Transcriptional Regulatory Mechanisms Of PPARγ Gene Promoter Of Dairy Goat

The unique nutritional value and flavor of goat milk mainly comes from its short-to-medium chain fatty acids and polyunsaturated fatty acids.Fatty acid metabolism of mammary tissue during lactation is a very important physiological process in determining the fatty acid composition of milk.Peroxisome proliferator-activated receptors gamma(PPARγ)is an important member of the nuclear receptor family.As a kind of ligand activated transcription factor,PPARγ binds on the promoter of target genes and plays important roles in the physiological processes of fatty acid metabolism,lipid droplet formation and secretion.Thus,by analyzing the structure and function of PPARγ,it is of great value to complete the regulation network of dairy goat lipid metabolism and improve the quality of goat milk.In the present research,the promoter region of PPARγ on dairy goat was cloned.Bioinformatics analysis was carried out on promoter fragments and the core regulation region was identified by deletion mutation.The transcriptional regulation mechanism of transcription factor C/EBPα was investigated by using site-deleted mutation,overexpression and interference techniques.At the same time,the auto-loop circuit regulation mechanism of PPARγ gene was studied.The main results are as follows:1.Goat PPARγ gene promoter cloning primers were designed according to bovine genome sequences.A 2328 bp of the 5’ flanking sequence of PPARγ gene was obtained by PCR amplification,including 2181 bp upstream of transcription start site.The online software was used to analysis the promoter fragment,and the transcription factor binding sites of SREBP,Sp1,LXR,C/EBPα and PPARγ were observed on the promoter,but the typical TATA-box was not found.Using deletion fragments activity analysis,we found that the region from-194 to-108 bp is the core regulation region of PPARγ gene,and there might be negative regulatory elements upon the promoter.2.Both C/EBPα gene expression vector and PPARγ promoter vector with C/EBPα binding sites mutation were constructed.Overexpression of C/EBPα up-regulated the mRNA expression level and promoter activity of PPARγ;however,inhibition of C/EBPα resulted in the down-regulation effects.Site-deleted mutation analysis revealed that deletion of 4 binding sites of C/EBPα at-1112,-734,-248 and-119 bp respectively can significantly reduced the basal promoter activity of PPARγ.Through the overexpression and interference experiments we found that mutation of the-119 bp C/EBPα binding sites completely abolished C/EBPα-induced PPARγ transcription.Thus,the C/EBPα gene can regulate the basic transcription activity of PPARγ gene through a C/EBPα binding element located in the core regulation region of PPARγ promoter.3.Using site-deleted mutation of three PPRE elements on the PPARγ gene promoter and analyzing the promoter activity,it is found that only mutation of the-1285 and-774 bp PPRE site significantly downregulated the promoter activity.Both PPARγ gene overexpression and specificity agonists ROSI treatment can enhance the wild type promoter activity.Furthermore,we overexpressed PPARγ gene in GMECs which were transfected with PPRE-mutation promoter,and found that only mutated PPRE element was at-774 bp,the promoter activity is not affected by overexpression treatment,suggesting that this element is the key element of PPARγ gene response auto-loop circuit regulation.In conclusion,the study cloned the 5’ flanking sequences of goat PPARγ gene on Xinong Saanen dairy goat,and by deletion analysis proved that the transcription core region of PPARγ gene promoter is located in-194 to-108 bp.There are multiple transcription factor binding sites on its promoter,and the C/EBPα binding element at-119 bp is the key to C/EBPα regulating PPARγ gene transcription.ROSI activated the transcription of PPARγ gene by a functional PPRE element on its promoter.