Ezh2 Inhibition is Bone-anabolic and Osteoprotective in Skeletally Mature Mice

We report genome-wide effects of Ezh2 inhibition (GSK126) in MC3T3 sc4 pre-osteoblasts by ChIP-Seq (H3K27me3) and mRNA-Seq (transcriptome). Treatment of cells for 24 hours with GSK126 suppresses genome-wide H3K27me3, most notably near the transcriptional start sites (TSSs). Genes that exhibit suppression of H3K27me3 near TSSs as a result of GSK126 tend to be up-regulated at the mRNA levels (mRNA-Seq) as well. The mRNA-Seq analysis demonstrates that osteogenic genes are up-regulated after the administration of GSK126. These pro-osteogenic effects are seen 3, 6, and 10 days after the initial GSK126 treatment. MC3T3 cells were seeded in maintenance medium (10,000 cells/cm2). The following day, maintenance medium was replaced with osteogenic medium (50 µg/ml ascorbic acid and 4 mM beta glycerol phosphate) containing vehicle or Ezh2 inhibitor (5µM GSK126). Three days later, vehicle or Ezh2 inhibitors were added again along with osteogenic medium. On day 6, Ezh2 inhibitor and vehicle were removed and fresh osteogenic medium was added with media changes scheduled every three days. 24 hours after the first drug/DMSO administration, ChIP assay was performed using a H3K27me3 (17-622, Lot 2213948, Millipore) antibody. The resulting DNA along with input DNA was submitted for ChIP-Seq analysis. RNA-seq was performed using RNA isolated at days 3, 6, and 10 from cells treated with vehicle or GSK126 in the presence of osteogenic differentiaiton.