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.

中文翻译：

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在脂肪细胞分化过程中，DNA甲基化调节含溴结构域的蛋白质2的表达。

肥胖的特征在于白色脂肪组织的过度积累。含溴结构域的蛋白质2（Brd2），属于蛋白质的溴结构域和末端外区域家族，可抑制脂肪细胞分化。DNA甲基化对于几种分化过程以及可能在脂肪细胞分化中至关重要。但是，DNA甲基化是否调节Brd2的表达尚不清楚。在我们的研究中，我们证明了DNA甲基化有助于前脂肪细胞分化过程中Brd2表达的调节。Brd2 mRNA水平在前脂肪细胞中较低，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前脂肪细胞向脂肪细胞分化过程中，Brd2表达是否以及如何受DNA甲基化调控，在甲基化抑制剂5-氮杂-2'-脱氧胞苷（5-氮杂）的存在下培养细胞。前脂肪细胞和暴露于5-Aza的脂肪细胞显示Brd2转录水平呈剂量依赖性增加，而只有成熟的脂肪细胞显示Brd2蛋白表达增加。随后，我们测试了Brd2启动子区域的DNA甲基化状态。亚硫酸氢盐测序分析显示，两个预测的Brd2启动子中的六个CpG位点在早期的脂肪细胞中被去甲基化，而在成熟的脂肪细胞中被高度甲基化。用BstU1（CGGC）消化3T3-L1细胞中Brd2启动子区域的亚硫酸氢盐转化PCR产物，发现Brd2启动子的去甲基化速率与分化3T3-L1细胞中Brd2 mRNA表达一致。结论，