有丝分裂细胞周期中核小体的解离模式与组蛋白甲基化的动态维持

【作者】 徐墨；

【导师】 朱冰；

【作者基本信息】 北京协和医学院 ， 生物化学与分子生物学， 2011， 博士

【摘要】 组蛋白八聚体与DNA包装形成的核小体结构是真核生物染色质的基本结构单元(Kornberg and Thomas,1974).成年动物由200多种不同类型的细胞构成,每种细胞具有各自独特的形态特征及生理功能(Allis et al.,2006)。除极少数例外,单个生物体内的所有细胞具有相同的遗传信息——DNA。多细胞生物使用各类染色质修饰将单一的基因组编辑成为数百种不同细胞特有的表观基因组。保证染色质状态在细胞世代之间以合适的方式传递,对生物个体发育以及成体的稳态具有重要意义,染色质状态继承的混乱可能导致多种发育紊乱或者疾病。由此表观遗传信息继承的分子机理受到研究者的普遍关注。除此之外,与遗传信息的高度稳定性不同,表观遗传信息具有一定程度的可变性和可逆性。表观遗传信息的继承或许无需如DNA复制一样具有精确性。DNA与组蛋白的修饰是表观遗传信息的重要组成部分。新合成的DNA链以类似半保留复制的方式重建DNA甲基化模式(Klose and Bird,2006)。然而组蛋白修饰是如何在有丝分裂细胞周期中传递给子代细胞的尚不清楚。为了解决这个问题,DNA复制偶联的核小体解离与重组模式必须首先被澄清。我们借助诱导表达体系,选择性地纯化含旧有FLAG-H3或者新生FLAG-H3的单核小体.利用基于稳定同位素标记的质谱定量技术,我们以高分辨率的模式检测了这些纯化到的单核小体中每种的组蛋白的新、旧构成。令人惊讶的是,尽管绝大多数(H3.1-H4)2四聚体在细胞周期中保持完整,有相当比例的(H3.3-H4)2四聚体发生解离。(H3.3-H4)2四聚体解离可以通过阻断DNA复制偶联的核小体组装得到抑制,这证明1.DNA复制不相关的H3.3组装途径主要通过同时装配两个H3.3-H4二聚体完成。2.尽管一定程度的(H3.3-H4)2四聚体解离不依赖DNA复制而发生,多数解离事件是DNA复制依赖的。组蛋白赖氨酸甲基化修饰在转录激活、基因沉默、以染色质形成以及DNA损伤修复等过程中发挥重要作用。然而对组蛋白甲基化整体水平变化的认识仍然缺乏。借助稳定同位素标记与质谱定量技术,我们对处于有丝分裂周期中组蛋白甲基化的重建以及非循环细胞中甲基化水平的保持进行了全面分析。我们的研究发现：1.组蛋白甲基化的整体水平在S期因DNA复制而打破,并在此后的整个细胞周期中逐步重建；2.尽管整体水平在细胞进入下一个S期前恢复,新、旧组蛋白上甲基化修饰的丰度具有明显差异；3.在非循环细胞中,各种甲基化水平会最终进入平台期；4.甲基转移酶活性的限制和甲基基团的动态置换均对非循环细胞中组蛋白甲基化整体水平的保持做出贡献。综上所述,我们的提出表观遗传修饰的保持具有一定的灵活性,而无需在特定位点精确重现。更多还原

【Abstract】 In eukaryotic cells, histones are packaged into octameric core particles with DNA wrapping around to form nucleosomes, the basic units of chromatin (Kornberg and Thomas,1974). An adult animal contains over 200 different cell types, each of them has specialized structure and distinct physiological function (Allis et al.,2006). With a few exceptions, all of these cells carry the same genetic information encoded by DNA. Multicellular organisms utilise chromatin marks to translate one single genome into hundreds of epigenomes for their corresponding cell types. During development and adult homeostasis, it is important to appropriately maintain the cromatin state after each cell division, as unscheduled compromise might lead to developmental disorder or disease. Thus, the molecular mechanism underlying epigenetic inheritance is highly interesting. Furthermore, unlike genetic information, which is a means of highly stable, epigenetic information stands for a certain level of plasticity and reversibility. The inheritance of epigenetic state may not be as faithful as that of genetic information.Histone and DNA modifications provide key epigenetic information. Newly synthesized DNA strand acquires DNA methylation pattern by copying the pre-existing DNA methylation signature from the template strand (Klose and Bird,2006). However, the mechanism by which patterns of histone modifications are passed on to daughter cells through mitotic divisions remains enigmatic. To understand this, the DNA replication-dependent nucleosome partition pattern must be unveiled first. Mono-nucleosomes containing either existing Flag-H3 from parental cells or newly deposited Flag-H3 were selectively purified. Stable isotope labeling-based quantitative mass spectrometry generated high-resolution, quantitative profiles for all co-purified individual native core histones. Surprisingly, significant amounts of H3.3/H4 tetramers split in vivo, while a vast majority of the H3.1/H4 tetramers remained intact. Inhibiting DNA replication-dependent deposition significantly reduced the level of splitting events, which suggest:1. the replication-independent H3.3 deposition pathway proceeds largely by cooperatively incorporating two new H3.3-H4 dimers; 2. detectable amounts of splitting events were seen during replication-independent deposition, but the majority of splitting events occurred during replication-dependent deposition.Histone lysine methylation has been implicated in transcription activation, gene silencing, heterochromatin formation and DNA repair. However, a thorough understanding of the dynamics of global methylation levels remains to be established. Using stable isotope labeling and quantitative mass spectrometry, we analyzed re-establishment of histone lysine methylation in dividing cells, and maintenance of methylation pattern in non-cycling cells experiencing an extended G1/S. Here, we report that:1) histone methylation levels are disturbed during S phase, and become gradually re-established at subsequent cell cycle stages; 2) despite the recovery of overall methylation levels before the next S phase, the methylation levels on parental and newly incorporated histones differ significantly; 3) in non-cycling cells, histone methylation levels eventually reach a plateau stage; 4) both restriction of methyltransferase activity and active turn over of methyl groups contribute to long-term equilibrium of methylation level in non-cycling cells arrested at G1/S. Taken together, we propose that the levels of epigenetic modifications are maintained in a flexible way, rather than in a local, precise manner.更多还原