Enhancer of zeste homolog 2 (Ezh2) controls bone formation and cell cycle progression during osteogenesis in mice

Abstract

Epigenetic mechanisms control skeletal development and osteoblast differentiation. Pharmacological inhibition of the histone 3 Lys-27 (H3K27) methyltransferase enhancer of zeste homolog 2 (EZH2) in WT mice enhances osteogenesis and stimulates bone formation. However, conditional genetic loss of <i>Ezh2</i> early in the mesenchymal lineage (<i>i.e.</i> through excision via <i>Prrx1</i> promoter-driven Cre) causes skeletal abnormalities due to patterning defects. Here, we addressed the key question of whether <i>Ezh2</i> controls osteoblastogenesis at later developmental stages beyond patterning. We show that <i>Ezh2</i> loss in committed pre-osteoblasts by Cre expression via the osterix/<i>Sp7</i> promoter yields phenotypically normal mice. These Ezh2 conditional knock-out mice (Ezh2 cKO) have normal skull bones, clavicles, and long bones but exhibit increased bone marrow adiposity and reduced male body weight. Remarkably, <i>in vivo Ezh2</i> loss results in a low trabecular bone phenotype in young mice as measured by micro-computed tomography and histomorphometry. Thus, <i>Ezh2</i> affects bone formation stage-dependently. We further show that <i>Ezh2</i> loss in bone marrow-derived mesenchymal cells suppresses osteogenic differentiation and impedes cell cycle progression as reflected by decreased metabolic activity, reduced cell numbers, and changes in cell cycle distribution and in expression of cell cycle markers. RNA-Seq analysis of <i>Ezh2</i> cKO calvaria revealed that the cyclin-dependent kinase inhibitor <i>Cdkn2a</i> is the most prominent cell cycle target of <i>Ezh2</i> Hence, genetic loss of <i>Ezh2</i> in mouse pre-osteoblasts inhibits osteogenesis in part by inducing cell cycle changes. Our results suggest that <i>Ezh2</i> serves a bifunctional role during bone formation by suppressing osteogenic lineage commitment while simultaneously facilitating proliferative expansion of osteoprogenitor cells.