Bioinformatics analysis and transcriptional regulation of TORC1 gene through transcription factors NRF1 and Smad3 in bovine preadipocytes

Intramuscular fat is the an important factor that defines meat quality; however, enhancing its deposition without increasing the other three adipose depots (subcutaneous, visceral, and intermuscular fat) is a challenge for animal science and the meat industry. The TORC1 is a key regulator of adipogenesis and its regulation in bovine intramuscular preadipocytes has not been studied. The TORC1 is a member of the gene family that codes for a binding proteins which regulate transcription of cAMP which, is a key regulator of adipogenesis. In the present study, expression and sub-cellular localization of the TORC1 gene was analyzed in bovine preadipocytes. Bioinformatics tools were applied to characterize TORC1. To investigate the molecular mechanism of bovine TORC1 gene regulation, we cloned a 1008 bp of the 5'UTR regulatory region into a luciferase reporter vector. Different fragments were amplified using 5'UTR unidirectional deletion of the TORC1 promoter. Site directed mutation, dual luciferase reporter assay, RNAi interference and DNA-protein interaction (EMSA) were used to validate the regulatory roles of Smad3 and NRF1 in the regulation of TORC1 gene in bovine preadipocytes. The core promoter region of the TORC1 gene was identified at a location -410 to -155 bp upstream of transcription start site. Different vectors were constructed through serial deletion of the 5'UTR flanking region and in combination with site directed mutations and transcription interference through siRNA or shRNA, two transcription factors of NRF1 and Smad3 were found to be repressors in the promoter of the TORC1 gene. These findings were further confirmed through Electrophoretic Mobility Shift Assay (EMSA) within nuclear extracts of bovine adipocytes. The core promoter region of the TORC1 gene, spanning from -410 to -155 bp upstream of the transcription start site was specified in this study and this information will provide opportunity for the improvement of intramuscular fat in cattle.