| Object:The process of spermatogenesis is regulated accurately. One of the most distinctive characteristics of chromatin remodeling during spermatogenesis is the expression of a large number of histone variants with complex modifications. In addition to all the somatic-type histone variants, spermatogenic cells express testis-specific histones.This study was designed to better understand the global dynamics of chromatin structure and functions during spermatogenesis by comprehensive analyses of histone variants and post-tranlational modifications during spermatogenesisMethods:In the present study, histones were extracted from spermatogonia, spermatocytes and round spermatids of Sprague-dawley rats, separated by high performance liquid chromatography (HPLC), treated with enzyme Glu-c and(or) Arg-c digestions, and subjected to LC-MS and LC-MS/MS analyses to identify histone H2A.H2B variations and post-translational modifications.Then, Identify novel proteins recognizing histone modifications using peptide pull-down assay coupled with immunocoprecipitation.Results:Seven histone H2A subtypes were observed, and histone TH2A was identified for the first time in spermatogonia. Several novel modification sites were also identified in the C-terminal region of H2A1, including methylation at K96 and K100, dimethylation at K119 and acetylation at K96.Six histone H2B variations were also detected in spermatogenesis. The results showed modification patterns of the intact histone TH2B during spermatogenesis. Acetylated TH2B was most abundant in spermatogonia (28.9%) as compared with the spermatocytes (8.3%) and round spermatids (11.2%).Several new PTMs of TH2B were identified. In spermatogonia, spermatocytes, and round spermatids, T116 and K117 were modified by phosphorylation and methylation(acetylation) respectively, forming two novel "phospho switch" site.Prtein TRRAP, CENP-E and PTP-BL were identified, recognising the T116 and K117 modified by phosphorylation and acetylation, which may play role in the chromatin segment during meiosis.In addition, linker histone H1 variants that participate in spermatogenesis were idntified by Mass.Conclusion:we identified several histone H2A.H2B subtypes and modification patterns in spermatogenic cells, providing a basis for future studies on histone coding during spermatogenesis and epigenetic regulation of germ cells development. |
PDF Full Text Request