PMID- 31487963 OWN - NLM STAT- MEDLINE DCOM- 20200114 LR - 20200114 IS - 1422-0067 (Electronic) IS - 1422-0067 (Linking) VI - 20 IP - 18 DP - 2019 Sep 4 TI - Function and Transcriptional Regulation of Bovine TORC2 Gene in Adipocytes: Roles of C/EBP, XBP1, INSM1 and ZNF263. LID - 10.3390/ijms20184338 [doi] LID - 4338 AB - The TORC2 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 TORC2 in bovine preadipocyte proliferation through cell cycle staining flow cytometry, cell counting assay, 5-ethynyl-2'-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 TORC2 in the regulation of bovine adipocyte differentiation. Furthermore, the transcription start site and sub-cellular localization of the TORC2 gene was identified in bovine adipocytes. To investigate the underlying regulatory mechanism of the bovine TORC2, we cloned a 1990 bp of the 5' untranslated region (5'UTR) promoter region into a luciferase reporter vector and seven vector fragments were constructed through serial deletion of the 5'UTR flanking region. The core promoter region of the TORC2 gene was identified at location -314 to -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 (CCAAT/enhancer-binding protein C/BEP, X-box binding protein 1 XBP1, Insulinoma-associated 1 INSM1, and Zinc finger protein 263 ZNF263) were identified as the transcriptional regulators of TORC2 gene. These findings were further confirmed through Electrophoretic Mobility Shift Assay (EMSA) within nuclear extracts of bovine adipocytes. Furthermore, we also identified that C/EBP, XBP1, INSM1 and ZNF263 regulate TORC2 gene as activators in the promoter region. We can conclude that TORC2 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. FAU - Khan, Rajwali AU - Khan R AD - College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, China. rajwalikhan@nwafu.edu.cn. AD - Department of Livestock Management, Breeding and Genetics, The University of Agriculture, Peshawar, 25130, Pakistan. rajwalikhan@nwafu.edu.cn. FAU - Raza, Sayed Haidar Abbas AU - Raza SHA AD - College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, China. haiderraza110@nwafu.edu.cn. FAU - Junjvlieke, Zainaguli AU - Junjvlieke Z AD - College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, China. yina645766253@nwafu.edu.cn. FAU - Xiaoyu, Wang AU - Xiaoyu W AD - College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, China. wangxiaoyu@nwsuaf.edu.cn. FAU - Garcia, Matthew AU - Garcia M AD - School of Animal Dairy and Veterinary Sciences, Utah State University. Logan, UT 84322, USA. matthew.garcia@usu.edu. FAU - Elnour, Ibrahim Elsaeid AU - Elnour IE AD - College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, China. ibravet83@yahoo.com. FAU - Hongbao, Wang AU - Hongbao W AD - College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, China. wanghongbao@nwafu.edu.cn. FAU - Linsen, Zan AU - Linsen Z AD - College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, China. zanlinsen@163.com. AD - National Beef Cattle Improvement Research Center, Northwest A&F University, Yangling, 712100, China. zanlinsen@163.com. LA - eng GR - 2013AA102505/National 863 Program of China/ GR - 2015BAD03B04/National Key Technology Support Program/ GR - CARS-38/National Beef and Yak Industrial Technology System/ GR - 2014KTZB02-02-01/Technical Innovation Engineering Project of Shaanxi Province/ PT - Journal Article DEP - 20190904 PL - Switzerland TA - Int J Mol Sci JT - International journal of molecular sciences JID - 101092791 RN - 0 (CCAAT-Enhancer-Binding Proteins) RN - EC 2.7.11.1 (Mechanistic Target of Rapamycin Complex 2) SB - IM MH - Adipocytes/cytology/*metabolism MH - Adipogenesis MH - Animals MH - CCAAT-Enhancer-Binding Proteins/genetics/*metabolism MH - Cattle MH - Cells, Cultured MH - Gene Expression Regulation, Developmental MH - Mechanistic Target of Rapamycin Complex 2/genetics/*metabolism MH - Promoter Regions, Genetic MH - Transcriptional Activation MH - Transcriptome PMC - PMC6769628 OTO - NOTNLM OT - DNA-Protein interaction OT - TORC2 OT - adipogenesis OT - bovine adipocytes OT - gene regulation OT - intramuscular fat OT - luciferase reporter assay OT - nuclear protein OT - preadipocytes proliferation and differentiation OT - transcription factors COIS- The authors declare no conflict of interest EDAT- 2019/09/07 06:00 MHDA- 2020/01/15 06:00 PMCR- 2019/09/01 CRDT- 2019/09/07 06:00 PHST- 2019/07/21 00:00 [received] PHST- 2019/09/01 00:00 [revised] PHST- 2019/09/01 00:00 [accepted] PHST- 2019/09/07 06:00 [entrez] PHST- 2019/09/07 06:00 [pubmed] PHST- 2020/01/15 06:00 [medline] PHST- 2019/09/01 00:00 [pmc-release] AID - ijms20184338 [pii] AID - ijms-20-04338 [pii] AID - 10.3390/ijms20184338 [doi] PST - epublish SO - Int J Mol Sci. 2019 Sep 4;20(18):4338. doi: 10.3390/ijms20184338.