Panel A shows a bar graph of predicted upstream transcriptional regulators enriched among differentially expressed genes in Cd11cCre positive Rxrab deficient mature alveolar macrophages versus control. Panel B shows a Venn diagram comparing overlaps between differentially expressed genes in Cd11cCre positive Rxrab deficient mature alveolar macrophages and published Cd11cCre positive Pparγ deficient alveolar macrophage datasets. Panel C shows a Venn diagram of overlapping downregulated genes shared between Cd11cCre positive Rxrab deficient and Cd11cCre positive Pparγ deficient alveolar macrophages. Panel D shows a Venn diagram of overlapping upregulated genes shared between Cd11cCre positive Rxrab deficient and Cd11cCre positive Pparγ deficient alveolar macrophages. Panel E shows heatmaps of apoptosis related genes differentially expressed across control, Cd11cCre positive Rxrab deficient mature alveolar macrophages, immature alveolar macrophages, and Cd11cCre positive Pparγ deficient alveolar macrophages. Panel F shows heatmaps of transcription factor identity genes differentially expressed across control, Cd11cCre positive Rxrab deficient mature alveolar macrophages, immature alveolar macrophages, and Cd11cCre positive Pparγ deficient alveolar macrophages. Panel G shows heatmaps of lipid metabolism related genes dysregulated across control, Cd11cCre positive Rxrab deficient mature alveolar macrophages, immature alveolar macrophages, and Cd11cCre positive Pparγ deficient alveolar macrophages. Panel H shows heatmaps of proliferation related genes differentially expressed across control, Cd11cCre positive Rxrab deficient mature alveolar macrophages, immature alveolar macrophages, and Cd11cCre positive Pparγ deficient alveolar macrophages. Panel I shows heatmaps of alveolar macrophage core identity genes differentially expressed across control, Cd11cCre positive Rxrab deficient mature alveolar macrophages, immature alveolar macrophages, and Cd11cCre positive Pparγ deficient alveolar macrophages. Panel J shows a bubble plot of significantly enriched Gene Ontology biological processes associated with inflammatory responses, metabolism, proliferation, and lipid regulation in Cd11cCre positive Pparγ deficient alveolar macrophages. Panel K shows motif enrichment analysis identifying transcription factor binding motifs enriched within PPARγ chromatin binding peaks. Panel L shows UCSC genome browser tracks displaying RXR, PPARγ, and H3K27ac chromatin peaks at representative alveolar macrophage genes including Spi1, Fabp1, Cd36, and Cidec. Panel M shows a Venn diagram comparing overlap between genes nearest to PPARγ and RXR chromatin binding peaks identified by HT-ChIPmentation. Panel N shows a donut chart illustrating genomic feature distribution of overlapping PPARγ and RXR chromatin binding peaks across promoters, introns, exons, untranslated regions, and intergenic regions. Panel O shows a bar graph quantifying associations between overlapping PPARγ-RXR peaks and proximal or distal H3K27ac chromatin regions.
Comparative transcriptomic analysis reveals RXR–PPARγ-driven co-regulation in AMs. (A) Selected upstream transcriptional regulators identified by IPA of DEGs from the comparison between Cd11cCre+Rxrabfl/fl and Rxrabfl/fl mAMs. Significant regulators were defined as –log10 (Benjamini–Hochberg P value) >1.3. (B) Venn diagram of overlaps between DEGs identified in Cd11cCre+Rxrabfl/fl mAMs (bulk RNA-seq) and DEGs from publicly available Cd11cCre+Pparγfl/fl AM transcriptomic data. (C and D) Venn diagrams showing overlapping downregulated (C) and upregulated (D) genes between our Cd11cCre+Rxrabfl/fl RNA-seq dataset and the publicly available Cd11cCre+Pparγfl/fl AM dataset. (E–I) Heatmap of dysregulated genes associated with selected biological processes in Rxrabfl/fl mAMs, Cd11cCre+Rxrabfl/fl mAMs, and Cd11cCre+Rxrabfl/fl ImmAMs, compared with Pparγfl/fl AMs and Cd11cCre+Pparγfl/fl AMs. (J) Significantly enriched GO terms derived from the comparison between Cd11cCre+Pparγfl/fl and Pparγfl/fl AMs (PPARγ array data), identified using the PANTHER statistical enrichment test tool (P < 0.05, Bonferroni correction). (K) HOMER motif enrichment analysis of PPARγ peaks. (L) UCSC genome browser tracks showing RXRα, PPARγ, and H3K27ac HT-ChIPmentation peaks at representative AM genes. Blue boxes indicate regions with peaks identified across datasets. (M) Overlap analysis of genes nearest to PPARγ and RXRα peaks identified by HT-ChIPmentation. (N) Genomic feature distribution of the 4,547 overlapping PPARγ–RXRα peaks. (O) Bar plot showing the association between the 4,547 overlapping PPARγ–RXRα peaks and H3K27ac regions classified as proximal or distal according to distance from the nearest gene.*P <; Test of equal or given proportions (prop.test function in R).