1 College of Animal Science and Technology, Northwest A&F University , Yangling, China .
2 National Beef Cattle Improvement Center , Yangling, China .
DNA Cell Biol. 2018 Jun;37(6):551-559. doi: 10.1089/dna.2018.4152. Epub 2018 Apr 19.
The transforming growth factor-β (TGFβ) pathway plays many key roles in regulating numerous biological processes. In addition, the effects of TGFβ are mediated by the transcription factor Smad3. However, the regulation of Smad3 activity is not well understood. In the present study, quantitative real-time PCR revealed that the Smad3 gene was expressed ubiquitously in 11 bovine tissues and displayed different expression patterns between muscle and adipose tissue. We further explored the expression and regulation of Smad3 gene by cloning the bovine Smad3 gene promoter; a dual-luciferase reporter assay identified that the core promoter region -337 to -41 bp was located in a CpG island. In addition, mutational analyses and electrophoretic mobility shift assays provided evidence that the KLF6, KLF15, MZF1, and KLF7 binding sites within the Smad3 promoter were responsible for the regulation of Smad3 transcription. These findings were confirmed by executing further RNA interference assays in bovine myoblasts and preadipocytes, which indicated that KLF6, KLF15, MZF1, and KLF7 are important transcriptional activators of Smad3 in both adipose and muscle tissue. These results will provide an important basis for an improved understanding of the TGFβ pathway and new insights in cattle breeding.
转化生长因子-β（TGFβ）途径在调节许多生物过程中发挥着许多关键作用。此外，TGFβ 的作用是由转录因子 Smad3 介导的。然而，Smad3 活性的调节机制尚不清楚。在本研究中，实时定量 PCR 显示 Smad3 基因在 11 种牛组织中广泛表达，并且在肌肉和脂肪组织之间表现出不同的表达模式。我们进一步通过克隆牛 Smad3 基因启动子来探索 Smad3 基因的表达和调节；双荧光素酶报告基因检测鉴定出核心启动子区域-337 到-41bp 位于 CpG 岛中。此外，突变分析和电泳迁移率变动分析提供了证据，证明 Smad3 启动子内的 KLF6、KLF15、MZF1 和 KLF7 结合位点负责 Smad3 转录的调节。这些发现通过在牛成肌细胞和前脂肪细胞中进一步进行 RNA 干扰实验得到了证实，表明 KLF6、KLF15、MZF1 和 KLF7 是脂肪组织和肌肉组织中 Smad3 的重要转录激活因子。这些结果将为深入了解 TGFβ 途径提供重要依据，并为牛的育种提供新的见解。
