Identification Of DNA Methylation Regions Associated With Early Gonad Development In Chickens And Analysis Of Their Regulation For Gene Expression

DNA methylation plays a key role in sex determination and sexual differentiation in vertebrates such as fish and mammals.However,studies on DNA methylation in chicken gonad development are rare,and most have focused on male hypermethylated regions(MHM).Genome-wide DNA methylation has not yet been analyzed in the early gonads of chickens,and it is unclear whether there are tissue-specific differentially methylated regions(DMRs)that regulate sex determination and sexual differentiation.Therefore,in this study,whole-genome bisulfite sequencing(WGBS)and RNA sequencing(RNA-seq)were used to investigate the relationship between DNA methylation and sex differential expression of genes related to gonadal development,and to analyze the regulation of DNA methylation on lncRNA expression at the tissue and cellular levels.This study provided a basis for revealing the epigenetic mechanisms of early gonadal development in chickens.The results obtained in this study are as follows:1.The whole genome DNA methylation map of chicken early gonad was constructed by WGBS,and its DNA methylation pattern was revealed:about 3.5%of the C sites in chicken embryo gonad DNA were methylated in the whole genome,of which the methylation rate in the CG context was 53-60%,while the methylation rate in the CHG and CHH context was only 0.13-0.18%,and the mCG context was the main methylation context.2.The principal component analysis and cluster analysis of the DNA methylome showed that the DNA methylation patterns were similar between males and females at E6,and there were large differences in methylation between males and females at E10.There were large differences in methylation between E6 and E10.At E6,1073 DMRs were found between males and females,of which 93 DMRs were located in promoter,120 DMRs were located in exons,and 518 DMRs were located in introns;1284 DMRs were found between males and females at E10,of which 124 DMRs were located in the promoter,167 DMRs were located in the exon region,and 714 DMRs were located in the intron region.At E6 and E10,DMRs with more than 30%difference in methylation levels between males and females were mainly located on chromosome Z.At E6,the differentially methylated genes between males and females were mainly enriched in biological processes such as phosphorylation,macromolecule modification,protein phosphorylation,transferase activity.At E10,the differentially methylated genes between males and females were mainly enriched in regulation of metabolic process,regulation of transcription,regulation of cellular metabolic,development process,regulation of macromolecule metabolic,regulation of biological process,transcription factor activity and other biological processes.3.The three differentially methylated in promoter and differentially expressed long non-coding genes(ENSGALG00000051419,ENSGALG00000050012 and ENSGALG 00000045887)were verified by BSP and qRT-PCR.Sex differences in DNA methylation and expression levels were consistent with those in gonadal tissues.In addition,in the sexreversed chicken embryo gonads treated with aromatase inhibitor(AI),the methylation of the ENSGALG00000051419 and ENSGALG00000050012 gene promoter regions was consistent with that of females,while the methylation level of the ENSGALG00000045887 gene promoter region increased;And compared with females,the expression levels of the three candidate genes were significantly decreased,but the expression levels were significantly higher than those of males.4.Treatment of DF-1 cells with the methyltransferase inhibitor 5-Aza-CdR resulted in an overall decrease in DNA methylation levels and quantitative PCR results showed a significant increase in the expression levels of three candidate lncRNAs compared to controls,suggesting that their regulation of gene expression is influenced by DNA methylation.5.We constructed truncated vectors for the promoter region of the candidate gene and found the active core promoter region by dual fluorescence reporter gene experiments in DF-1 cells and chicken primary gonadal cells.We found the region which may have transcription factor binding sites that play an important regulatory role in the transcriptional activation of the gene.We used JASPAR software to predict transcription factors that may be affected by DNA methylation in the active core promoter segment,and found that there was differential DNA methylation in the binding site sequences of transcription factors such as YY1,NRF1,ZEB1,Lhx8 and E2F1.Based on the results of genome-wide DNA methylation analysis of chicken early gonads,we found that DNA methylation dynamics difference between sexes and may play an important role in the regulation of sexual differentiation-related genes.In addition,the results of studying the MHM region with the sex inversion model and demethylation treatment indicated that the MHM region may be involved in the process of cellautonomous sex identity in chicken.