Analysis Of DNA Methylation Patterns In Skin Tissues At Different Stages Of Hair Follicle Development In Subo Merino Sheep

Subo Merino sheep is China’s super fine wool sheep new breeds,its wool growth and hair follicle development has been the concern of researchers.As an important epigenetic mechanism,DNA methylation plays a role in many biological processes such as gene expression and genomic imprinting.Also,DNA methylation can play a role by regulating the expression of genes related to hair follicle development.Therefore,in this study,the Subo Merino sheep skin at 6 stage were collected at embryonic days 65d(G1),85d(G2),105d(G3),135d(G4),born days at 7(G5)and 30(G6)were selected as the research objects,using MeDIP-seq technology to explore the changes of DNA methylation status during the hair follicle development.This study aimed to reveal the regulatory mechanism of the development process from the perspective of epigenetics,thus providing a basis for further research on candidate genes and laying the foundation for the construction of a complete regulatory network for the hair follicle development of Subo Merino sheep.Through the MeDIP-seq,we obtained the genome-wide DNA methylation profiles of Subo Merino sheep,and the number of Peaks scanned by each individual ranges from 75,514 to 129,618,which most distributed in the intergenic region and the promoter region.Comparing the DNA methylation regions of the neighboring comparison group in the six stages of Subo Merino hair follicle development,a total of 596 differentially methylated genes(DMGs)were noted.Cluster analysis of differentially methylated genes showed that the GO terms mainly includs:hair follicle development,hair cycle process,skin epidermal development,and cell differentiation,etc.The KEGG pathways analysis results include related to hair follicle development such as ECM-receptor interactions,MAPK signaling pathways,TNF signaling pathways,TGF-beta signaling pathways,etc.We intergrated analysis of transcriptome data in our previous study and DNA methylation data to find potential regulatory realationships between epigentetic regulation and gene expression.A total of 65 genes that both differentially methylated and differentially expressed were found,which could be the results of the DNA methylation regulation.PPI analysis of the protein interaction network found that proteins encoded by genes such as FURIN,TIMP2,MMP14,EDN1,IGF1,and GHR were related to each other,and using the Cytoscape software to find the hub gene,EDN1,IGF1,and B2M genes may be important targets in the hair follicle development.Five genes that were differentially methylated and differentially expressed were selected:EDN1,LAMC2,NR1D1,RORB,and MYOZ3 for validation using BSP of differences in DNA methylation levels.The results showed that EDN1,NR1D1,RORB,MYOZ3 genes are hypermethylated genes,and LAMC2 genes are hypomethylated genes Compared with the sequencing results,the MeDIP-seq and BSP results were basically consistent,indicating that the sequencing results were more accurate.DNMT3a,and DNMT3b as the encoding genes of methylation transferase,are involved in the regulation of target gene methylation,which affects its transcription.Analysis of the expression levels of and genes showed that DNMTs genes were expressed in six stages of hair follicle development During the critical period of secondary hair follicle development,the expression of EDN1 gene was significantly higher in embryonic days 105d than in 135d,while the expression trends of DNMT3a and DNMT3b genes in embryonic days 105d and 135d stages are similar to the changes in methylation of EDN1 gene,so it is speculated that DNMTs gene regulates the level of EDN1 gene methylation and participates in the regulation of hair follicle development cycle.In summary,this study uses DNA methylation immunoprecipitation and high-throughput sequencing technologies to select a series of differentially methylated genes and relate to various hair follicle development metabolic pathways.The study provided crucial candidate genes for the hair follicle development research,at the same time,has important significance for explaining the regulation network of hair follicle development from molecular and epigenetic aspect.