【作者】 杨飞；

【导师】 李立家；

【作者基本信息】 武汉大学 ， 遗传学， 2010， 博士

【摘要】 染色质表观遗传修饰包括组蛋白翻译后修饰和DNA甲基化修饰。核小体核心组蛋白H2A、H2B、H3、H4以及核小体连接组蛋白H1均能发生各种翻译后修饰,如甲基化、乙酰基化、磷酸化、泛素化等。DNA序列本身能在胞嘧啶位点发生甲基化修饰。近些年来,表观遗传修饰的生物学功能受到广泛关注。研究表明,组蛋白翻译后修饰和DNA甲基化通过直接影响或者募集其他蛋白影响核小体稳定性,改变染色质状态,从而在基因转录、DNA复制修复和细胞周期进程中发挥重要的作用。目前,关于玉米有丝分裂中染色质表观遗传修饰间相互作用未见报道。本论文研究了玉米有丝分裂过程中表观遗传修饰的动态变化,通过药物处理,研究表观遗传修饰之间的相互作用,并分析表观遗传修饰变化对玉米有丝分裂的影响。本研究有助于深入理解遗传修饰在细胞分裂和细胞周期中的作用,为进一步阐明高等植物的细胞分裂分化和生长发育奠定基础。具体结果如下：曲古霉素A(trichostatin A, TSA)是组蛋白脱乙酰基化酶抑制剂,能提高组蛋白乙酰基化水平；5-氮杂胞苷(5-azacytidine,5-AC)是胞嘧啶(cytosine)类似物,但不能被甲基化,能阻止DNA甲基化酶活性,减少基因组甲基化水平。流式细胞术显示,TSA处理后玉米根尖细胞核的G0/G1期和G2/M期的荧光强度值均变大,峰向右移,统计发现,TSA处理引起细胞阻滞在G2期；5-AC处理后G0/G1期和G2/M期的荧光强度值均变小,峰向左移,统计发现,5-AC处理引起细胞阻滞在G1期。微球菌核酸酶敏感性实验显示与对照组相比,TSA和5-AC处理后玉米根尖细胞染色质对微球菌核酸酶敏感性增强,酶切后,更早出现DNA ladder,这说明TSA和5-AC处理引起染色质脱浓缩。在正常玉米根尖细胞中,免疫荧光染色表明,从有丝分裂前期到中期,随着染色质的凝缩发生了组蛋白H4脱乙酰基化,H3K9二甲基化水平降低和DNA甲基化水平升高,从中期到末期,随着染色质的脱浓缩,组蛋白H4乙酰基化水平重建,H3K9二甲基化水平增高和DNA甲基化水平降低。与正常的有丝分裂过程相比,TSA处理引起玉米根尖细胞有丝分裂过程中各个时期的组蛋白H4高乙酰基化,同时也检测到了组蛋白H3K9二甲基化和DNA甲基化水平的降低,这说明组蛋白H4高乙酰基化,直接或间接导致了H3K9me2的低甲基化和DNA低甲基化。与正常的玉米根尖细胞相比,免疫荧光分析表明5-AC处理引起玉米根尖细胞有丝分裂各个时期的DNA甲基化水平的降低,同时也检测到了组蛋白H3K9二甲基化水平的降低和组蛋白H4高乙酰基化,这说明,DNA低甲基化,导致了组蛋白H4高乙酰基化和H3K9me2的低甲基化。Western-blot和DNA点杂交表明TSA和5-AC处理都引起组蛋白H4总的乙酰基化水平升高,H3K9总的二甲基化和DNA总的甲基化水平降低。统计分析发现,TSA和5-AC处理后,玉米根尖细胞有丝分裂指数降低,进入有丝分裂后期和末期的细胞数量明显减少,TSA和5-AC处理引起细胞阻滞在有丝分裂中期,.这说明组蛋白H4四乙酰基化,组蛋白H3K9二甲基化和DNA甲基化的正常水平含有维持细胞有丝分裂所必须的表观遗传信息。基于以上结果即TSA和5-AC处理均引起组蛋白H4四乙酰基化水平的升高,组蛋白H3K9二甲基化水平的降低和DNA甲基化水平的降低,全基因组范围内组蛋白H4乙酰基化,组蛋白H3K9二甲基化和DNA甲基化之间相互作用的模型被提出。在这个模型中,H4高乙酰基化,可以调控H3K9me2的低甲基化和DNA低甲基化；而DNA低甲基化,可以调控组蛋白H4高乙酰基化和H3K9me2的低甲基化。更多还原

