1. Volume 47, Issue 2, Pages 298-309.e5 (August 2017)
Anti-Inflammatory Chromatinscape Suggests Alternative Mechanisms of Glucocorticoid Receptor Action 
Kyu-Seon Oh, Heta Patel, Rachel A. Gottschalk, Wai Shing Lee, Songjoon Baek, Iain D.C. Fraser, Gordon L. Hager, Myong-Hee Sung 
Immunity 
Volume 47, Issue 2, Pages e5 (August 2017)
DOI: /j.immuni
Copyright © Terms and Conditions

2. Immunity 2017 47, 298-309.e5DOI: (10.1016/j.immuni.2017.07.012)
Copyright © Terms and Conditions

3. Figure 1
Transcriptomic Profiling of LPS Responses in Macrophages with Varied Timing of Dex Treatment
(A) Experimental design for BMDM treatment and sample collection for RNA-seq. The timeline is with respect to the onset of LPS stimulation (t = 0).
(B) Genome-wide expression patterns from RNA-seq data. Scatterplots show mean log2 (FPKM + 0.1) values from biological duplicates. The red and blue points mark highly induced and repressed genes, respectively, i.e., genes with fold change greater than 4 for at least one post-LPS time point. The pink and sky blue points mark modestly induced and repressed genes, respectively, i.e., those with fold change between 2 and 4 for at least one post-LPS time point. The black points show the remaining genes. Green dashed line indicates the diagonal. The color coding of genes is the same for all the plots to keep track of the LPS-regulated genes in plots comparing Dex-treated and Dex-untreated samples. See also Figure S1.
Immunity  , e5DOI: ( /j.immuni )
Copyright © Terms and Conditions

4. Figure 2
Classification of Genes Based On Sensitivity to Dex and the Treatment Timing
(A) Heatmap of three gene classes from RNA-seq obtained by a robust K-means clustering method, partitions around the medoids (see STAR Methods). The largest cluster (“Transrepression by GR”) is shown on top. The other two clusters are further distinguished as highly LPS-induced genes and moderately LPS-induced genes, both of which show little effects from Dex treatments (“Dex-insensitive”). The color scale displays log2 FPKM ratios with respect to the LPS 0 h values. Values from biological duplicates are shown in separate columns for each sample.
(B) Gene ontology of genes sensitive to the timing of Dex treatment. For the lists of genes, see Tables S1–S3. See also Figure S2.
Immunity  , e5DOI: ( /j.immuni )
Copyright © Terms and Conditions

5. Figure 3
Dex Treatments Globally Inhibit Genomic Occupancy of RelA Subunit of NF-κB
See Figure S3A for experimental scheme of BMDM treatment and chromatin sample collection.
(A–C) Genome browser shots of RelA ChIP-seq on representative genes from the clusters: Tnf (cluster 2) (A), Ccr7 (cluster 3) (B), Sfpi1 (cluster 1) (C). Data tracks show ChIP density normalized for sequencing depth. Genomic coordinates in mm9.
(D) Scatterplots of ChIP-seq density showing global loss of RelA binding in Dex-treated BMDMs.
(E) Occurrences of RelA binding near genes grouped by the clusters from RNA-seq analysis. Violin plots show the number of 3 h RelA ChIP-seq sites within 20 kb of TSS for genes in the indicated clusters or subsets. “LPS-induced_1” corresponds to the first cluster of genes from the top of heatmap in Figure 2A, etc. The thick box inside each violin indicates the top 25 percentile and bottom 25 percentile marks, and the median is labeled by a white dot. ∗, ∗∗, ∗∗∗ indicate statistically significant differences between the two groups with Welch two-sample t test p values of 1.3 × 10−7, 5.9 × 10−11, 2.3 × 10−14, respectively. See also Figures S3 and S4.
Immunity  , e5DOI: ( /j.immuni )
Copyright © Terms and Conditions

