RNA-sequencing and ChIP-sequencing in murine mesenchymal stem cells treated with Ezh2 inhibitor GSK126 or ß-catenin siRNA

During mesenchymal stem cell (MSC) differentiation, both Wnt signaling and the development of a rigid cytoskeleton promote commitment to the osteoblastic over adipogenic lineage. ß-catenin is thought to play a critical role in Wnt effects. We show that ß-catenin was additive with cytoskeletal signals to prevent adipogenesis, and ß-catenin knockdown promoted adipogenesis even when the actin cytoskeleton was depolymerized. ß-catenin also prevented osteoblast commitment in a cytoskeletal-independent manner, with ß-catenin knockdown enhancing lineage commitment. Chip-seq showed that ß-catenin associated with the promoter of EZH2, a key component of the PRC2 complex that governs genome methylation. Knocking down ß-catenin lowered EZH2 levels and H3K27me3 at osteogenic loci. Further, when EZH2 was inhibited, ß-catenin 's anti-differentiation effects were lost. These results indicate that regulating EZH2 activity is key to ß-catenin effects on MSC to preserve MSC multipotentiality. Overall design: RNA-sequencing of 3 groups (non-targeting siRNA, ß-catenin siRNA, and GSK126 treated) each with 3 biological replicates. ChIP-sequencing for H3K27me3 in one biological replicate from each group (non-targeting siRNA, ß-catenin siRNA, and GSK126 treated).