Studies in CRISPR-generated Mediterranean G6PD variant rats reveal G6PD orchestrates genome-wide DNA methylation and gene expression in vascular wall

BACKGROUND: Recent advances have revealed the importance of epigenetic modifications to gene regulation and transcriptional activity. DNA methylation, a determinant of genetic imprinting and de novo silencing of genes genome-wide, is known to be controlled by DNA methyltransferases (DNMT) and demethylases (TET) under disease conditions. However, the mechanism(s)/factor(s) influencing the expression and activity of DNMTs and TETs, and thus DNA methylation, in healthy vascular tissue is incompletely understood. Based on our recent studies, we hypothesized that glucose-6-phosphate dehydrogenase (G6PD) is a modifier of DNMT and TET expression and activity and an enabler of gene expression. METHODS: In aorta of CRISPR-edited rats with the Mediterranean G6PD variant we determined DNA methylation by whole-genome bisulfite sequencing, gene expression by RNA sequencing, and large artery stiffness by echocardiography. RESULTS: Here, we documented higher expression of Dnmt3a, Tet2, and Tet3 in aortas from Mediterranean G6PD(S188F) variant (a loss-of-function single nucleotide polymorphism) rats than their wild-type littermates. Concomitantly, we identified 17,618 differentially methylated loci genome-wide (5,787 hypermethylated loci, including down-regulated genes encoding inflammation- and vasoconstriction-causing proteins, and 11,827 hypomethylated loci, including up-regulated genes encoding smooth muscle cell differentiation- and fatty acid metabolism-promoting proteins) in aorta from G6PD(S188F) as compared to wild-type rats. Further, we observed less large artery (aorta) stiffness in G6PD(S188F) as compared to wild-type rats. CONCLUSIONS: These results establish a noncanonical function of the wild-type G6PD and G6PD(S188F) variant in the regulation of DNA methylation and gene expression in healthy vascular tissue and reveals G6PD(S188F) variant contributes to reduce large artery stiffness.