6. Figure 4 GR Binding Is Greatly Enhanced in LPS-Activated BMDMs
(A and B) Genome browser shots of RelA and GR ChIP-seq on Serpine1 (A) and Ccr7 (B) loci. Boxed regions show highly increased GR binding co-localized with LPS-induced RelA binding. Data tracks show ChIP density normalized for sequencing depth. Genomic coordinates in mm9.
(C) Comparison of the two GR binding repertoires in BMDMs in the absence or presence of LPS. The Venn diagram is drawn with areas proportional to the sizes of subsets shown.
(D) Distance from de novo GR binding sites or from pre-existing (before LPS) GR sites to LPS 3h RelA ChIP-seq sites. The two sets of GR binding sites have significantly different distributions (Kolmogorov-Smirnov two-sample test p value = 2.2 × 10−16).
(E) Occurrences of GR binding near genes grouped by the clusters from RNA-seq analysis. Violin plots show the number of post-LPS GR ChIP-seq sites within 20 kb of TSS for genes in the indicated clusters or subsets. The violin plots were generated in the same manner as in Figure 3E. The “Dex-regulated” set consists of genes which were expressed independently of LPS but differentially regulated by Dex. See also Figures S3, S5, and S6.
Immunity  , e5DOI: ( /j.immuni )
Copyright © Terms and Conditions

7. Figure 5
Chromatin Accessibility Near Dex-Sensitive Genes after Dex Treatments
(A–D) Genome browser shots of DNase-seq and RelA and GR ChIP-seq at Klf6 (A), Tnf (B), Lta (B), Ltb (B), Il12b (C), Clec5a (D). The gene names have been color-coded to indicate their gene cluster membership in Figure 2. Boxed regions show whether the DNase density is decreased in Dex-treated conditions, as indicated at the bottom of browser shots. Data tracks show ChIP or DNase density normalized for sequencing depth. Genomic coordinates in mm9.
(E) DNase density in BMDMs pre-treated (left) or late-treated (right) with Dex versus DNase density from Dex untreated BMDMs (8 h LPS alone). Colored points mark distal (> 1 kb from TSS) DHSs within 10 kb of LPS-induced genes. Logratios of RNA-seq FPKM values are color-coded as indicated in the scale bar. The ratios FPKM(LPS 10 h) / FPKM(LPS 10 h, Dex pre-treated) (left) and FPKM(LPS 10 h) / FPKM(LPS 10 h, Dex late-treated) (right) are shown to match the DNase conditions. DNase density data are representative of two independent experiments. See also Figures S5 and S7.
Immunity  , e5DOI: ( /j.immuni )
Copyright © Terms and Conditions

8. Figure 6
Chromatin Accessibility Is Similarly Modulated by Dex Pre- and Late-Treatments
(A) Genome browser shots of Ccl2-Ccl7-Ccl11-Ccl12-Ccl8 locus.
(B) Genome browser shots of Glul and Wee1 loci. Data tracks show density normalized for sequencing depth. Genomic coordinates in mm9. DHSs whose intensity change after Dex treatment are marked with colored boxes as indicated.
(C) Violin plots of Dex-specific changes in DNase-seq intensity at DHSs within 10 kb of genes in the indicated groups. White dots mark median values. Thick bars end at 25 percentile and 75 percentile values. Dex-induced genes are defined as those whose expression is at least 2-fold increased in Dex pre-treated BMDMs (LPS 0 h or LPS 4 h). Asterisks mark statistically significant difference of the group with respect to any of the other two groups (Welch two-sample t test p < 2.2 × 10−16). See also Figure S7.
Immunity  , e5DOI: ( /j.immuni )
Copyright © Terms and Conditions

9. Figure 7
Binding of GR to Anti-Inflammatory Target Genes Does Not Require LPS Stimulation
(A–D) Genome browser shots of GR and RelA ChIP-seq at genes encoding negative regulators of NF-κB and AP-1 signaling: Nfkbia, Tnfaip3, Dusp1, Tsc22d3. Data tracks show ChIP density normalized for sequencing depth. Genomic coordinates in mm9.
Immunity  , e5DOI: ( /j.immuni )
Copyright © Terms and Conditions