Epigenetic Regulation of Endothelial Extracellular Matrix Components is Critical for Murine Lung Development

ABSTRACT Background The chromatin remodeling enzymes BRG1 (brahma-related gene 1) and CHD4 (chromodomain helicase DNA binding protein 4) independently regulate transcription of genes critical for vascular development, but their coordinated impact on vessels in late- stage embryos has not been explored. Methods In this study we genetically deleted endothelial Brg1 and Chd4 in mixed background mice ( Brg1 fl/fl ;Chd4 fl/fl ;VE-Cadherin-Cre + ), and littermates that were negative for Cre recombinase were used as controls. Perinatal lung tissue was analyzed by immunostaining, immunoblots, and flow cytometry. Quantitative reverse transcription PCR was used to determine gene expression, and chromatin immunoprecipitation revealed gene targets of BRG1 and CHD4 in cultured endothelial cells (ECs). Results We found that Brg1/Chd4 double mutants died soon after birth with small and compact lungs. Despite having normal cellular composition, distal air sacs of the mutant lungs displayed diminished ECM (extracellular matrix) components and TGFβ (transforming growth factor beta) signaling, which typically promotes matrix synthesis. Transcripts for collagen- and elastin-related genes and the TGFβ ligand Tgfb1 were decreased in mutant lung ECs, but genetic deletion of endothelial Tgfb1 failed to recapitulate the small lungs and ECM defects seen in Brg1/Chd4 mutants. We instead found several ECM genes to be direct targets of BRG1 and CHD4 in cultured ECs. Conclusions Collectively, our data highlight essential roles for ECs in promoting ECM deposition at late stages of embryonic lung development. Moreover, this endothelial ECM production is epigenetically regulated. Abstract Figure HIGHLIGHTS Genetic deletion of the chromatin remodeling enzymes BRG1 and CHD4 in endothelial cells of late-stage mouse embryos ( Brg1/Chd4-ECdko ) results in small and compact lungs at birth. Mutant embryos display reduced collagen IV deposition, dysregulated elastin fibers, and diminished TGFβ1 in the distal air sacs. Our combined in vitro and in vivo analyses indicate that BRG1 and CHD4 epigenetically regulate collagen IV- and elastin-related gene expression in embryonic ECs to promote proper lung development.