精子发生过程中组蛋白H2A.H2B编码及功能研究
发布时间：2018-08-19 12:19
【摘要】：研究目的: 哺乳动物精子发生过程(Spermatogenesis process)是一被精确调控的复杂的细胞分化过程。原始生殖细胞经有丝分裂,两次减数分裂和变态过程最终发育成成熟精子。该过程中最显著的一个特点就是染色体重建(Chromatin remolding),其机制主要从两个方面来解读,第一,精子发生过程中有大量不同种类的组蛋白合成;第二,这些组蛋白的翻译后修饰可能呈现时间及空间特异性。“生精细胞特异的组蛋白编码”这一概念也随之提出,而目前还没有这方面系统的研究。 为了更好地理解精子发生过程中染色体重建。本研究应用质谱技术分析了参与精子发生的组蛋白H2A.H2B的亚型种类,组蛋白H2A.H2B翻译后修饰的动态变化,初步探索了相应翻译后修饰的功能。 研究方法: 首先应用密度梯度沉降的方法分离精原细胞,精母细胞和圆形精子细胞;分别纯化各细胞的组蛋白,应用HPLC进一步分离纯化组蛋白各组分(H1,H2A,H2B,H3,H4)。纯化后的组蛋白H2A和H2B经蛋白酶酶切,应用LC-MS;LC-MS/MS分析H2A.H2B的亚型种类及其翻译后修饰。 联合应用Pull-down和免疫共沉淀的方法来鉴定识别组蛋白修饰基团的伴侣分子。 研究结果: 精子发生过程中,一共鉴定了7种H2A亚型,睾丸组织特异的组蛋白TH2A首先出现在精原细胞中。发现了一些新的位于H2A1 C末端的翻译后修饰,包括K99和K100的甲基化,K119的二甲基化和K96的乙酰化。 同时鉴定了6种H2B亚型。在此期间,睾丸组织特异的组蛋白TH2B的翻译后修饰呈动态变化:精原细胞中乙酰化TH2B的相对丰度最高,约有28.9%的TH2B为乙酰化状态;精母细胞乙酰化的TH2B降到了最低(8.3%),到了圆形精子细胞阶段乙酰化的TH2B约为11.2%。 同时也鉴定了几种TH2B的翻译后修饰,TH2B的N端呈高乙酰化修饰状态;在TH2B的C端,我们发现了T116的磷酸化和K117的甲基化(乙酰化),形成了两个新的“磷酸化开关”(phospho switch)。 蛋白-蛋白相互作用的结果提示,蛋白质TRRAP,CENP-E和PTP-BL可能是识别H2B PhT_(116)/AcK_(117)的蛋白因子,提示该组合修饰可能参与了减数分裂过程中的染色体分离。 另外,应用质谱技术描述了参与精子发生的组蛋白H1的多样性。 研究结论: 本研主要应用质谱技术描述了精子发生过程中组蛋白H2A和H2B的亚型种类及其翻译后修饰;鉴定了识别其特异修饰基团的蛋白质。该研究尽管还不尽完善,为进一步理解精子发生过程中染色体的重建奠定了基础。
[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.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R321
本文编号：2191631
[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.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R321
【共引文献】
相关期刊论文 前5条
1 瞿颖,刘炳亚;癌症高表达蛋白——Hec1在纺锤体组装检查点中的作用[J];生命科学;2004年05期
2 王春梅,吴燕婉,李伟雄;哺乳动物精子发生过程中阶段特异性基因表达[J];生殖医学杂志;1999年04期
3 焦海燕,墙克信;染色体着丝粒—动粒复合体研究进展[J];宁夏医学院学报;2001年05期
4 鲁爽;程露阳;汪卓群;李欣;石心泉;贾孟春;马旭;;质谱法分析大鼠精子发生过程中组蛋白H1变异体的多样性[J];中国实验动物学报;2009年05期
5 曹波;人类染色体着丝粒点结构及功能的研究[J];中国优生与遗传杂志;2003年06期
相关博士学位论文 前10条
1 张清华;mSpindly在小鼠卵母细胞减数分裂进程中的定位与功能[D];山东师范大学;2010年
2 李彦锋;重组鼠精子抗原疫苗株的构建及其避孕效应研究[D];第三军医大学;2001年
3 倪成志;拟南芥类动蛋白AtKP1的亚细胞定位研究[D];中国农业大学;2005年
4 李绪彦;拟南芥动蛋白异型体AtKP1基因功能的研究[D];中国农业大学;2005年
5 姜世玲;拟南芥中两种动蛋白异型体KatB/KatC生化性质及组织分布研究[D];中国农业大学;2005年
6 符传孩;细胞核PML body动力学及PRC1中体微管构架的分子与蛋白质组学解析[D];中国科学技术大学;2006年
7 刘辉贤;大鼠精子发生阶段特异表达基因的分离和表达[D];中国协和医科大学;1999年
8 柯玉文;Aurora激酶生化通路对细胞有丝分裂可塑性的调节作用[D];中国科学技术大学;2007年
9 牟长军;拟南芥菜基因Arabidillo-1结构和功能的初步研究[D];兰州大学;2007年
10 都建;纺锤体微管与动点粘连的分子解析[D];中国科学技术大学;2007年
相关硕士学位论文 前3条
1 董梅;果蝇精子生成过程中新基因的鉴定及哺乳动物甲基转移酶3a/3b作用研究[D];东北师范大学;2004年
2 李素彦;CENP-E基因表达下调增强宫颈癌细胞对紫杉醇的敏感性[D];重庆医科大学;2009年
3 闫琛;两种CENP-E mRNA形式在不同肿瘤组织中的表达差异及其功能研究[D];重庆医科大学;2010年
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