Evidence that miR-152-3p is a positive regulator of SETDB1-mediated H3K9 histone methylation and serves as a toggle between histone and DNA methylation

Abstract

SETDB1 is a histone methyltransferase that converts H3K9me2 to H3K9me3. SETDB1 activity and H3K9me3 are crucial for the formation of obligately silenced heterochromatin such as that of centromeres. Here we show that a microRNA, miR-152-3p, is involved in the regulation of SETDB1 protein levels, but surprisingly, miR-152-3p plays a positive regulatory role for SETDB1 expression. Inhibition of miR-152-3p by anti-miR treatment resulted in a robust reduction in SETDB1 protein levels, though SETDB1 mRNA levels were unaffected. This was also accompanied by a blockade of the biochemical pathway proceeding from H3K9me2 to H3K9me3 as evidenced by quantitative nucleosome ELISA assays that showed that H3K9me2 accumulates in cells treated with an anti-miR that targets miR-152-3p. In addition, the action of a miR-152-3p mimic increased flux of the reaction leading to H3K9me3. We also performed site-directed mutagenesis of three predicted miR-152-3p target recognition sequences to yield three precise deletions. Deletion of one of the three sites recapitulated the positive regulatory aspect of the action of miR-152-3p upon SETDB1 expression in a luciferase reporter assay. Previous studies have shown that miR-152-3p negatively regulates DNMT1, the sole maintenance DNA methyltransferase which is required for levels of 5-methylcytosine levels within DNA. Our results shown that miR-152-3p positively regulates the production of H3K9me3 by regulating the production of SETDB1. Therefore, our findings provide strong evidence that miR-152-3p can serve as a toggle switch that regulates the balance between DNA methylation and H3K9 histone methylation in constitutive heterochromatin.