| Peroxisome proliferator-activated receptor beta/delta(PPARD)is a ligand-activated transcription factor that plays an important role in many biological processes.Our previous studies identified a missense mutation in the PPARD gene that significantly reduces its transcription activity and consequently causes enlarged external ears in pigs.However,how PPARD regulates the growth of pig auricle cartilage and the mechanisms underlying the causality have remained largely unknown.To this end,we first measured the size of external ears in Erhualian and Duroc pigs at five time points.Erhualian showed a significantly faster growth rate of external ears than Duroc.We speculated that the composition of the auricular cartilage may be different between Erhualian and Duroc pigs.We then collected auricular cartilage tissues from the two breeds at the same age and conducted histochemical staining on the collected tissues.The results showed that Erhualian pigs had much more isogenous groups that were formed after chondroblasts divided continuously.In contrast,mature chondrocytes accounted for a majority of cartilage cells and matrix showed a certain level of degradation in Duroc pigs.We next isolated the primary pig auricle chondrocytes in vitro,and tested the composition of these primary cells in commercial hybrid pigs using flow cytometry analysis.The activation of PPARD gene by its agonist(GW0742)accelerated the apoptosis of cartilage stem/progenitor cells(CSPCs)and induced the differentiation of CSPCs into chondrocytes.We further performed transcriptome sequencing to identify differentially expressed genes(DEGs)in the primary pig auricle chondrocytes after the activation of PPARD.A total of98 DEGs were identified.These genes were most significantly enriched in the PPAR signaling pathway,and DEGs enriched biological processes were all related to the development of auricular cartilage,which is perfectly consistent with our phenotypic observation that PPARD inhibits the growth of auricular cartilage.Next,we performed Ch IP-seq experiments to search for the target genes of the PPARD in primary auricular chondrocytes.A total of 87 target genes were identified,none of which overlapped with DEGs.This indicates that PPARD may regulate DEGs via cross-regulatory networks.To test this possibility,we identified a target gene of PPARD(PPARG),which is a key gene for cartilage development.Both fluorescence reporter assays and RT-q PCR confirmed that PPARD inhibited the transcriptional activity of PPARG,up-regulated the expression of CEBPB and IGFBP3 that promote CSPCs apoptosis and differentiation,and also of MMP1 and MMP13 that degrade cartilage matrix and mineralize cartilage.Taken together,PPARD G32 E attenuates the depression effect of PPARD on PPARG and delays the functions of proapoptotic and degradative factors,thus causes larger ears in pigs.This study illustrates the inhibitory role of PPARD in the development of ear cartilage and uncovers the molecular mechanisms of PPARD G32 E causing large external ears in pigs.Liver plays an extremely important role in the metabolism of lipids,carbohydrates and proteins in mammals,associated with a large number of traits.Promoters and enhancers in the genome are important functional regulatory elements with great effects on gene expression.The vast majority of GWAS tag SNPs lie in intergenic or intronic regions,which often harbour potential functional regulatory elements.Here,a preliminary study was conducted to seek how SNPs in promoter and enhancer enriched regions regulate hepatic gene expression in pigs.In this study,we firstly collected liver tissues form F6 heterogeneous pig population which is multigenerational outbred population consists of 8 pig breeds(four Chinese native pig breeds-Laiwu pig,Erhualian pig,Tibetan pig and Bamaxiang pig;and four western pig breeds-white Duroc,Pietrain,Large white and Landrance).And chromatin immunoprecipitation sequencing(Ch IP-seq)with H3K27 ac antibody were carried out to capture enhancers in the porcine genome.On average,27,474 active enhancers were identified with an average length of 2,782 bp in the liver,accounting for ~2.9% of the pig reference genome.About 53.0% of the enhancers were located in introns,which is consistent with previous reports in human and mouse.Next,a large-scale transcriptome sequencing was performed on liver samples from109 individuals,expression quantitative trait loci(e QTL)analysis was performed based on whole genome sequencing data.We annotated the significant e QTL using hepatic enhancers and promoters reported by Villar(2015).The results showed that about 72.0%and 37.6% of cis-e QTL and trans-e QTL were located in enhancers and promoters.These e QTL associated with genes that involved in biological processes including lipid metabolism,carbohydrate transport,innate immune activation,vitamin metabolism and oxidative phosphorylation.We calculated the distances between cis-e QTL and physical location of their target genesand found that SNPs from promoter and enhancer regions tend to associate with their adjacent genes.This was especially the case for e QTL in promoters,most of which located within 75 kb to their associated genes.In comparison,e QTL in the enhancer region were mostly found beyond 75 kb from their associated genes,corresponding to more SNP-gene pairs,which consistent with the long-distance regulation pattern of enhancers.In this study,we have also identified e QTL in promoter and enhancer regions that were significantly associated with genes associated with liver diseases in human.Among them,a mutation(chr3:75494557 A>G)greatly down-regulated the expression of PCYOX1(P = 1.21e-87).The expression level of ABCB4 located on chromosome 9 was regulated by a SNP from chromosome 15.These findings not only provided a reference for the study of liver disease in human,but also laid a foundation for the large-scale study of how SNPs in the functional regulatory regions eventually cause phenotypic changes via affecting gene expression regulations. |
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