Ablation of Dnmt3b in chondrocytes suppresses cell maturation during embryonic development

DNA methylation is a major mode of epigenetic regulation in the mammalian genome and is essential for embryonic development. The three catalytic DNA methyltransferases (Dnmts), Dnmt1, Dnmt3a, and Dnmt3b, catalyze the methylation of cytosine. Dnmt3b is highly expressed in chondrocytes and global knockout of Dnmt3b led to skeletal deformations and embryonic lethality, suggesting an essential role of Dnmt3b in endochondral bone formation. To further define the role of Dnmt3b in skeletal development, Dnmt3b was deleted in Col2 positive chondrocyte lineage cells. Both axial and appendicular skeletal size were reduced and bone mineralization was delayed in Col2Cre ⁺ ;Dnmt3b ^f/f (Dnmt3b ^Col2 ) mice at E14.5 and E18.5. While Alcian Blue Hematoxylin/Orange G (ABH/OG) staining showed normal chondrocyte columns in control growth plates, the length of hypertrophic chondrocyte zone and type X collagen expression were decreased in E18.5 growth plates from Dnmt3b ^Col2 mice. TUNEL and PCNA staining demonstrated that the delay in chondrocyte maturation observed in the Dnmt3b ^{Col 2} growth plates was not secondary to altered chondrocyte apoptosis or proliferation. Complementary in vitro experiments were performed on primary sternal chondrocytes isolated from control and Dnmt3b ^Col2 mice. Gene expression studies confirmed delayed terminal maturation as Mmp13 and Col10a1 expression was down-regulated in Dnmt3b ^Col2 chondrocytes. In addition, alkaline phosphatase (ALP) and Alizarin Red staining confirmed that Dnmt3b deletion in chondrocytes delays in vitro chondrocyte hypertrophic differentiation and matrix mineralization. Mechanistically, Dnmt3b gene deletion resulted in decreased BMP signaling through reduction of Smad1 phosphorylation. These findings show that epigenetic factor, Dnmt3b is necessary for normal chondrocyte hypertrophic maturation and limb development.