The Role And Mechanism Of MBD4and TDG Gene In Porcine Preadipocyte Differentiation

The quantity and distribution of fat in the porcine carcass are determinants of thepork’s quality and flavor. Adipocytes are produced by the differentiation ofpreadipocytes formed from mesodermal cells during various stages of embryonicdevelopment. The formation of adipocytes is a dynamic process, and thedifferentiation of preadipocytes is regulated by hormones and growth factors.Epigenetic plays an important role in cell differentiation, which the gene expressionchanges under the genomic DNA sequence remains intact. In the preadipocytedifferentiation process, the epigenome changes as the differentiation proceeds. Themethylation and demethylation of genomic DNA are important aspects of epigenetics,and the two processes balance each other to maintain the stability of methylationprofiles. The MBD4and TDG gene encodes an important glycosylase that can identifyT-G mismatches and complete DNA demethylation processes through base excisionrepair which play an important role in preadipocyte differentiation. However, the rolesof the MBD4and TDG gene in the differentiation of preadipocytes and the impact ofthis gene on the expression levels of associated genes have not yet been verified. Inthis study, porcine preadipocytes were examined to analyze how knocking down theMBD4and TDG gene affected lipogenesis and altered the expression levels andpromoter methylation levels of the C/EBPα, PPARγ, and aP2genes, which encodeadipocyte differentiation-related transcription factors.Dorsal subcutaneous adipose tissue from the One-to seven-day-old Junmu-1pigs’necks was collected to isolate, culture, and induce to differentiation. Relative to thecorresponding mRNA expression levels of the control group cells, the mRNAexpression levels of the adipose differentiation-associated genes C/EBPα, PPARγ, andaP2changed over the course of the differentiation process in porcine preadipocytesthat were induced to differentiate. In particular, the highest expression levels ofC/EBPα, PPARγ, and aP2were reached at two, four, and one days after inducingdifferentiation, respectively. The highest expression levels of DNMT1gene wasreached at three days, otherwise, the DNMT3β and GADD45α gene were no change.The highest expression levels of DNMT3α gene was reached at one day. The mRNAexpression level of TET1, TET2, TET3, MBD4, and TDG gene changed over thecourse of the differentiation process, which the highest expression levels were reached at three, two, four, two, two days, respectively. The protein expression levels ofMBD4and TDG changed over the course of the induced differentiation of the porcinepreadipocytes, with the highest expression levels reached at two days afterdifferentiation induction according to the Western blot.The mRNA expression levels of the differentiation-related transcription factorsC/EBPα, PPARγ, and aP2were decreased in the MBD4transfection group aftertreated with MBD4-siRNA relative to the control group. The mRNA expression levelsof PPARγ and aP2gene were decreased in the TDG-siRNA transfection group and theC/EBPα gene was no change. The protein expression levels of MBD4or TDG weresignificantly decreased after treated with MBD4-siRNA or TDG-siRNA according tothe Western blot.The methylation of the C/EBPα, PPARγ, and aP2gene promoters changed afterthe MBD4gene was knocked down; in particular, the methylation levels of these threepromoters were1.4%,70%, and54%, respectively, in the control group but2.27%,92.5%, and84%, respectively, in the MBD4-siRNA group. The methylation levels ofthese three promoters were0.9%,80%, and76%, respectively, in the control groupbut0.5%,67.5%, and58%, respectively, in the TDG-siRNA group by bisulfitesequencing method.The transfected cells were induced to differentiate after transfection, the cellbiology and OD values at500nm after staining with oil red O revealed that theformation of lipid droplets in adipocytes was significantly reduced in theMBD4-siRNA or TDG-siRNA transfected group relative to the blank and negativecontrol groups. These results suggested that knocking down MBD4or TDG geneinhibited the differentiation of porcine preadipocytes into adipocytes.Although the MBD4and TDG genes possessed the base excision repair functions,after knocking down MBD4and TDG gene, separately, the different degree of mRNAexpression and methylation levels of promoter region were displayed, Which meanstwo genes exhibited different effect on the differentiation of preadipocyte. The effectof MBD4or TDG genes in preadipocyte differentiation was carried on the analysis ofmorphological and molecular regulation mechanism in this research. In addition,studies had confirmed that changes to these hormones or growth factors affectedpreadipocyte differentiation，providing genetic markers for genetics and breeding toimprove the quality of pork.