Loss of Histone H3 K79 Methyltransferase Dot1l Facilitates Kidney Fibrosis by Upregulating Endothelin 1 through Histone Deacetylase 2

Significance Statement If and how endothelin 1, a potent vasoconstrictor with proinflammatory and profibrotic properties, is upregulated to promote CKD is largely unknown. Emerging evidence has suggested that histone H3 K79 methyltransferase Dot1l exerts an antifibrotic effect by repressing the gene encoding endothelin 1 ( Edn1 ) in the connecting tubule/collecting duct. The authors demonstrate in connecting tubule/collecting duct–specific Dot1l conditional knockout mice that disruption of Dot1l facilitates CKD development by upregulating endothelin 1, and that Dot1l and Edn1 double-knockout mice have a significantly attenuated CKD phenotype. Dot1l and histone deacetylase 2 mutually inhibit their association with the Edn1 promoter to regulate endothelin 1 production. This study is the first to define Dot1l as an epigenetic modifier of CKD, establishes a new CKD mouse model, and reveals a novel mechanism regulating Edn1 transcription. Background The progression rate of CKD varies substantially among patients. The genetic and epigenetic contributions that modify how individual patients respond to kidney injury are largely unknown. Emerging evidence has suggested that histone H3 K79 methyltransferase Dot1l has an antifibrotic effect by repressing Edn1 , which encodes endothelin 1 in the connecting tubule/collecting duct. Methods To determine if deletion of the Dot1l gene is a genetic and epigenetic risk factor through regulating Edn1, we studied four groups of mice: wild-type mice, connecting tubule/collecting duct–specific Dot1l conditional knockout mice ( Dot1lAC ), Dot1l and Edn1 double-knockout mice ( DEAC ), and Edn1 connecting tubule/collecting duct–specific conditional knockout mice ( Edn1AC ), under three experimental conditions (streptozotocin-induced diabetes, during normal aging, and after unilateral ureteral obstruction). We used several approaches (colocalization, glutathione S-transferase pulldown, coimmunoprecipitation, yeast two-hybrid, gel shift, and chromatin immunoprecipitation assays) to identify and confirm interaction of Dot1a (the major Dot1l splicing variant in the mouse kidney) with histone deacetylase 2 (HDAC2), as well as the function of the Dot1a-HDAC2 complex in regulating Edn1 transcription. Results In each case, Dot1lAC mice developed more pronounced kidney fibrosis and kidney malfunction compared with wild-type mice. These Dot1lAC phenotypes were ameliorated in the double-knockout DEAC mice. The interaction between Dot1a and HDAC2 prevents the Dot1a-HDAC2 complex from association with DNA, providing a counterbalancing mechanism governing Edn1 transcription by modulating H3 K79 dimethylation and H3 acetylation at the Edn1 promoter. Conclusions Our study confirms Dot1l to be a genetic and epigenetic modifier of kidney fibrosis, reveals a new mechanism regulating Edn1 transcription by Dot1a and HDAC2, and reinforces endothelin 1 as a therapeutic target of kidney fibrosis.