【作者】 周国岭；

【导师】 梁植权； 刘德培； 吴雪松；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 细胞分裂中染色质活性和转录状态记忆机制的研究高等真核生物分化发育中要产生多种细胞型，每种细胞型都建立了它特异的基因表达谱，这些特异的基因表达谱在随后的细胞分裂中被有效地保持，以保证生物体分化和发育的稳定型。既然每种细胞型通过DNA复制和有丝分裂都保持着相同的遗传物质—基因组DNA，那么与基因表达密切相关的表观遗传学信息可能在特异细胞型的基因表达谱的传递和保持中起重要作用。因而研究表观遗传学信息在细胞周期进程中如何变化并记忆基因的活性和转录状态是阐明生物体分化发育机制的一个重要课题。为了研究组蛋白修饰是否在细胞分裂中染色质活性和基因表达状态的记忆中发挥作用，利用针对四种活性组蛋白修饰(H3乙酰化、H4乙酰化、H3-K4双甲基化、H3-K79双甲基化)的抗体，采用免疫荧光实验分析了四种修饰在小鼠的红白血病(Murine erythroleukemia，MEL)细胞细胞周期中的变化。结果表明，在间期的细胞核中，H3和H4乙酰化、H3—K4双甲基化三种修饰主要集中在常染色质区，而H3—K79双甲基化修饰除了集中在常染色质区外，在异染色质区也有一些分布，当细胞进入有丝分裂期，染色质固缩为染色体，四种修饰在中期染色体上都有保留，有丝分裂末期，子代细胞核分裂，四种修饰在细胞核内的亚核定位又恢复了。在有丝分裂染色质固缩过程中，大部分由RNA聚合酶介导的转录过程也停止了，有丝分裂后，染色质解固缩，核结构重建，停止的转录又继续了。那么四种活性组蛋白修饰在有丝分裂染色质凝集时有无变化，是否能介导染色质活性和基因转录的保持?为了研究这个问题，我们用有丝分裂的同步化试剂Nocodazole将MEL细胞同步化到有丝分裂期，比较了同步化和未同步化的MEL细胞中四种活性组蛋白修饰在两种细胞群体中的保留情况，探讨了有丝分裂染色质失活时这些活性修饰在基因表达状态记忆中的作用。Nocodazole处理将95％以上的MEL细胞同步化到了G2／M期，在未同步化的MEL细胞中则包含了大约50％的G0／G1，45％S和少于5％的G2／M期细胞。用Westernblotting分析四种修饰在两个细胞群体中总的变化，发现与未同步化的MEL细胞相比，尽管H3和H4乙酰化、H3—K4双甲基化三种修饰在同步化到有丝分裂期的MEL细胞中降低了20-30％，但仍有保留，而H3—K79双甲基化修饰在两种细胞群体中无太大变化。为了研究有丝分裂时这些修饰在染色体上特定位点的变化，分析他们与基因转录状态记忆的关系，我们选择了具有各种转录状态的基因，用染色质免疫沉淀的方法分析了有丝分裂染色质失活时这些修饰在基因启动子区的变化，结果显示四种活性组蛋白修饰可以以不同的组合模式修饰不同转录状态的基因的启动子区，尽管某些种类的修饰在有丝分裂染色质失活时有所降低，但这些修饰仍有保留，而且保留的水平依赖于原来水平的高低，说明这些组蛋白修饰在有丝分裂时可以作为基因活性和转录状态的标记信号，记忆它们以前的表达状态。远距离调控元件是高等真核生物调控基因表达的独特和重要的因素，通过所在区域的染色质结构变化并结合特异的转录因子调控基因的转录状态的变化。为了探讨远距离调控元件在基因表达状态记忆中的作用，我们选择了被广泛作为模型研究远距离基因激活的小鼠的α—和β—珠蛋白基因簇作为模型。通过分析同步化和未同步化MEL细胞中四种活性组蛋白修饰在α—和β—珠蛋白基因簇远端高敏位点上的变化，发现四种活性组蛋白修饰仍以不同的组合修饰不同的高敏位点，尽管某些种类的修饰在有丝分裂染色质失活时有所降低，但这些修饰仍有保留，而且保留的水平依赖于原来水平的高低。这说明在有丝分裂时活性组蛋白修饰可以在远距离调控元件上保留不同的组合模式，保持所在区域的染色质状态和所调控基因的转录状态。上面的结果充分说明了在基因表达调控中起重要作用的组蛋白修饰可以在有丝分裂时充当分子记忆信号，保持染色质活性和基因的转录状态，便于在有丝分裂结束时大量基因转录状态的迅速恢复。为了分析与基因转录密切相关的特异转录激活子在有丝分裂转录记忆中的作用，我们选择了两个对珠蛋白基因表达起重要作用的转录因子GATA-1和NF-E2p45，同时选择有丝分裂染色质凝集时与染色体分离的RNA polⅡ做对照。首先用免疫荧光分析了细胞有丝分裂时它们在细胞核中的变化，结果表明，有丝分裂染色质凝集时RNApolⅡ与染色体无共定位，弥散到整个细胞中。红系特异的激活子GATA-1与之有类似又不同的变化，在间期的细胞核中GATA-1只分布在核区，染色质凝集时，GATA-1在细胞中消失，当有丝分裂后期，姊妹染色单体分开，GATA-1开始在核区出现，有丝分裂末期，子代细胞核分裂时，GATA-1在细胞核中的分布又重新恢复。这说明与RNA polⅡ一样，红系特异的激活子GATA-1在有丝分裂时与染色体分离了。但是有丝分裂时另一个红系特异的激活子NF-E2p45与染色体存在共分布。为了更精确地验证有丝分裂时NF-E2p45与染色体的关系，我们进行了同步化和未同步化MEL细胞的比较染色质免疫沉淀实验，结果显示NF-E2p45与珠蛋白基因簇远端调控元件的两个高敏位点可以特异结合，而且在有丝分裂染色质失活时也可以特异保留在它的结合位点。说明尽管有丝分裂染色质失活时大部分转录因子都与染色体分离了，仍有某些特异的蛋白因子可以保留在中期染色体上充当某种分子记忆信号，并可能在下一轮基因转录状态恢复时起重要作用。已有的研究表明组蛋白H3异构体H3.3在核小体中的整合和移出与转录过程密切相关。为了分析它在染色质中的分布与转录记忆的关系，我们用针对内源H3.3的抗体进行了下面的实验，用免疫荧光分析它在细胞核中的变化时，发现有丝分裂染色质凝集时一部分的H3.3蛋白成团聚集在细胞膜附近区域，但在核区的染色体分区也有分布。为了验证H3.3在中期染色体上是否还有特异位点的保留，比较染色质免疫沉淀分析揭示它在活性和非活性基因位点上都有一定的分布，而且有丝分裂时仍然保留。因而尽管H3.3在核小体中的整合和移出与转录过程密切相关，但它在染色质中的分布是否与基因活性密切相关，是否介导转录记忆需要进一步验证更多的基因和染色质位点。以上结果表明：(1)．活性组蛋白修饰可以以不同的组合模式标记基因不同的转录状态，尽管有些修饰在有丝分裂时有所降低，但这些修饰的组合模式在有丝分裂染色质凝集时仍然保持，从而在中期染色体上标记了基因不同的转录状态，从而有利于有丝分裂后大规模基因表达的重新继续。(2)．活性组蛋白修饰可以以不同的组合模式标记与基因表达密切相关的远距离调控序列，在有丝分裂时也可以保留这些修饰的模式，从而保持调控元件的活性状态和所调控基因的转录状态。(3)．有丝分裂中活性组蛋白修饰介导的基因转录状态的表观记忆模式可能是保持染色质活性和转录记忆的普遍和有效机制。(4)．虽然有丝分裂中大部分与基因转录相关的特异转录因子都与染色体分离了，但仍有某些特异的蛋白因子如NF-E2p45可以保留在染色体上，充当一种分子记忆信号，有助于有丝分裂后所在区域染色质活性的迅速恢复。(5)．虽然H3.3在核小体中的置换和移出与转录过程密切相关，但它在染色体中的分布与基因活性和转录记忆的关系需要进一步的实验验证。更多还原

