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

Abstract

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/BEPγ, 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/EBPγ, 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.

Genetic variants in the TORC2 gene promoter and their association with body measurement and carcass quality traits in Qinchuan cattle

“…In silico analysis revealed that SNP3 neither gained nor loss of any new transcription factor binding site in the DNA sequence; however, this locus already contained binding sites for the MZF1, KLF1 and ZNF263 transcription factors, which remained intact as no loss occurred at this position. Moreover, previously, we confirmed the role of ZNF263 transcription factor in the regulation of TORC2 gene as transcriptional activator [10] Furthermore, SNP3 is located in the proximal minimal promoter region; therefore, these transcription factors may have a substantial role in the regulation of the TORC2 gene. In previous experiments conducted by our research group in the same breed of Qinchuan cattle, we demonstrated the role of the KLF transcription factor family and the MZF1 transcription factor in the regulation of genes responsible for adipogenesis and myogenesis [45,46].…”

“…Additionally, through the CREB pathway in coordination with peroxisome proliferator-activated receptor γ (PPARγ), coactivator 1α (PGC1α) and NR4A (nuclear receptor subfamily 4 group A), CRTC2 promotes gluconeogenesis, adipogenesis [7] and myogenesis [8]. In adipose tissue, TORC2 plays a role in the anabolic processes of adipocytes in lipid metabolism, including lipogenesis, adipogenesis and lipid esterification [9,10]. TORC2 is one of the main inhibitors of lipolysis by its regulation of PKA and HSL activities [11].…”

Bta‐miR‐149‐5p inhibits proliferation and differentiation of bovine adipocytes through targeting CRTCs at both transcriptional and posttranscriptional levels

“…Previously, we explored several important transcription factors in the transcriptional regulation of the CRTC2 and CRTC1 genes (Khan, Raza, Junjvlieke, et al, 2019; Khan, Raza, Schreurs, et al, 2019). The transcription factors CEBP/G, XBP1, INSM1 , and ZNF263 were found to be the core regulatory elements responsible for transcription regulation of the CRTC2 gene, while NRF1 and SMAD3 were found to be the key regulators of the CRTC1 gene.…”

“…Next, we targeted the transcription factors located in the promoter of CRTC1 and CRTC2 with bta-miR-149-5p. In our previous studies(Khan, Raza, Junjvlieke, et al, 2019;Khan, Raza, Schreurs, et al, 2019), these transcription factors including C/EBPγ, XBP1, INSM1, and ZNF263 regulated the CRTC2 gene, while NRF1 and SMAD3 regulated the CRTC1 gene. Obviously, most of the genes are synergistically regulated through a network of transcription factors and miRNAs.…”

“…SEM, SEM, standard error of mean; qPCR, quantitative polymerase chain reaction. *p < .05, **p < .01, and ***p < .001 3.4 | Bta-miR-149-5p regulate CRTC2 and CRTC1 through transcriptional regulation of TFs present in their core promoter Previously, we explored several important transcription factors in the transcriptional regulation of the CRTC2 and CRTC1 genes(Khan, Raza, Junjvlieke, et al, 2019;Khan, Raza, Schreurs, et al, 2019). The transcription factors CEBP/G, XBP1, INSM1, and ZNF263 were found…”