College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China.
College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China; National Beef Cattle Improvement Center of Northwest A&F University, Yangling 712100, China.
Gene. 2019 Feb 5;684:39-46. doi: 10.1016/j.gene.2018.10.052. Epub 2018 Oct 22.
Sirtuin 5 (SIRT5) belongs to the mitochondrial sirtuin family, which constitutes a highly conserved family of nicotinamide adenine dinucleotide NAD-dependent deacetylases and ADP-ribosyltransferases that play important regulatory roles in stress resistance and metabolic homeostasis. SIRT5 was shown to have deacetylase, desuccinylase, and demalonylase activities. However, the mechanisms regulating SIRT5 transcription remain unclear. To explore the molecular regulation of bovine SIRT5 expression, we obtained a 500-base pair fragment of the 5'-regulatory region of bovine SIRT5 by molecular cloning, which contained a region with 3 CpG islands. Electrophoretic mobility shift assays and luciferase reporter assays revealed the E2F transcription factor 4 (E2F4) and Kruppel-like factor 6 (KLF6) binding sites as transcriptional activators or repressors in the promoter region of SIRT5. We further verified that E2F4 and KLF6 bind to the SIRT5 promoter by chromatin immunoprecipitation assays. Additionally, methylation and luciferase report assays showed that SIRT5 promoter activity was enhanced by demethylation, and transcriptional activation by E2F4 and transcriptional inhibition by KLF6 of SIRT5 expression was strengthened by demethylation during adipocytes differentiation. This study focused on the mechanism underlying the methylation and transcriptional regulation of SIRT5 expression in bovine adipocytes.
Sirtuin 5（SIRT5）属于线粒体 sirtuin 家族，该家族由高度保守的烟酰胺腺嘌呤二核苷酸（NAD）依赖性去乙酰化酶和 ADP-核糖基转移酶组成，在应激抵抗和代谢稳态中发挥重要的调节作用。SIRT5 具有去乙酰化酶、脱琥珀酰酶和脱丙二酰化酶活性。然而，调节 SIRT5 转录的机制尚不清楚。为了探索牛 SIRT5 表达的分子调控机制，我们通过分子克隆获得了牛 SIRT5 5'调控区的 500 碱基对片段，其中包含一个含有 3 个 CpG 岛的区域。电泳迁移率变动分析和荧光素酶报告基因分析显示，E2F 转录因子 4（E2F4）和 Kruppel 样因子 6（KLF6）作为转录激活子或抑制剂结合在 SIRT5 启动子区域。我们进一步通过染色质免疫沉淀实验验证了 E2F4 和 KLF6 与 SIRT5 启动子的结合。此外，甲基化和荧光素酶报告基因实验表明，SIRT5 启动子活性在去甲基化后增强，E2F4 的转录激活和 KLF6 的转录抑制作用在脂肪细胞分化过程中增强了 SIRT5 的表达。本研究主要关注牛脂肪细胞中 SIRT5 表达的甲基化和转录调控机制。