【Abstract】 Higher eukaryote contains several hundreds of cell types, each with a distinctive set of gene expression profile. These gene expression profiles are set up and propagated during cell differentiation and ontogeny. To maintain the products of cell differentiation and ontogeny, higher eukaryote must have evolved some elegant mechanisms to remember the cell phenotypes. Every cell maintain the same genetic material-genome DNA, which is replicated during S phase and transmitted to their daughter cells through mitosis. So the epigenetic information such as nucleosome structure, histone modification, non-histone proteins, interactions between DNA and protein, protein and protein etc., which is closely related to gene expression regulation, may play an important role in the maintenance of gene expression states during cell division. Exploring the roles of epigenetic information in the maintenance of gene expression states during cell division is an significant task to elucidate the mechanisms of differentiation and development of higher organisms.Employing the immunofluorescence assay, we analyzed the changes of four active histone modifications in mitotic MEL (murine erythroleukemia) cells by using the antibodies against H3 and H4 acetylation, H3-K4 dimethylation and H3-K79 dimethylation. In interphase cell nucleus, H3 and H4 acetylation, H3-K4 dimethylation concentrat on the euchromatin compartment; H3-K79 dimethylation is mainly localized in euchromatin but some are in heterochromatin compartment. Notablely, four active histone modifications remain on mitotic chromosomes even though chromtin is compacted into higher order chromosomes. Their localizations are restored coupling the appearance of nuclear envolope and the division of daughter cell nuclei at mitotic telophase. It has reported that most of transcriptions mediated by three RNA polymerase are ceased accompanying the chromatin condensation and the ceased transcriptions are resumed accompanying chromatin decondensation at mitotic exit. To further explore wether these active histone modifications mediate transcriptional memory during mitotic chromatin inactivation, we synchronized MEL cells into mitosis by treatment with nocodazole and then comparatively analyzed the changes of them in asynchronous and synchronized mitotic MEL cell populations. The more than 95% cells in synchronized MEL population were synchronized into G2/M phase after 16h treatment with nocodazole. There are about 50% G0/G1, 45% S and less than 5% G2/M cells in asynchronous MEL cell population. The total histone proteins were extracted from asynchronous and synchronous MEL cells and were blotted by antibodies against active histone modifications. The results indicated that the global amounts of H3 and H4 acetylation, H3-K4 dimethylation decreased 20-30% in mitotic cell population compared to asynchronous cell population but H3-K79 dimethylation is stable in two populations. Next we performed the comparative chromatin immunoprecipitation (CHIP) assay and analyzed the levels of four active histone modifications at the promoters of genes with different transcription states. The results indicated that these genes with different transcription states are marked by the different histone modification patterens and these modifications are reserved at gene promoters during mitosis. The reserved levels depend on the previous gene expression states even though some modifications are lowered in mitotic cells compared to those in asynchronous interphase cells. These results suggested that the preserved active histone modifications can function as epigenetic memory marks to maintain the previous gene expression states.The distal regulatory sequences, which is a unique and significant regulatory factor in higher eukaryotes, control the expression of many tissue- or development-specific