Effection Of NCOA3 Promotor Methylation On Its Differential Expression Profile Between Intramuscular And Subcutaneous Fat Tissue In Pigs

The regulation of intramuscular fat deposition has important implications for improving animal productivity.The deposition of intramuscular fat affects meat quality.Through understanding the mechanisms of fat difference deposition in the subcutaneous fat and intramuscular fat,we can improve the pork quality and reduce the economic waste by increasing intramuscular fat deposition,while not affecting and even reducing subcutaneous fat deposition.Nuclear receptor coactivator 3(NCOA3)is one of the steroid coactivator protein family members,which affected the transcriptional activity of PPARy2 through interaction with C/EBP? and C/EBPa,so it plays an important role in the adipocyte differentiation and fat metabolism.In order to investigate the differences in subcutaneous fat and intramuscular fat,we analysis the NCOA3 protein using bioinformatics method to learn its structure and function.Then we built deletion reporter vectors of NCOA3 promoter and detected the core promoter.Meanwhile we compared the NC.OA3 mRNA expression in two fat and detected the methylation level of the CpG island in NCOA3 promoter in two tissue by sodium bisulfite genomic sequencing.The results are as follows:1.Bioinformatic analysis of porcine NCOA3 geneIts cDNA contains 4173bp open reading frame,encoding 1390 amino acid which protein molecular weight is 151.2kDa.There are no signal peptide and transmembrane structure in NCOA3 proteins,and has extremely hydrophilic and complex spiral structure,however its stability is not good,which may be related to the complexity of its structure and function.By comparing the amino acid sequence of NCOA3 in human,murine and porcine,we found it very high similarity(88.16%)in this three species.Therefore,we can speculate the function of NCOA3 in adipocyte differentiation based on its studies in human and murine.2.Construct deletion reporter vectors of 5,flanking sequence of porcine gene and analysis of the promoter activityThe porcine NCOA3 gene of six dual luciferase reporter vectors were built.We detected the promoter activity by transient transfection into PK15 cells and livers cells in porcine.The results showed that the core promoter located in-150?-3bp region.The only CpG island was found in the upstream of NCOA3 promoter which located in-930?-699bp.Simultaneously,the result of transcription factor predicting,showed that theCpG island region was containing Spl,Jun,CREEP1,C/EBP?and so on.Among that transcription factor,Splwas methylation-sensitive sites.Therefore we speculated that the CpG island in NCOA3 promoter may affect its promoter activity.3.The relationship between the methylation level of the nuclear coactivator3(NCOA3)promoter and its different expression patternsThe mRNA expression of NCOA3 in intramuscular and subcutaneous adipose tissue was determined by RT-PCR.The result showed that the mRNA expression of NCOA3 in intramuscular adipose tissue was significantly lower than in subcutaneous adipose tissue(P<0.01).Methylation level was detected by sodium bisulfite genomic sequencing for the CpG island in NCOA3 promoter.The whole methylation level of CpG island shows no differences between intramuscular(90%)and subcutaneous(83.5%)adipose tissue,However,the methylation level of-904 CG site in intramuscular fat(55%)is higher than that in subcutaneous fat(20%)(P<0.05).To detect whether NCOA3 gene promoter activity was affected by the methylation in the CpG island,we treated NCOA3 promoter region(contains CpG island)by methyltransferase,which increased methylation level of CpG island,lead to significant depression of gene activities(P<0.05).Meanwhile,the result of transcription factor predicting showed that there has a transcription factor C/EBPb? in-904 site nearby.We compared the activity of mutated vector(the C/EBPb? binding site was mutated)to the wide vector and found the activity of mutated vector was markly decreased(p=0.004<0.05).The result suggested that the methylation level of NCOA3 promoter affected its promoter activity and the methylation of NCOA3 promoter CpG island(-904 site)might have effect on its differential expression in intramuscular and subcutaneous adipose tissue.