Fig 2: EGR1 represses activity of inflammatory enhancers and their target genes in mature macrophages.(A) ATAC-seq data (21) show that enhancers repressed by EGR1 during macrophage differentiation increase their accessibility upon mature macrophage activation with LPS. (B) Immunoblot and RNA-seq display EGR1 overexpression obtained by infecting macrophages with a FLAG-EGR1 pLenti vector after 4 days of differentiation. (C) ChIP-qPCR for H3K27ac: In condition of EGR1 overexpression and after LPS treatment, EGR1-gaining enhancers associated to inflammatory genes are significantly less acetylated than in the EGR1–wild-type (WT) condition (*P < 0.05, **P < 0.005, and ***P < 0.0005). (D) From the publicly available list of human LPS-responsive genes (21), we selected a subset of 271 genes that were LPS-responsive in our system (FPKM fold change of >1.5 between –LPS and +LPS) and targets of EGR1-gaining enhancers (i.e., representing the closest gene to an EGR1-gaining enhancer). RNA-seq indicates that, in EGR1 overexpression condition, the 271 genes are not properly up-regulated after LPS treatment when compared to EGR1-WT condition. (E) Volcano plot depicting the loss of gene expression in FLAG-EGR1 versus EGR1 wild-type macrophages as determined by RNA-seq in biological duplicates. The blue dots are down-regulated genes, and the red dots are up-regulated genes (fold change > 2, FDR < 0.1). Orange dots highlight either up-regulated or down-regulated genes that are regulated by interferon-γ (INF-γ genes) according to ingenuity pathway analysis. FC, fold change.

Fig 3: EGR1 down-regulates enhancer activity and gene expression during macrophage differentiation.(A) Immunoblot confirms depletion of EGR1 protein levels upon treatment with short hairpin RNA (shRNA). (B) Average profiles and boxplots of ChIP-seq data for H3K27ac suggest that EGR1 depletion only affects acetylation in regions of the “repressed site” cluster. Loss of EGR1 produces increase of acetylation in these regions. P values were determined via Wilcoxon’s rank-sum test. n.s., not significant. (C) RNA-seq conducted in shEGR1 condition shows that depletion of EGR1 results in more than 900 differentially expressed genes (FDR < 5%). (D) 481 of the 893 genes differentially expressed upon EGR1-knockdown (EGR1-KD) are also the closest gene to an EGR1-gaining enhancer. FPKM, fragments per kilobase of exon per million fragments mapped. (E) Screening of H3K27ac levels in the EGR1-gaining enhancers associated to the 481 genes reveals that the enhancers associated to the up-regulated genes significantly gain acetylation upon EGR1-KD (Fisher’s exact test, P < 2.2 × 10−16).

Fig 4: EGR1 binding correlates with both enhancer activation and enhancer repression.ChIP-seq for H3K27ac (21) and ATAC-seq data (this study) suggest that the EGR1-gaining distal regions can be divided into three main clusters: (A) regions not active in monocytes that gain acetylation and accessibility following EGR1 binding (ACTIVATED cluster), (B) regions active in monocytes that lose acetylation and accessibility following EGR1 binding (REPRESSED cluster), and (C) a small cluster of 900 regions not acetylated and with accessibility not affected by EGR1 binding. (D) Sequence-based motif analysis for the three clusters of EGR1-gaining distal regions. The activated cluster is enriched for the EGR1/2 motif, while the same motif is not present in the repressed cluster, suggesting that EGR1 may be recruited at these regions by other cofactors. All heatmaps depict one replicate per condition and are normalized by sequencing depth across conditions.

Fig 5: EGR1 blunts the inflammatory response in human macrophages.(A) Absorbance values of a cytokine panel ELISA show reduced secretion of select cytokines in EGR1-overexpressing macrophages upon stimulation with LPS. Of the cytokines assayed, IL-6 levels were significantly decreased by ~13%, while IL-12 and TNFα levels were decreased by ~40 and 80%, respectively, as compared to control macrophages after LPS stimulation (**P < 0.01, ***P < 0.001, and ****P < 0.0001). (B) Flow cytometry results of EGR1-overexpressing macrophages exhibit a lack of surface expression of the costimulatory signaling marker CD86 as compared to control macrophages after LPS stimulation. (C) Flow cytometry further shows a distinct population of PDL1(CD274)+- and CD80+-coexpressing cells in EGR1-overexpressing macrophages as compared to control macrophages. (D) Diagrammatic representation of the role of EGR1 in monomacrophagic cells. EGR1 associates with enhancers of both developmental and proinflammatory genes. At developmental enhancers, EGR1 is recruited by its DNA motif and promotes activation, especially during early monocytic commitment (12). Conversely, EGR1 is indirectly recruited at proinflammatory enhancers (likely via repressive TFs) during macrophage differentiation. EGR1 recruitment at inflammatory enhancers leads to deacetylation and loss of accessibility by way of the NuRD-repressive complex.