genes through programming their chromatin accessibility and binding the specific activators or repressors. By taking the well-studied mouseα- andβ-globin gene clusters as a model, we compared active histone modifications at the distant hypersensitive sites (HSs) of globin gene clusters in asynchronous and mitotic cells. The results demonstrated that these distant regulatory elements are marked by the different histone modification combinations and also these modifications persist during mitosis in spite of some decreases. The preserved levels are also corresponding to their previous levels. The above results strongly suggested that during mitotic chromatin inactivation, active histone modifications can function as epigenetic memory marks to maintain the active chromatin state and gene expression states.Two erythroid-specific transcriptional activators GATA-1 and NF-E2p45, which play important roles in globin gene expressions, were taken as models to probe their roles in transcription memory. As is known, RNA polⅡis displaced from chromosomes during mitosis. Immunofluorescence analysis showed that RNA polⅡare not colocalized with chromosomes in mitotic cells. The signals of GATA-1 are disappeared in mitotic cells, indicating that GATA-1 may be abrogated during mitosis. However, some signals of NF-E2p45 remain at chromosomes compartments. The further ChIP analysis showed that NF-E2p45 specifically binds to the distant HS26 and HS2 of globin gene clusters and are still retained at its binding sites during mitosis, suggesting that certain specific protein factor can also serve as molecular memory mark in favor of the maintenance of transcription state and transcription reactivation.The deposition and removal of H3 variant H3.3 is closely related to transcription activation. To elucidate the relationship between the distribution of H3.3 at chromatin and transcription memory, we analyzed the changes of H3.3 protein in mitoic cells using the antibodies against the endogenous H3.3. The immunofluorescence analysis showed that during mitosis some H3.3 proteins are dispersed at periphery of cell membrane in conglobation and some remain at chromosomes compartments. The comparative ChIP analysis indicated that H3.3 variants are distributed at the promoters of the active and inactive genes and reserved during mitosis. However, it needs analyzing more genes and heterochromatin sites to disclose the correlation between its distribution and gene expression and its possible roles in transcription memory.Conclusions: (1) the preserved active histone modifications at mitotic chromosomes can imprint the previous gene expression states in favor of the transcriptional resumption of a large scale genes at mitoic exit. (2) The preserved active histone modification combinations at mitotic chromosomes can mark the active chromatin states of distant regulatory sequences and the transcriptional state of its controlled genes to maintain their previous chromatin activity. (3) The reserved histone modifications at mitotic chromosomes is a universal and efficient epigenetic memory mechanism to maintain active chromatin state and gene expression states. (4) Some specific transcription factors can function as molecular memory marks during mitosis to help the rapid restoration of active chromaton state at mitotic exit. (5) It needs further investigation to verify the relationship between the distribution of H3.3 variant and transcription memory.更多还原