Panel A shows a dot plot correlating RNA sequencing differential gene expression with ATAC sequencing chromatin accessibility changes in Cd11cCre positive Rxrab deficient immature alveolar macrophages. Panel B shows bar graphs of genomic distances between differentially accessible chromatin regions and nearest transcription start sites in control and Cd11cCre positive Rxrab deficient immature alveolar macrophages. Panel C shows donut charts illustrating genomic feature distributions of chromatin regions closed or opened in Cd11cCre positive Rxrab deficient immature alveolar macrophages. Panel D shows a Venn diagram comparing genes associated with chromatin regions opened or closed in Cd11cCre positive Rxrab deficient mature alveolar macrophages. Panel E shows a bubble plot of significantly enriched Gene Ontology biological processes associated with genes linked to open and closed chromatin regions. Panel F shows genomic heatmaps of RXR chromatin binding signals around transcription start sites in control and Cd11cCre positive Rxrab deficient mature alveolar macrophages. Panel G shows a bubble plot of enriched Gene Ontology biological processes associated with RXR controlled genes involved in apoptosis, lipid metabolism, proliferation, and differentiation. Panel H shows MA plots of RXR chromatin immunoprecipitation peaks highlighting proximal and distal H3K27ac associated binding regions in alveolar macrophages. Panel I shows an MA plot illustrating overlap between differentially accessible ATAC sequencing peaks and RXR chromatin binding peaks in mature alveolar macrophages.
ATAC-seq and genome-wide binding show the importance of RXR signaling for AM function, identity, and chromatin regulation. (A) Dot plot showing the correlation between the logFC values of RNA-seq–based DEGs and the logFC values of differentially accessible regions (DARs) obtained by ATAC-seq in Cd11cCre+Rxrabfl/fl ImmAMs versus Rxrabfl/fl mAMs. DARs and DEGs were paired according to the nearest gene detected for each DAR. (B) Distribution of nearest gene TSS distances for DARs in Cd11cCre+Rxrabfl/fl ImmAMs and Rxrabfl/fl mAMs. (C) Distribution of genomic feature associations for DARs in Cd11cCre+Rxrabfl/fl ImmAMs and Rxrabfl/fl mAMs. (D) Venn diagram comparing the collections of genes identified as nearest to open and closed DARs, as identified by ATAC-seq. (E) Significantly enriched GO terms detected with the PANTHER statistical enrichment test tool (P < 0.05 with Bonferroni correction) for the collections of genes exclusively associated with closed regions (green) or open regions (orange), as described by the Venn diagram in E. (F) Genomic heatmaps of RXRα binding signals located in TSS-flanking regions (±5 kb) in sorted mAMs from 9-wk-old Rxrabfl/fl and Cd11cCre+Rxrabfl/fl mice (n = 2 per genotype) (FC ≤ −2 or ≥2, FDR <0.05). (G) Significantly enriched GO terms from RXR-controlled genes detected with the PANTHER statistical overrepresentation test tool (P ≤ 0.05 with Bonferroni correction). (H) MA plots for RXRα ChIPmentation peaks. Orange and blue dots represent 6,365 peaks immunoprecipitated with anti-RXRα antibody exclusively in Rxrabfl/fl mAMs. Dots outlined in black represent peaks that overlap with H3K27ac regions classified as proximal to genes (left plot) or distal (right plot). (I) MA plot showing the overlap between differentially accessible ATAC-seq peaks (FDR <0.05) with RXRα HT-ChIPmentation peaks in mAMs. Colored dots correspond to differentially accessible ATAC-seq peaks with a Log2(FC)xLog2(concentration) value ≥0.65; light red, differentially open peaks with no RXR peak; light blue, differentially closed peaks with no RXR peak; dark red, differentially open peaks colocalizing with RXR peaks; dark blue, differentially closed peaks colocalizing with RXR peaks.