Sun Ruixin, Wu Yi, Wang Yuxiong, Zang Kun, Wei Huanhuan, Wang Fangnian, Yu Min
The Key Laboratory of Molecular Medicine, The Ministry of Education, Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, P.O. Box #238, No. 138 Yi Xue Yuan Road, Shanghai, 200032, China.
Mol Cell Biochem. 2015 Apr;402(1-2):23-31. doi: 10.1007/s11010-014-2310-1. Epub 2015 Jan 10.
Obesity is characterized by excessive accumulation of white adipose tissue. Bromodomain-containing protein 2 (Brd2), which belongs to the bromodomain and extraterminal domain family of proteins, suppresses adipocyte differentiation. DNA methylation is critical for several differentiation processes and possibly in adipocyte differentiation. However, whether DNA methylation regulates the expression of Brd2 is not clear. In our study, we demonstrated that DNA methylation contributes to the regulation of Brd2 expression during pre-adipocyte differentiation. Brd2 mRNA levels were low in pre-adipocytes, increased in early adipocytes, and declined in mature adipocytes. To test whether and how Brd2 expression is regulated by DNA methylation during the differentiation of 3T3-L1 pre-adipocytes to adipocytes, cells were cultured in the presence of the methylation inhibitor 5-aza-2'-deoxycytidine (5-Aza). Pre-adipocytes and adipocytes exposed to 5-Aza exhibited a dose-dependent increase in Brd2 transcription levels, while only mature adipocytes exhibited increased expression of Brd2 protein. Subsequently, we tested the DNA methylation status of the Brd2 promoter region. Bisulfite-sequencing analysis revealed that six CpG sites in two predicted promoters of Brd2 were demethylated in early adipocytes and highly methylated in mature adipocytes. Digestion of bisulfite-converted PCR products of the Brd2 promoter region from 3T3-L1 cells with BstU1 (CGGC) revealed that the demethylation rate of the Brd2 promoter was consistent with Brd2 mRNA expression in differentiating 3T3-L1 cells. In conclusion, DNA demethylation of the Brd2 promoter region induced Brd2 expression during differentiation of 3T3-L1 cells into adipocytes.
肥胖的特征是白色脂肪组织过度积累。含溴结构域蛋白2（Brd2）属于含溴结构域和额外末端结构域蛋白家族，可抑制脂肪细胞分化。DNA甲基化对多个分化过程至关重要，可能也参与脂肪细胞分化。然而，DNA甲基化是否调节Brd2的表达尚不清楚。在我们的研究中，我们证明DNA甲基化在脂肪前体细胞分化过程中有助于调节Brd2的表达。Brd2 mRNA水平在脂肪前体细胞中较低，在早期脂肪细胞中升高，而在成熟脂肪细胞中下降。为了测试在3T3-L1脂肪前体细胞向脂肪细胞分化过程中Brd2表达是否以及如何受到DNA甲基化的调节，细胞在甲基化抑制剂5-氮杂-2'-脱氧胞苷（5-Aza）存在的情况下进行培养。暴露于5-Aza的脂肪前体细胞和脂肪细胞中Brd2转录水平呈剂量依赖性增加，而只有成熟脂肪细胞中Brd2蛋白表达增加。随后，我们检测了Brd2启动子区域的DNA甲基化状态。亚硫酸氢盐测序分析显示，Brd2两个预测启动子中的六个CpG位点在早期脂肪细胞中去甲基化，而在成熟脂肪细胞中高度甲基化。用BstU1（CGGC）消化3T3-L1细胞Brd2启动子区域的亚硫酸氢盐转化PCR产物，结果显示Brd2启动子的去甲基化率与分化的3T3-L1细胞中Brd2 mRNA表达一致。总之，在3T3-L1细胞向脂肪细胞分化过程中，Brd2启动子区域的DNA去甲基化诱导了Brd2的表达。
