Wang Haifei, Feng Haiyue, Sun Juan, Zhou Yajing, Zhu Guoqiang, Wu Shenglong, Bao Wenbin
Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design, College of Animal Science and Technology, Yangzhou University.
College of Veterinary Medicine, Yangzhou University.
Genes Genet Syst. 2018 Dec 22;93(5):191-198. doi: 10.1266/ggs.18-00016. Epub 2018 Nov 23.
DNA methylation is an important mediator of gene expression regulation and has been shown to be closely linked to aging. Immune-related genes tend to be influenced by DNA methylation at different ages. To explore DNA methylation changes in the porcine TNFα gene and analyze their potential effects on gene expression, we measured the methylation level of the TNFα promoter and TNFα mRNA expression in the spleen of Meishan piglets at six developmental stages (1, 7, 14, 21, 28 and 35 days old) by bisulfite sequencing PCR and quantitative PCR. The results revealed a trend for TNFα promoter methylation level to increase and mRNA expression to decrease with age. Correlation analysis showed a significant negative association between methylation level and mRNA expression (Pearson's r = -0.775, P = 4.87E-07). In addition, the transcription factor Sp1 was revealed to bind with the TNFα promoter and regulate TNFα expression. DNA methylation in the TNFα promoter was found to decrease the promoter's activity, and methylation inhibition could enhance the expression level of TNFα, providing functional evidence that promoter methylation controls TNFα expression. Together, our data provide insights into age-associated changes in promoter methylation of the TNFα gene in the spleen and contribute to our understanding of regulatory mechanisms for TNFα expression in the immune system of pigs.
DNA甲基化是基因表达调控的重要介导因子，且已被证明与衰老密切相关。免疫相关基因在不同年龄往往会受到DNA甲基化的影响。为了探究猪TNFα基因的DNA甲基化变化并分析其对基因表达的潜在影响，我们通过亚硫酸氢盐测序PCR和定量PCR检测了梅山仔猪六个发育阶段（1、7、14、21、28和35日龄）脾脏中TNFα启动子的甲基化水平和TNFα mRNA表达。结果显示，TNFα启动子甲基化水平随年龄增长而升高，mRNA表达随年龄增长而降低。相关性分析表明甲基化水平与mRNA表达之间存在显著负相关（Pearson's r = -0.775，P = 4.87E-07）。此外，还发现转录因子Sp1与TNFα启动子结合并调节TNFα表达。TNFα启动子中的DNA甲基化被发现会降低启动子活性，甲基化抑制可提高TNFα的表达水平，这为启动子甲基化控制TNFα表达提供了功能证据。总之，我们的数据为脾脏中TNFα基因启动子甲基化的年龄相关变化提供了见解，并有助于我们理解猪免疫系统中TNFα表达的调控机制。
