NFIB–MLL1 complex is required for the stemness and Dlx5-dependent osteogenic differentiation of C3H10T1/2 mesenchymal stem cells

Abstract

Despite significant progress in our understanding of the molecular mechanisms of mesenchymal stem cell (MSC) differentiation, less is known about the factors maintaining the stemness and plasticity of MSCs. Here we show that the NFIB/MLL1 complex plays key roles in osteogenic differentiation and stemness of C3H10T1/2 MSCs. We find that depletion of either NFIB or MLL1 results in a severely hampered osteogenic potential and failed activation of key osteogenic transcription factors, such as Dlx5, Runx2, and Osx, following osteogenic stimuli. In addition, the NFIB/MLL1 complex binds directly to the promoter of Dlx5, and exogenous expression of Myc-Dlx5, but not the activation of either the BMP- or the Wnt-signaling pathway, is sufficient to restore the osteogenic potential of cells depleted of NFIB or MLL1. Moreover, ChIP and ChIP-seq analysis showed that the NFIB/MLL1 complex mediates the deposition of trimethylated histone H3K4 (H3K4me3) at both Dlx5 and Cebpa, key regulator genes that function at the early stages of osteogenic and adipogenic differentiation, respectively, in uncommitted C3H10T1/2 MSCs. Surprisingly, the depletion of either NFIB or MLL1 leads to decreased H3K4me3 and results in elevated H3K9me3 at those developmental genes. Furthermore, gene expression profiling and ChIP-seq analysis revealed lineage-specific changes in chromatin landscape and gene expression in response to osteogenic stimuli. Taken together, these data provide evidence for the hitherto unknown role of the NFIB/MLL1 complex in the maintenance and lineage-specific differentiation of C3H10T1/2 mesenchymal stem cells and support the epigenetic regulatory mechanism underlying the stemness and plasticity of mesenchymal stem cells.