Integrated Multiomics To Identify Key Genes And Regulatory Elements In Porcine Skeletal Muscle Development

The growth and development of skeletal muscle determine the yield and quality of pork.Due to the influence of geographical environment and genetic background,there are some differences in skeletal muscle growth and development between local and commercial pigs.The growth rate of local pigs is slow,fat content is high,lean meat rate is low.Commercial pigs grow fast and have a high lean meat rate.It has been found that there are significant differences in the expression of skeletal muscle related genes between local and commercial pigs,but the chromatin status affecting gene expression has not been thoroughly studied.Therefore,we systematically compared the different chromatin accessibility patterns and regulatory element profiles of skeletal muscle growth and development at epigenome(ATAC-seq,ChIP-seq)and transcriptome(RNA-seq)levels in local and commercial pigs using published data.The results provide data support for analyzing the regulation mechanism of skeletal muscle growth and development between local and commercial pigs.In this study,local pigs(Enshi pigs,Meishan pigs)and commercial pigs(Duroc pigs,Large white pigs)were selected as the research objects to conduct epigenome and transcriptome analysis of gene expression regulation in skeletal muscle tissues of 2-week-old pigs.The results are as follows:1.A total of 1,799 diffierentially expressed genes(DEGs)were identified in Duroc and Enshi pigs,Duroc and Meishan pigs,Large white and Enshi pigs and Large white and Meishan pigs.GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)analysis showed that DEGs were mainly enriched in the biological processes and signaling pathways related to skeletal muscle development,such as glucose metabolism,skeletal muscle cell differentiation,myoblast differentiation,etc.Based on the results of functional enrichment,we screened some key genes for skeletal muscle development,such as ANGPT1,CEBPB,IGF2,KLF5,MYF6,MYOD1,PDK4,SOX8 and VEGFA.2.The chromatin openness of Duroc and Enshi pigs and Large white and Enshi pigs was compared and analyzed.4,090 and 3,527 Open Chromatin Regions(OCRs)with significant differences were selected from the two comparison groups,respectively.Motif enrichment analysis showed that 189 transcription factors were common in the two comparison groups,including MEF2 family,MYOD,MYF5,MYOG,FOXO1/3 and SIX1/4,which are closely related to skeletal muscle development.Functional enrichment results showed that the adjacent genes differential OCRs were mainly involved in biological processes such as skeletal muscle system development,muscle organ development and myoblast fusion.Combined with the results of DEGs analysis,a total of 205 differential OCRs and associated DEGs were found in the two comparison groups,and the correlation coefficient of signal strength was greater than 0.8,indicating that the openness of OCRs was closely related to gene transcription.Further prediction was made on the transcription factors potentially regulated by differential OCRs associated with DEGs.Finally,transcription factors in17 differential OCRs were identified to be involved in the regulation of 61 genes related to skeletal muscle development in the two comparison groups.3.The histone modification levels of Duroc and Enshi pigs,Duroc and Meishan pigs,Large white and Enshi pigs and Large white and Meishan pigs were compared.In the 4 groups,93,105,80 and 150 differential active promoters and 277,346,305 and789 differential active enhancers were identified,respectively.Functional enrichment analysis of differentially active regulatory elements using GREAT showed that these differentially active regulatory elements were mainly involved in biological processes such as muscle fiber development,skeletal muscle differentiation,MAPK and JAKSTAT cascade regulation.Combined with the results of DEGs analysis,a total of 42 DEGs regulated by differential active promoters and 64 DEGs potentially regulated by differential active enhancers were screened in the 4 groups.These genes include IGF2,IYD,MLC1,MYH7 B,PDK4,PVALB,RDH16,SIK1 and SNAI3,which may be affected by active regulatory elements.4.Through combined RNA-seq,ATAC-seq and ChIP-seq analysis,several genes related to skeletal muscle development were screened in this study,which are regulated by chromatin accessibility or histone modification.6 candidate target genes(CEBPB,LDHB,IGF2,KLF11,PDK4 and RDH16)were randomly selected for RT-qPCR validation.The results showed that the gene expression patterns were similar to those of RNA-seq analysis,which confirmed the reliability of data analysis.In conclusion,through the comparative analysis of local and commercial pig skeletal muscle regulation of gene expression pattern,screening of skeletal muscle development affected to a group of candidate genes,transcription factors,and the active regulatory elements,to explore the molecular mechanism of skeletal muscle growth and development to provide valuable reference,and enhance the understanding of the genetic basis of skeletal muscle growth and diversity.