Study On The Spatiotemporal Variations Of Pig Methylomes And The Function Of Methylated PRE1s

DNA methylation is of paramount importance for mammalian growth and development,and it has numerous functions that involve exquisite spatiotemporal regulation of genes.Pig is not only the main meat supply animal,but also gradually become the model animal for human medical research.This study focuses on the methylome from pigs’different tissues with large age span,systematically analyzed the effects of different tissues,ages,breeds,genders,pedigrees on pig methylome,and clarified the structure and the methylation-associated regulatory functions of PRE1s(Porcine Repetitive Element 1s)that are the most widespread repeat elements.In this study,eight Bamaxiang boars and four Large White pigs at age of 7 months to9 years old were used as the research animals,and the longissimus dorsi(LD)and testis were selected as the research objects.High-depth whole-genome bisulfite sequencing(WGBS)was performed on 20 samples,and a total of 2.24Tb clean data was obtained from which55,685,213 autosomal Cp Gs were detected.The genomes of all adults’muscle and testis tissues were heavily methylated with global m CG levels of 64.79-73.39%,some local regions of genome showed hypomethylation,especially CGI promoters.Using binomial mixed model or Beta distribution model,we identified 5.30M,0.20M,1.20M,and 0.16M differentially methylated Cp Gs(P<0.01)between tissues,ages,breeds,and genders,respectively.Between tissues,7.54%of differentially methylated regions(DMRs)between LDs and testes from Bamaxiang pigs are co-localized with promoters,which potentially regulate gene expression.RNA-seq analysis revealed that 23.42%Cp Gs are significantly correlated with gene expression(mean|r|=0.58,P<0.01),most of which are enriched in tissue-specific functions.For example,SPACA1 and MEI1 are involved in acrosome formation and meiosis which are the testicular tissue-specific functions,and TNNT3 and MYBPC1 have related to the contraction of fast and slow muscle fibers.Among ages,the Cp Gs of age-related methylation changes showed discrete distribution,but a fraction of differentially methylated Cp Gs are enriched in genes that relate to aging.We also established two robust age predictors based on methylation level of Cp Gs(R~2=0.9988,MAD=0.099 year)using elastic net regression,and the predictor for adult LD and testes include 197 and 163 Cp Gs,respectively.Between breeds,the HOXB and HOXC gene clusters contain a large number of differentially methylated Cp Gs between LDs from Bamaxiang and LW pigs,which play important regulatory roles in myocytes hypertrophy and intermuscular fat deposition.Dramatically,higher similarity of methylation pattern was observed within pedigree than across pedigrees,which indicates the existence of heritable methylation regions.This study found that the widespread PRE1s were located in the DMRs.To explore the methylation-associated functions of PRE1,we first reconstructed the structure and the evolution history of PRE1,and found that PRE1 was originated from ancestral CHR-1family by three successive partial duplications.Splicing sites enrichment analysis showed that PRE1s harbored a bunch of splicing signal motifs,which mediated exonization and alternative splicing.We hence proposed a splicing model to explain its duplication,deletion,and degenerative direct repeat in evolution history of PRE1.Co-differential analyses of methylation and RNA-seq data detected 364 significant differentially methylated proximal PRE1s significantly correlated with expressions of their neighboring genes between adult muscle and testis tissues.Epigenomic analysis reveals that demethylation of proximal PRE1s of HORMAD1 and HACD3 in testis act as promoter of HORMAD1 and enhancer of HACD3 respectively,while methylation of proximal PRE1 of TCEA3 offers muscle-specific enhancer for TCEA3.These results demonstrated the unique origin and evolution processes of PRE1,and it provided a structural basis for diverse regulatory functions.In conclusion,a part of Cp Gs in genome can change its methylation pattern in different age stages and tissues,and play a remarkable regulatory function for gene expression in different physiological or natural environments.