Function and Transcriptional Regulation of Bovine <i>TORC2</i> Gene in Adipocytes: Roles of <i>C/EBPγ</i>, <i>XBP1</i>, <i>INSM1</i> and <i>ZNF263</i>

• Main Authors: Rajwali Khan, Sayed Haidar Abbas Raza, Zainaguli Junjvlieke, Wang Xiaoyu, Matthew Garcia, Ibrahim Elsaeid Elnour, Wang Hongbao, Zan Linsen
• Format: Article
• Language: English
• Published: MDPI AG 2019-09-01
• Series: International Journal of Molecular Sciences
• Subjects: TORC2; adipogenesis; preadipocytes proliferation and differentiation; transcription factors; luciferase reporter assay; bovine adipocytes; nuclear protein; intramuscular fat; DNA-Protein interaction; gene regulation
• Online Access: https://www.mdpi.com/1422-0067/20/18/4338

The <i>TORC2</i> gene is a member of the transducer of the regulated cyclic adenosine monophosphate (cAMP) response element binding protein gene family, which plays a key role in metabolism and adipogenesis. In the present study, we confirmed the role of <i>TORC2</i> in bovine preadipocyte proliferation through cell cycle staining flow cytometry, cell counting assay, 5-ethynyl-2&#8242;-deoxyuridine staining (EdU), and mRNA and protein expression analysis of proliferation-related marker genes. In addition, Oil red O staining analysis, immunofluorescence of adiponectin, mRNA and protein level expression of lipid related marker genes confirmed the role of <i>TORC2</i> in the regulation of bovine adipocyte differentiation. Furthermore, the transcription start site and sub-cellular localization of the <i>TORC2</i> gene was identified in bovine adipocytes. To investigate the underlying regulatory mechanism of the bovine <i>TORC2</i>, we cloned a 1990 bp of the 5&#8217; untranslated region (5&#8242;UTR) promoter region into a luciferase reporter vector and seven vector fragments were constructed through serial deletion of the 5&#8242;UTR flanking region. The core promoter region of the <i>TORC2</i> gene was identified at location &#8722;314 to &#8722;69 bp upstream of the transcription start site. Based on the results of the transcriptional activities of the promoter vector fragments, luciferase activities of mutated fragments and siRNAs interference, four transcription factors (<i>CCAAT/enhancer-binding protein C/BEP&#947;, X-box binding protein 1 XBP1, Insulinoma-associated 1 INSM1,</i> and <i>Zinc finger protein 263 ZNF263</i>) were identified as the transcriptional regulators of <i>TORC2</i> gene. These findings were further confirmed through Electrophoretic Mobility Shift Assay (EMSA) within nuclear extracts of bovine adipocytes. Furthermore, we also identified that <i>C/EBP&#947;, XBP1, INSM1</i> and <i>ZNF263</i> regulate <i>TORC2</i> gene as activators in the promoter region. We can conclude that <i>TORC2</i> gene is potentially a positive regulator of adipogenesis. These findings will not only provide an insight for the improvement of intramuscular fat in cattle, but will enhance our understanding regarding therapeutic intervention of metabolic syndrome and obesity in public health as well.