Figure 8.

SUV39H1 negatively regulated osteoblast differentiation. (A) C2C12 cells were transfected with the indicated siRNAs and incubated with BMP2. SUV39H1 knockdown was verified by immunoblotting (top panel). Osteoblast differentiation was monitored by ALP stainingand and Alizarin staining for the indicated times. (B) The expression levels of the osteogenic genes were analyzed by RT-qPCR and presented as relative values to the day-0 level. Each point represents the mean ± SD (n = 3). ^* denotes P < 0.05 vs the si-Con value on the corresponding day. (C) C2C12 cells were transfected with OG2 promoter-luciferase reporter, β-gal, the indicated siRNAs, and/or Myc-Runx2 plasmid. Each bar represents the mean ± SD (n = 4) for luciferase activity. (D) C2C12 cells, which had been transfected as indicated, were incubated with BMP2 for 24 h. Each bar represents the mean ± SD (n = 4), ^* and n.s. denote P < 0.05 and non-significant, respectively. The protein levels were immunoblotted (bottom panel). (E-F) C2C12 cells were transfected with SUV39H1 plasmid or siRNA, and incubated with BMP2 for 2 days. ChIP with anti-Runx2 was performed to identify the binding of Runx2 to OCN promoter. Precipitated DNA segments were quantified by real-time PCR. The results (mean ± SD, n = 3) are expressed as percentages of the input level. (G) The proposed mechanism underlying Runx2 regulation via lysine methylation. SUV39H1 inhibits the transcriptional activity of Runx2 by methylating Runx2 at Lys 245 [off]. Conversely, PHF2 activates Runx2 by removing the methyl group from Runx2 [on]. PHF2 and SUV39H1 counterbalance osteoblast differentiation by reciprocally regulating Runx2 methylation.