【Abstract】 Chromatin epigenetic modificaiongs includes histone post-translational modifications (PTMs) and DNA methylation. The core histones, H2A, H2B, H3, and H4, and the H1 family of linker histones are subjected to various PTMs such as methylation, acetylation, phosphorylation, and ubiquitination. DNA itself may be modified by methylation of cytidine. Genetic and immunochemical analyses focusing on the function of individual histone PTMs and DNA methylation have revealed that they play key roles in controlling the accessibility of DNA to regulatory factors and complexes responsible for transcription, replication, repair and cell cycle process through direct effects on nucleosome stability and chromatin compaction and by recruiting other proteins which affect these properties. Currently, the cross-talk among epigenetic modifications during mitosis in maize is poorly understood. Exploring the roles of epigenetic information during mitosis in maize will lay the foundation for elucidating the mechanisms of cell differentiation and development of higher plants.In the present study, trichostatin A (TSA), an inhibitor of HDACs, which can induce increase histone acetylation of chromatin and 5-azacytidine (5-AC), an analog of 5-cytosine, which can not be methylated and inhibit DNA (5-cytosine) methylases to reduce the level of overall DNA methylation of chromatin were used to treat maize root tip cells. Flow cytometric analysis showed that TSA treatment arrested cells at G2 phase, while 5-AC treatment arrested cells at G1 phase. MNase assay showed that treatment with 5-AC or TSA caused the chromatin decondensation in maize root tip cells. The immunostaing assay showed that in control maize root-tip cells, histone H4 was deacetylated, H3K9me2 was demethylated and DNA was hypermethylated from prophase to metaphase. And from metaphase to telophase, the inverse progression of epigenetic modifications changes was associated with chromatin condensation. In the TSA treated cells, immunostaing assay showed that an increase of acetylated histone H4 accompanied with a decrease in global H3K9 dimethylation and DNA methylation during mitosis compared to control maize root tip cells. In the 5-AC treated maize root tip cells, immunostaing assay showed that 5-AC mediated an increase of H4 actylation and a decrease of H3K9me2 and DNA methylation compared to the control maize root tip cells. Western-blotting and DNA methylation dot blotting confirmed these cytological observations. Moreover, the mitotic index was significantly decreased and cells were arrested at metaphase after TSA and 5-AC treated.The above results indicated that histone acetylation might directly or indirectly affect DNA and histone methylation and a decrease in DNA methylation could causes a reduction in H3K9 dimethylation and an increase in H4 acetylation. And TSA and 5-AC treatment that caused the epigenetic modification changes resulted in the cells atrrested at metaphase during mitosis, suggesting that H4 deacetylation and DNA and H3K9 methylation might contain necessary information for mitosis in maize root tips.According to the effect of 5-AC and TSA on H4 acetylation, DNA methylation and H3K9 dimethylation, a mutual re-enhancing cross-talk relationship between histone acetylation, DNA methylation and H3K9me2 is proposed. Histone H4 hyperacetylation may cause a global loss of DNA methylation, and DNA hypomethylation may mediate histone H4 acetylation. Not only may histone H4 hyperacetylation and DNA hypomethylation mediate decreased H3K9me2, but also it is possible that a reduction of H3K9me2 may feed back on the extent of H4ac and DNA methylation.更多还原