Department of Orthopaedic Surgery, School of Medicine, Washington University in St. Louis, St. Louis, Missouri.
Institute of Orthopaedics and Traumatology, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China.
J Cell Biochem. 2018 Jul;119(7):5852-5863. doi: 10.1002/jcb.26775. Epub 2018 Apr 10.
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 (Dnmt3b ) 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 mice. TUNEL and PCNA staining demonstrated that the delay in chondrocyte maturation observed in the Dnmt3b 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 mice. Gene expression studies confirmed delayed terminal maturation as Mmp13 and Col10a1 expression was down-regulated in Dnmt3b 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.
DNA 甲基化是哺乳动物基因组中表观遗传调控的主要模式，对胚胎发育至关重要。三种催化 DNA 甲基转移酶（Dnmts），Dnmt1、Dnmt3a 和 Dnmt3b，催化胞嘧啶的甲基化。Dnmt3b 在软骨细胞中高度表达，而 Dnmt3b 的全局敲除导致骨骼畸形和胚胎致死，表明 Dnmt3b 在软骨内骨形成中起着至关重要的作用。为了进一步确定 Dnmt3b 在骨骼发育中的作用，在 Col2 阳性软骨细胞谱系细胞中删除了 Dnmt3b。在 E14.5 和 E18.5 时，Col2Cre；Dnmt3b（Dnmt3b）小鼠的轴向和附肢骨骼大小减小，骨矿化延迟。虽然 Alcian Blue Hematoxylin/Orange G（ABH/OG）染色显示对照生长板中的软骨细胞柱正常，但在 E18.5 生长板中，Dnmt3b 小鼠的肥大软骨细胞区带长度和 X 型胶原表达减少。TUNEL 和 PCNA 染色表明，在 Dnmt3b 生长板中观察到的软骨细胞成熟延迟不是由于软骨细胞凋亡或增殖改变所致。在从对照和 Dnmt3b 小鼠分离的原代胸骨软骨细胞上进行了补充的体外实验。基因表达研究证实，Mmp13 和 Col10a1 的表达下调导致终末成熟延迟，这表明 Dnmt3b 软骨细胞中的基因表达下调。此外，碱性磷酸酶（ALP）和茜素红染色证实，软骨细胞中 Dnmt3b 的缺失延迟了体外软骨细胞肥大分化和基质矿化。从机制上讲，Dnmt3b 基因缺失通过减少 Smad1 磷酸化导致 BMP 信号转导减少。这些发现表明，表观遗传因子 Dnmt3b 对于正常软骨细胞肥大成熟和肢体发育是必要的。
