Dynamic Changes In N6-methyladenine(6mA)and Its Mediated Transcriptional Regulation In Chicken Embryonic Skeletal Muscle

DNA N(6)-methyladenine(DNA-6mA)is a novel epigenetic marker in eukaryotes,and its distribution and function in genomic DNA are not fully understood.Although recent studies have shown that 6mA is present in a variety of model organisms and is dynamically regulated during development,the genomic signature of 6mA in avian species remains to be elucidated.In this study,we analyzed the distribution and dynamics of 6mA in muscle genomic DNA at eight time points(E5,E7,E9,E12,E15,E18,E20,D1)during chick embryo development(embryonic 5 days of age to postnatal 1 day of age)using 6mA immunoprecipitation sequencing.Then the 6mA immunoprecipitation sequencing data were analyzed jointly with transcriptome sequencing data to reveal the role of 6mA in the regulation of gene expression.Finally,the results of previous studies were combined to explore the possible pathways of 6mA involvement in muscle development.The main results are as follows:1.The overall pattern of genome-wide 6mA methylation of skeletal muscle in chickens showed a dynamic change from E5 to D1 during growth and development from5 days of embryonic age to 1 day of postnatal life.Meanwhile the transcriptome also showed continuous changes during embryonic skeletal muscle development.2.Analysis of 6mA methylome data showed that 6mA was significantly enriched in intergenic regions compared to promoters and gene bodies(P < 2.2e-16);6mA was significantly enriched in protein-coding genes compared to other types of genes(P <2.2e-16);short autosomes tended to have higher peaks coverage compared to longer autosomes,the highest peaks coverage was found in mitochondrial DNA(23.82%);6mA modification was region-specific and enriched downstream of the gene transcription termination site.3.The coverage of 6mA peaks in the whole genome tended to decrease with embryonic skeletal muscle development(Spearman’s r =-0.69,P = 0.058),but the distribution pattern was relatively stable across gene elements and repeat elements.4.6mA modifications were significantly associated with the expressed genes encoding proteins,and the presence of 6mA in the promoter may have inhibited gene expression.These genes modified by 6mA were mainly enriched in embryonic organ development,regulatory,and other related pathways.5.N6AMT1 and DNMT3 B are potential 6mA methyltransferases in chickens.seven genes(MATK,ENSGALG00000005956,HSPB8,OASL,LRATD1,XKR9,and ENSGALG00000051278)showed a significant negative correlation between 6mA levels and gene expression(P < 0.05).6mA may be involved in muscle development and immune processes through down-regulation of HSPB8 and OASL expression.In summary,this study provides evidence that 6mA modification is widespread throughout the chicken genome and presents preliminary data on the genome-wide distribution of this epigenetic marker.6mA modification in the promoter region represses gene expression,while promoters of some developmentally relevant genes are modified by 6mA,suggesting that 6mA may be involved in chicken embryonic development.In addition,6mA may be involved in muscle development and immune function by regulating HSPB8 and OASL expression.These results will help us to further understand the epistatic modifications of the avian genome and their roles,and provide a reference for avian developmental studies.