The Molecular Cloning And Characterization Of Porcine Sirtuin Genes And The Study Of SIRT3Function During Adipogenesis

The Sirtuin family of class III histone deacetylases (HDACs) is named after their homology to yeast silent information regulator2(Sir2). SIR2and its mammalian derivatives (SIRT1-7) play a central role in gene silencing, cell cycle, aging and metabolism. Here we reported cDNA cloning, chromosome mapping, expression and evolutional analysis of Sirtuin genes in Sus scrofa (Tongcheng pig). Sequence analysis showed that porcine Sirtuin genes contain7members designated SIRT1-7. Tissue distribution analysis indicated porcine Sirtuin genes ubiquitously expressed but with the highest abundance in brain, spinal cord and genital tissue. In silico and radiation hybrid mapping analysis mapped porcine SIRT1-7to the chromosome14q23,6ql1-12,2q29,14q19,7p12,2q11,12p15, respectively. We also isolated and characterized genomic sequence of porcine SIRT1, which spaned a region of31,834bp comprising9exons ranging in size from80bp to2,121bp. The5’flanking genomic region preceding an open reading frame of SIRT1has a TATA box, a small300bp CpG island and several putative Spl and p53transcription factor binding sites. Moreover, we isolated two novel splicing SIRT6variants with346bp (variant2) in-frame deletions from lung and327bp (variant3) in-frame deletions from spleen and brain. This is the first systematic report of molecular cloning and characterization of Sirtuin genes in pigs, which will be helpful for a better understanding of the physiological role of Sirtuin proteins in pigs.SIRT3belongs to yeast SIR2protein family and mitochondria localized NAD+dependent protein deacetylase. It is involved in erergy metabolism including the respiratory chain, TCA cycle, fatty acid β-oxidation and ketogenesis. Mitochondria are multi-functional cellular organelles involved in oxidative metabolism, ion homeostasis, signal transduction, apoptosis and contribute to human disease such as diabetes, heart dysfunction, cancer and aging by a variety of mechanisms. Previous studies reported that SIRT3and Beclin-1localized in the mitochondria, while the mitochondria can be eliminated by autophagy. In this study, we detected the ROS levels and the mitochondria amount alternation during the3T3L1adipocyte differentiation process, we found that the ROS and the mitochondria began to peak off after differentiated4-6days. Quantative PCR analysis showed that SIRT3, Beclin-1, Atg5and Atg7were upregulated during the late stage of differentiation. While the mRNA and protein expression profiles of SIRT3and Beclin-1were similar with PPAR-y, which is the key trasncriptional factor for the adipogenesis. Dual luciferase analysis, PPAR-y overexpressio, sh-RNA target to PPAR-y and ChIP experiment indicate that PPAR-y can bind to SIRT3promoter and upregulate its expression during adipogenesis. Also we showed that SIRT3can interact with Beclin-1by Co-IP, this suggested that Beclin-1is an deacetylate suberstrate during the adipogenesis. Based on those results, we speculated that SIRT3might promote the deacetylation of Beclin-1, which can enhanced the clearence of mitochondris during the late stage of adipogenesis. The next, we transfected the SIRT3, Beclin-1overexpression vector or RNAi vector into the differentiating(Day4to Day6)3T3L1cells, we found that overexpression SIRT3or Beclin-1can decrease the mitochondria amount, while interfered the expression of both can increase the mitochondria compared to the control.