Competitive regulation by transcription factors and DNA methylation in the bovine SIRT5 promoter: Roles of E2F4 and KLF6.

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.