Runx2 Binding Protein and the Regulation of Osteogenesis
In the developmental process of osteogenesis, bone is formed, laid down, and repaired in a highly regulated process (Wu et al., 2014a). This organized formation of bone tissue is controlled by the nucleic acid binding protein Runx2 (Wu et al., 2014a). Runx2 regulates transcriptional mechanisms in osteoblast cells, or bone forming cells, that are vital to the formation of bone tissue and to the maintenance of bone mass (Wu et al., 2014a). Osteoblasts phenotypically express certain genes depending upon the differentiation process they are regulated to undergo (Wu et al., 2014a). The commitment of osteoblasts to a particular stage-specific phenotype is dependent upon the expression of Runx2 protein (Wu et al., 2014a).
Chromatin immunoprecipitation studies have demonstrated the expression of Runx2 binding protein in over 80,000 regions of the mouse genome at both promoter and non-promoter regions (Wu et al., 2014a). Runx2 binding and intensity varies at the three stages of osteoblast differentiation, proliferation, matrix deposition, and mineralization (Wu et al., 2014a). Runx2 binds to cis-regulatory DNA elements and the Runx motif is located in or near promoter regions (Wu et al., 2014a). Runx2 interacts with co-regulatory factors to upregulate osteoblast genes, such as Osx, Ocn, and Bsp, while it represses the expression of non-osteoblast genes like PPAR-γ and MyoD (Wu et al., 2014a). Runx2 has not only shown its influence on transcriptional pathways, but also has an influence on epigenetic mechanisms of gene expression, such as serving as an identity marker on mitotic chromosomes after division (Wu et al., 2014a).
Because Runx2 has been shown to control skeletal gene expression, when there is a mutation that influences Runx2, one will observe consequential effects on osteogenesis. For example, in mice where the Runx2 protein has been ablated, there was an absence of a mineralized skeleton (Li et al., 2013). Another study focused on Runx2 overexpression in osteosarcoma bone cancer cells (van der Deen et al., 2012). In this particular study, researchers used whole genome chromatin immunoprecipitations for Runx2 along with promoter microarrays and utilized siRNAs to study Runx2 in osteosarcoma cells (van der Deen et al., 2012). The results of this study demonstrated that Runx2 regulates genes and pathways that involved processes like cell migration in osteosarcoma cells (van der Deen et al., 2012).
To conduct the chip- on-chip assay for this study on tumor migration, SAOS-2 osteosarcoma cells were grown and cross-linked in formaldehyde solution. Anti-Runx2 antibodies were added to the chromatin along with polymerase II antibody to mark genes that were being transcribed. IgG served as a negative control. To reverse cross-linking, the suspensions were incubated with RNase A and proteinase K. DNA was then purified and underwent DNA precipitation with ethanol. DNA concentrations...