Insights into negative regulation by the glucocorticoid receptor from genome-wide profiling of inflammatory cistromes

Abstract

How the glucocorticoid receptor (GR) activates some genes while potently repressing others remains an open question. There are three current models for suppression: transrepression via GR tethering to AP-1/NF-κB sites, direct GR association with inhibitory elements (nGREs), and GR recruitment of the corepressor GRIP1. To gain insights into GR suppression, we used genomic analyses and genome-wide profiling of GR, p65, and c-Jun in LPS-stimulated macrophages. We show that GR mediates both activation and repression at tethered sites, GREs, and GRIP1-bound elements, indicating that motif classification is insufficient to predict regulatory polarity of GR binding. Interestingly, sites of GR repression utilize GRIP1's corepressor function and display reduced histone acetylation. Together, these findings suggest that while GR occupancy confers hormone responsiveness, the receptor itself may not participate in the regulatory effects. Furthermore, transcriptional outcome is not established by sequence but is influenced by epigenetic regulators, context, and other unrecognized regulatory determinants.

Figure 1. GR, AP-1 and NF-κB cistromes intersect

a) Area-proportional Venn diagram of GR cistromes in unstimulated (orange) and stimulated (red) macrophages. b) Motif analysis of the GR cistrome in macrophages treated with Dex only. c) Area-proportional Venn diagram showing the overlap between GR, c-Jun and p65 ChIP-Seq peaks in macrophages treated with LPS and Dex. Genomic regions with significant peak scores were merged and given a 100bp margin for co-occurrence. d) Globally enriched motifs within the GR, c-Jun and p65 cistromes in response to LPS. Tables show the top five to six statistically overrepresented binding sites.

Figure 2. Genomic localization of GR, AP-1 and NF-κB binding in macrophages

ChIP-sequencing tracks of regulatory regions of GR target genes in macrophages treated with LPS and/or Dex, normalized to 10 million reads. a) IL6 locus with overlapping GR, p65 and c-Jun occupancy. b) IL6 proximal promoter displaying GR binding only in the presence of c-Jun and p65. c) Ccl2 locus with overlapping GR, p65 and c-Jun binding. GR binding is LPS-independent. d) Irf1 locus with GR binding induced by LPS, but independent of p65 and c-Jun (arrow).

Figure 3. Correlating gene expression and recruitment in response to LPS and Dex

a) Heat map cluster showing the numbers and overlap of microarray probe sets that were upregulated (red) or downregulated (light green) at least 1.5fold in macrophages treated with LPS and/or Dex. b) Graph showing ChIP peaks assigned to differentially expressed genes and correlating gene expression with occupancy by GR, c-Jun and p65. Percentages and numbers reflect fractions of GR ChIP peaks.

Figure 4. Activation versus repression by GR

a) Luciferase assays using reporters containing ChIP peak sequences that were linked to activated genes. Black bars represent percent induction in response to Dex. Bottom legend lists occupancy of GR/c-Jun/p65 as determined by ChIP-Seq in primary macrophages. b) De novo motif discovery on ChIP peak sequences that were assigned to genes activated by Dex. c) Luciferase assays using reporters containing ChIP peak sequences that were assigned to repressed genes. Black bars represent percent repression in response to Dex. Bottom legend lists occupancy of GR/c-Jun/p65 as determined by ChIP-Seq in primary macrophages. d) De novo motif discovery on ChIP peak sequences that were assigned to genes repressed by Dex. e) Luciferase assays using reporters that were repressed by GR in c) and that now contain a point mutation in the GRE motif. f) Luciferase assays performed in CV-1 cells (devoid of endogenous GR) using GR point mutants that abolish DNA binding (C421G and C441G). Error bars represent S.E.M.

Figure 5. GR recruits GRIP1 to activated and repressed genes

a) Area-proportional Venn diagram of GRIP1 cistromes in LPS-stimulated (light blue) and Dex-treated (cyan) macrophages. b) Area-proportional Venn diagram showing the overlap between GRIP1 and GR ChIP-Seq peaks in macrophages treated with LPS and Dex. Arrows and numbers on bottom right represent ChIP peaks associated with GR-repressed versus –activated genes. c) Globally enriched motifs found in the GRIP1 cistrome in response to LPS+Dex. d) ChIP-sequencing tracks of regulatory regions of GR target genes in macrophages treated with LPS plus or minus Dex, normalized to 10 million reads. Ligand-dependent GRIP1 recruitment can be observed at activated (Tlr2, Per1) and repressed (IL6, Ccl2) enhancers.

Figure 6. GR repression includes GRIP-1 corepressor function and opposition of IRF3 activity

a) siRNA knockdown of GR and GRIP-1 in RAW264.7 macrophages treated with LPS and/or Dex. Reduction of both GR and GRIP-1 expression levels impairs repression of LPS-induced genes Il1a, Il6, Nos2, Mmp13 and Tnfsf10 by Dex, but only GR knockdown affects the positively regulated gene Gilz. Brackets above bars show p-values for differential expression in LPS vs. LPS+Dex treated cells, * p <0.01, n.s. not significant. b) siRNA knockdown experiments in RAW264.7 cells. Knockdown of IRF3 affects expression of LPS-induced and Dex-repressed GR target genes, but not Dex-activated genes such as Gilz. Error bars represent S.T.D. c) Representative examples of IRF3 ChIP-Seq tracks. IRF3 binding co-occurs with the presence of GR and GRIP1 at repressed genes such as Il1a, but not at activated ones like Gilz.

Figure 7. Repression by GR involves selective HDAC recruitment and histone deacetylation

a) ChIP-Seq experiments in primary macrophages show a reduction in acetylated histone H3K9 marks at sites repressed in response to Dex, in both unstimulated and in TLR4-activated conditions. b) At repressed enhancers such as Nos2, Il6 etc., Dex treatment leads to increased recruitment of HDAC2 and/or HDAC3, as determined by ChIP experiments. c) Conversely, HAT activity is increased in response to Dex at activated enhancers (i.e. Dusp1), as determined by ChIP experiments using α-CBP and α-p300 antibodies, but not at repressed loci. Error bars represent S.T.D.