| The rapid development of sequencing technology has been contributing to the increasing improvement of the pig reference genome.However,the lack of information regarding cis-regulatory elements embedded in the pig genome has limited the genetic improvement or manipulation of pigs as an animal protein supply and biomedical model.In this study,the cis-regulatory elements and their functions in 12 diverse tissues from four pig breeds were systematically characterize to fill this gap by adopting similar strategies as the Encyclopedia of DNA Elements(ENCODE)and Roadmap Epigenomics projects,which include transcriptome sequencing(RNA-seq),Assay for Transposase-Accessible Chromatin using sequencing(ATAC-seq),and Chromatin immunoprecipitation followed by sequencing(Ch IP-seq)of histone modification(H3K27ac and H3K4me3).Moreover,the High-throughput chromosome conformation capture(Hi-C)experiments were performed to further explore the three dimension structure of pig genome.In addition,most single nucleotide polymorphisms(SNPs)are detected in noncoding region of pig genome,such that it is difficult to interpret the functions of these SNPs.Therefore,the functional annotation of SNPs were conducted based on pig epigenomic features.The main results of this study are as follows:(1)In this study,199 datasets were generated through self-constructed libraries.Based on sus Scr11 genome assembly,3316 new transcripts that without annotation in pig genome were identified,including 1713 long non-coding RNAs(lnc RNAs).Moreover,220,723 potentially non-redundant cis-regulatory sequences were identified,including37,838 promoters,146,399 enhancers and 137,838 open chromatin regions.The accuracy and reliability of the cis-regulatory elements identified in this study were confirmed by comparison with the results of a previous study and experimental validation of Dual-Luciferase Reporter Assay System.(2)In the present study,4510 tissue-specific genes and 15,753 tissue-specific enhancers were identified according to gene expression or H3K27 ac intensities of enhancers across different tissues and confirmed by the functional enrichment analysis.In addition,414-1306 super-enhancers,418-1899 broad H3K4me3 peaks and 13,971-20,138 active promoters were separately identified in each tissue of each breed.Furthermore,these types of cis-regulatory elements were found to be associated with gene expression in pigs.(3)The Hi-C contact map were built for pig skeletal muscle.Additionally,2305 topologically associating domains(TADs)and 11,838 loops were identified at 40 kb resolution.The significant enhancer-gene pairs(R>0.5),enhancer-enhancer pairs(R>0.5),and gene-gene pairs(R>0.8)embedded in the same TAD were identified based on their Spearman correlation coefficient values.In addition,79.15% of pig TAD boundaries were indicated to be usage conserved with humans in this study,which suggested that the TAD structure is strongly conserved between pigs and humans.(4)The conservation comparisons between different species indicated a higher conservation of cis-regulatory elements between human and pig genomes than those between human and mouse genomes.There were similar expression patterns of pig-human orthologous genes(one-to-one)within variously concordant tissues between pig and human.Further,Spearman correlation coefficient values of H3K27 ac intensities in the vicinity of the pig-human orthologous gene pairs(±500 kb)were significantly higher than those between the non-orthologous genes.These results can provide an important reference value for pigs as animal models to study human diseases.(5)The differential expression analysis of genes between four breeds were performed separately in five tissues.In total,7708 non-redundant differentially expressed(DE)genes were identified with FDR<0.05.Furthermore,the changes in H3K27 ac intensities of active promoters(or enhancers)were consistent with their DE genes(or their significantly correlated DE genes).These results indicated that differences in gene expression might be associated with differences in histone modifications of promoters or enhancers between different breeds.(6)Totally,251,361 SNPs with differential allele frequencies were localized in active promoters or enhancers based on the comparison of Western commercial pigs with Chinese local pigs.A T/C SNP(Chr1:190035161)and a G/C SNP(Chr12:5451199)both with differential allele frequencies were supposed to associated with the activities of cis-regulatory elements corresponding to SIX1,SIX4,and ACOX1,which with differential expression between Large White and Enshi Black pigs.In addition,enhancers were significantly enriched around published genome-wide association studies(GWASs)significantly-associated SNPs,indicating the potential regulatory function of these enhancers in genetic regulation of pig complex traits.(7)In this study,22,926,176 SNPs(minor allele frequency of at least 0.0407)were conducted annotation and classification based on the locations of SNPs in functional region and their possibility of affecting transcription factor binding.Finally,6,071,960 putative functional SNPs were identified and that provided an important reference resource for screening key regulatory loci affecting economically important traits or disease phenotypes in pigs.A T/C SNP located in Chr14:23914097 were identified as a crucially candidate site for pig melanoma research. |
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