Inactivation of the polycomb repressive complex 2 (PRC2) in osteoblasts increases post-natal trabecular bone mass and mechanical bone strength.

The epigenetic methyltransferases Ezh1 or Ezh2 each interact with two structural proteins (Eed and Suz12) to form Polycomb Repressive Complex 2 (PRC2). Inhibition of Ezh2 stimulates osteoblast differentiation and bone formation, but Ezh2 conditional knockout (cKO) mice have variable skeletal phenotypes. Ezh1 null mice are phenotypically normal. Here, we show that combined homozygous ablation of both Ezh1 and Ezh2 alleles results in a pronounced bone phenotype, suggesting genetic redundancy between Ezh1 and Ezh2. Transient loss of Eed, Suz12 or Ezh2 by siRNA depletion or treatment with distinct PRC2 inhibitors (A395, EED226 and GSK126) enhances differentiation of MC3T3-E1 pre-osteoblasts (i.e., increased Bglap, Ibsp and mineralization). We compared Ezh2 cKO mice with Ezh1 null (KO) mice, and mice with both Ezh2 cKO and Ezh1 KO alleles (dKO mice) in the presence of the Osterix/Sp7-Cre recombination driver. During active bone development (post-natal 8 weeks), both male and female dKO mice exhibit significantly increased L5 trabecular bone mass, femoral cortical thickness and the relative midshaft bone volume (μCT), as well as bone strength (3-point bending) compared to Ezh1 KO or Ezh2 cKO mice. Suppression of Cre recombination using doxycycline (DOX) for 8 weeks prevents the dKO phenotype, but subsequent DOX removal increases trabecular bone mass in dKO mice by 14 weeks. Conditional Eed ablation (which inactivates PRC2/Ezh1 and PRC2/Ezh2) results in a similar high- bone and increased mechanical strength phenotype in male and female Eed cKO. Our findings identify PRC2 as a suppressor of post-natal bone accrual in Osx/Sp7 lineage cells, and a potential target for strategies to improve bone mass accrual.