Regulation Of GR And FOXA1 On Transcription Of ANGPTL4 Gene In Qinchuan Beef Cattle

The marble pattern is an important indicator for evaluating the beef quality–the richer it is,the higher the quality.Angiopoietin-like protein family(ANGPTLs)is an important class of secreted proteins that are involved in physiological processes such as angiogenesis,wound repair,lipid metabolism,and glucose metabolism.Among them,ANGPTL3,ANGPTL4,and ANGPTL8 play important roles in triglyceride metabolism.ANGPTL4 controls triglyceride deposition in adipocytes by inhibiting the activity of lipoprotein lipase,which plays a pivotal role in promoting fat liberation and reducing fat storage.Studies have shown that bovine ANGPTL4 has become an important candidate related to meat quality and growth traits.Despite improvements in functional studies,the transcriptional regulation of bovine ANGPTL4 has yet to be elucidated.Therefore,this study further investigated the transcriptional regulation of Qinchuan beef cattle(bred by Northwest A & F University)ANGPTL4 at the cellular level and obtained the following experimental results:1.Tissue expression of ANGPTL4 in Qinchuan beef cattle.q RT-PCR was used to detect the m RNA expression level of ANGPTL4 in different tissues and organs.Among them,the liver had the highest relative expression level(P <0.01),followed by subcutaneous fat,whereas the abomasum and spleen had the lowest expression level.2.The transcription start site of ANGPTL4 was identified in Qinchuan beef cattle.The rapid amplification of 5?c DNA was used to determine that bovine ANGPTL4 exhibited three different 5? ends,i.e.,at 210,206,and 187 bp upstream of the translational start site(ATG).The guanine residue(G)proximal to 5? untranslated regions(UTR)was designated as +1,which advanced 18 bp compared to the reported transcription start site of the ANGPTL4 m RNA sequence(NM?001046043.2).3.The minimal functional region of the ANGPTL4 promoter in Qinchuan beef cattle was determined.Seven 5' unidirectional deletion fragments of ANGPTL4 promoter were obtained by PCR cloning.After transfection,the result showed that the dual-luciferase activity significantly decreased 46%(P <0.01)when the promoter was deleted from-568 to-261 bp,which means that it is a minimal functional promoter region of bovine ANGPTL4 gene.4.GR and FOXA1 are positive regulatory elements necessary for ANGPTL4 transcription in Qinchuan beef cattle.We further analyzed regulatory elements in the core promoter region of the ANGPTL4 using the online software.As a result,two important transcription factor recognition sites GR and FOXA1 were found.Dual-luciferase experiments showed that the activity of the mutated GR and FOXA1 fragment was significantly decreased by 38% and 31% respectively(P <0.01).RNA interference experiments showed that co-transfection of si-GR or si-FOXA1 with p GL-568/+35 also exhibited 46% and 45% reduction(P<0.05).EMSA was used to further confirm the specific binding of GR and FOXA1 to the ANGPTL4 promoter in vitro.The above results indicated that the transcription factors GR and FOXA1 are necessary positive regulatory elements to maintain the basic transcriptional activity of the bovine ANGPTL4 promoter.In addition,silencing of GR and FOXA1 genes reduced m RNA expression of bovine ANGPTL4(P <0.05).In conclusion,GR and FOXA1 play a positive role in regulating transcriptional activity by binding to the core region of the ANGPTL4 promoter in Qinchuan beef cattle.Our results lay the foundation for further studies on the regulatory mechanism of ANGPTL4 and also provide a reference for improving beef quality.