High-throughput Sequencing Method To Study The Epigenetic Changes Of Histone H2A Variants And H3K79me3 Modification During Mouse Spermatogenesis

Spermatozoa are the terminally differentiated cells of the male germ cells.The chromosomes of the spermatozoa undergo unique changes to achieve sperm function.Unlike somatic cells,sperm chromosomes are mainly composed of protamine-bound DNA,which helps the DNA to be highly compressed in sperm nucleus.Only about 5%-15%of histones remains in mammalian sperm compared to somatic cells.During spermatogenesis,spermatogonia go through mitosis,meiosis of primary spermatocytes and secondary spermatocytes,and spermiogenesis to finally achieve sperm maturation.Accompanied by this process,sperm chromosomes also undergo dramatic changes.In the meiotic stage,chromatins undergo synapsis and two successive meiotic processes.During spermiogenesis,chromatin undergoes histone-transition protein replacement and transition protein-protamine replacement.The processes of replacement are accompanied by dramatic epigenetic changes.In this study,we elucidate the dynamic changes and biological functions of histone H2A variants in the pachytene primary spermatocytes and round spermatids.We also uncover the function of histone modification H3K79me3,which is localized in the pachytene spermatocytes during mouse spermatogenesis by the high-throughput sequencing method.The STA-PUT method was used to sperate the pachytene primary spermatocytes and round spermatids from mouse testises.To construct the high-throughput sequencing libraries of histone H2A variants and H3K79me3 in the pachytene spermatocytes and round spermatids,we performed the Native-ChIP.Bioinformatics analysis was used to investigate the distribution of histone H2A variants in pachytene spermatocytes and round spermatids on their host genes,chromosome locations and the transcription levels with the high-throughput sequencing data and the datasets that reported in public databases.Also,to dig the information on the H3K79me3 modified DNA sequences in the pachytene spermatocytes,we combined multiple analyses on the loci relationship between different histone variants and modifications with the transcriptional differences.Furthermore,we analyzed the gene ontology(GO)of H3K79me3 occupied gene.Native-ChIP libraries of histone H2A variants H2A.Z,?H2A.X and macroH2A in pachytene spermatocytes and round spermatids and the H3K79me3 library in pachytene spermatocytes were successfully constructed.We used the ChIP-seq method,that is,on mono-nuclesome level resolution,for the first time to describe the distribution and localization of the various variants involved in the study in pachytene spermatocytes and round spermatids,and the chromosomal distribution status of H3K79me3 in pachytene spermatocytes.We found that little gene was located on the histone H2A variants loci in the pachytene spermatocytes and round spermatids,and the histone H2A variants were distributed majorly in the distal intergenic region and intron.Besides,we found histone H2A variants do not directly contribute to the reactivation of X chromosome in the round spermatids.The functions of the histone H2A variants involved in the pachytene spermatocytes and round spermatids are not directly related to transcriptional regulation,but may participate in the reorganization of chromatin structure and chromatin remodeling.In addition,we found that during the round sperm stage,there are many overlapping between the H2A variants loci,which implicated the chromatins undergo dynamic replacement of the histone H2A variants.We verified the crosstalk between H3K4me3 and H3K79me3 in the spermatogenesis system by bioinformatic method.Combined with RNA-seq data,we found that the appearance of H3K79me3 in pachytene stage is correlated with transcriptional activation,especially with the pachytene spermatocyte and round spermatid specific genes.This detection is different from other reports.