精子發(fā)生過(guò)程中組蛋白H2A.H2B編碼及功能研究
[Abstract]:Objective: mammalian spermatogenesis (Spermatogenesis process) is a complex cell differentiation process regulated precisely. Through mitosis, meiosis and metamorphosis, the primordial germ cells develop into mature spermatozoa. One of the most significant characteristics of this process is that the mechanism of chromosome reconstruction (Chromatin remolding), is interpreted from two aspects: first, there are a large number of different types of histone synthesis during spermatogenesis; second, Posttranslational modifications of these histones may present temporal and spatial specificity. The concept of specific histone coding for spermatogenic cells has been proposed, but no systematic research has been done. In order to better understand the process of spermatogenesis chromosome reconstruction. In this study, the subtypes of histone H2A.H2B involved in spermatogenesis and the dynamic changes of post-translational modification of histone H2A.H2B were analyzed by mass spectrometry, and the function of the corresponding post-translational modification was preliminarily explored. Methods: firstly, spermatogonia, spermatocytes and round spermatocytes were isolated by density gradient sedimentation, histone of each cell were purified and further purified by HPLC. The purified histone H2A and H2B were digested by protease, and the subtypes of H2A.H2B and its posttranslational modification were analyzed by LC-MS / MS / MS. Pull-down and immunoprecipitation were used to identify the chaperones that recognize histone modified groups. Results: during spermatogenesis, seven H2A subtypes were identified, and testis histone TH2A was first found in spermatogonia. Some new post-translational modifications at the C-terminal of H2A1 were found, including the dimethylation of K99 and K100 methylation of K119 and the acetylation of K96. At the same time, 6 subtypes of H2B were identified. During this period, the posttranslational modification of testicular histone TH2B showed dynamic changes: the relative abundance of acetylated TH2B was the highest in spermatogonia, and about 28.9% of TH2B was acetylated; The TH2B of acetylation of spermatocytes was the lowest (8.3%), and the TH2B of acetylation of spermatozoa was about 11.2% at the stage of round spermatozoa. At the same time, several kinds of TH2B were identified as highly acetylated N-terminal modification of TH2B. At the C end of TH2B, we found phosphorylation of T116 and methylation (acetylation) of K117, which formed two new "phosphorylation switches" (phospho switch). The results of protein-protein interaction suggest that CENP-E and PTP-BL may be the protein factors that recognize H2B PhT116 / AcK117, suggesting that the combined modification may be involved in chromosome segregation during meiosis. In addition, the diversity of histone H 1 involved in spermatogenesis was described by mass spectrometry. Conclusion: the subtypes and posttranslational modifications of histone H2A and H2B during spermatogenesis were described by mass spectrometry, and the proteins that recognized their specific modification groups were identified. This study, though imperfect, lays a foundation for further understanding of chromosome reconstruction during spermatogenesis.
【學(xué)位授予單位】:中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】:博士
【學(xué)位授予年份】:2009
【分類號(hào)】:R321
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