respective tag distributions under the same locations in p65_KD cells. (E) GR and p65 ChIP-seq data illustrate binding of GR and p65 at gained GR sites co-occupied by p65, detected upon the indicated treatments, in WT and p65_KD cells. Data were viewed in the UCSC Genome Browser. The maximum number of overlapping tags, representing peak height, is indicated on the y-axis. (F) Model of GR and p65 interaction at gained GR sites co-occupied by p65. (G) Enrichment of gained GR sites co-occupied by p65 that are assigned to the genes of each cluster. Peak enrichment fold ratio represents the ratio of the nonrandom enrichment (ratio of assigned binding sites to the total number of genes in each cluster) to the random enrichment (parallel enrichment calculation for 100 random sets of nonregulated genes, performed to assess the statistical significance of the peak enrichment analysis).