The regulatory landscape of osteogenic differentiation

During embryogenesis, body growth and repair the bone substance is formed by osteoblasts, which are derived from mesenchymal stem cells. Bone marrow-derived mesenchymal stroma cells (MSCs) have many of the properties of multipotent stem cells, and can be differentiated towards osteoblasts in vitro, making these cells a convenient tool for investigation of osteogenesis. We here describe the pattern of gene expression, histone modification and DNA binding of the bone master regulator RUNX2 during osteogenic differentiation of an immortalized human bone marrow-derived MSC cell line. By sequencing of immuno¬precipi¬tated chromatin we generated high resolution maps of enrichment of five histone H3 modifications associated with different functional states of chromatin, and identified differentiation-induced epigenetic changes. By the same approach, several thousand binding sites for the transcription factor RUNX2 was mapped to around 1900 target genes in differentiated osteoblasts. RUNX2 target genes were associated with cellular growth, movement, apoptosis and PDGF BB and p53 signaling. Surprisingly, RUNX2 was found to bind both at the 5’ and 3’ ends of genes, and also at distal regulatory elements, and was co-localized with activating histone modifications at approximately half of bound sites. This is to our knowledge the first genome-wide description of RUNX2 targets in bone biology. Further, we identified GTF2I and TEAD2 as regulators of differentiation by computational prediction of over-represented transcription factor motifs in regions with differential histone modification enrichment. The predictions were confirmed by siRNA knockdown of the transcription factors which lead to inhibition of differentiation. Comparison of DNA methylation patterns of iMSC#3 before (Undiff) and after (Day 28) osteogenic differentiation