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

Abstract

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 <i>Edn1</i>, which encodes endothelin 1 in the connecting tubule/collecting duct. To determine if deletion of the <i>Dot1l</i> gene is a genetic and epigenetic risk factor through regulating <i>Edn1,</i> we studied four groups of mice: wild-type mice, connecting tubule/collecting duct-specific <i>Dot1l</i> conditional knockout mice (<i>Dot1l^AC</i> ), <i>Dot1l</i> and <i>Edn1</i> double-knockout mice (<i>DE^AC</i> ), and <i>Edn1</i> connecting tubule/collecting duct-specific conditional knockout mice (<i>Edn1^AC</i> ), 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 <i>Dot1l</i> splicing variant in the mouse kidney) with histone deacetylase 2 (HDAC2), as well as the function of the Dot1a-HDAC2 complex in regulating <i>Edn1</i> transcription. In each case, <i>Dot1l^AC</i> mice developed more pronounced kidney fibrosis and kidney malfunction compared with wild-type mice. These <i>Dot1l^AC</i> phenotypes were ameliorated in the double-knockout <i>DE^AC</i> mice. The interaction between Dot1a and HDAC2 prevents the Dot1a-HDAC2 complex from association with DNA, providing a counterbalancing mechanism governing <i>Edn1</i> transcription by modulating H3 K79 dimethylation and H3 acetylation at the <i>Edn1</i> promoter. Our study confirms <i>Dot1l</i> to be a genetic and epigenetic modifier of kidney fibrosis, reveals a new mechanism regulating <i>Edn1</i> transcription by Dot1a and HDAC2, and reinforces endothelin 1 as a therapeutic target of kidney fibrosis.