GSE196583
Inhibition of the epigenetic regulator Ezh2 redistributes bivalent domains within transcriptional regulators associated with Wnt and Hedgehog pathways
Osteoporosis is characterized by bone loss, reduced bone strength, and increased fracture risk. Studies have shown that epigenetic mechanisms play a role during bone formation and bone-related disorders, including osteoporosis.  However, the contribution of bivalent domains, which are chromatin regions that are co-occupied by the gene activating Histone 3 lysine 4 trimethylation (H3K4me3) and the gene suppressive Histone 3 lysine 27 trimethylation (H3K27me3) marks, has not been elucidated during osteogenesis. Previous work demonstrated that the enhancer of zeste homolog 2 (Ezh2), a histone 3 lysine 27 (H3K27) methyltransferase, suppressed differentiation of osteogenic progenitor cells and that a small molecule inhibitor of Ezh2 (GSK126) enhanced osteogenic commitment of osteoblast progenitors in vitro and bone formation in vivo. Tazemetostat (or EPZ6438) is an Ezh2 inhibitor (iEzh2) that is FDA approved for the treatment of epithelioid sarcoma and might be repurposed to mitigate bone loss. The present study aimed to elucidate mechanisms by which Tazemetostat-mediated H3K27me3 reduction regulates bivalent domain associated enhancers critical for bone formation. Tazemetostat enhanced the expression of bone-related genes (e.g., Runx2-p57, Bglap, Ibsp) as measured by qPCR analysis and stimulated extracellular matrix deposition as revealed by alizarin red staining of cell cultures. Western blotting analysis revealed that Tazemetostat reduced H3K27me3 levels and concomitantly enhanced H3K27Ac, as well as increased protein levels of the osteoblastic master regulator Runx2. Integrative analysis of RNA-seq and ChIP-seq datasets revealed that upregulation of group of genes that are associated with bivalent domains. Importantly, gene ontology analysis showed that genes associated with Wnt, Pth and Hh signaling pathways are enriched within these up-regulated genes. Follow-up mechanistic-based studies revealed that the pro-osteogenic effects induced by Ezh2 inhibition are attenuated by inhibition of Hh signaling and enhanced by disruption of Wnt signaling. Our data demonstrate that the genes controlled by bivalent domains are associated with key osteogenic signaling pathways and are induced upon Ezh2 inhibition and subsequent H3K27me3 reduction. Our data also demonstrate that iEzh2-induced osteogenic differentiation is modulated by Wnt and Hh signaling pathways. Overall design: MC3T3-E1 subclone 4 cells Passage 9 were maintained in ?-minimal essential medium without ascorbic acid (Gibco) containing 10% fetal bovine serum (Atlanta Biologicals), 100 units/ml penicillin and 100 ?g/ml streptomycin (Gibco). Cells were seeded in maintenance medium at a density of 10,000 cells/cm2. Next day, cells were treated with vehicle (DMSO) or 2.5µM EPZ6438 (MedChem, HY-13803) in osteogenic medium (maintenance medium supplemented with 50?g/ml ascorbic acid (Sigma) and 10mM ?-glycerol phosphate (Sigma). Three day later, the cells were harvested by trypsin and analyzed by ChIP assay using H3K27me3 (H3K27me3, Cell Signaling, #9733 Lot 8) and H3K4me3 (H3K4me3, Abcam, ab8580 Lot GR3264490-1) antibodies. DNA fragments were isolated using QIAquick PCR Purification Kit (Qiagen). Total RNA was isolated on days three of osteogenic differentiation using the Direct-zol? RNA kit (Zymo Research) and TRIzol reagent (Invitrogen)