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植物核小体组装蛋白1(NAP1)家族成员的功能鉴定
中文摘要

真核生物基因组以高度折叠的染色质(Chromatin)形式贮存于细胞核内。染色质的基本结构和功能单位是核小体(Nucleosome),其核心部分由大约146-147bp的DNA围绕着由各两个分子的H2A,H28,H3和H4构成的组蛋白八聚体组成。核小体的正确组装对于重建有功能的染色质结构十分重要,影响着包括DNA复制,修复,重组,转录,细胞增殖与分化以及个体发育等诸多生物过程。核小体组装需要组蛋白分子伴侣的参与,组蛋白分子伴侣不仅影响着染色质的结构形成及动态调节,而且参与到组蛋白的储存,核质运输以及组蛋白在染色质组装过程中的掺入和替换。 NAP1(Nucleosome Assembly Protein 1)是目前被确认的在各物种(酵母,动物和植物)中都保守存在的组蛋白H2A-H28分子伴侣。通过去除/替换染色质上的细蛋白H2A/H28,或者通过调节核小体的滑动,NAP1可以改变染色质的结;陶及调节染色质的代谢,进而影响细胞分化和个体发育等过程。果蝇和老鼠中的遗传学研究结果表明一个NAP1同源基因的缺失会导致胚胎致死,说明了NAP1蛋白在胚胎发育中的重要功能。NAP1蛋白在植物中也是高度保守的,而目前有关植物NAP1蛋白的结构和功能研究还处于起步阶段。我的博士论文主要利用分子生物学、生物化学、细胞生物学以及遗传学等研究手段分析了高等植物NAP1家族成员的功能及其对植物生长发育的影响。 我博士论文的第一部分工作主要对水稻和烟草NAP1家族成员进行了分子和生化分析。我们实验室利用筛选cDNA文库的方法,分离到3个水稻NAP1基因和4个烟草NAP1基因(Dong et al.,2003.Planta 216,561-570)。我们证明了烟草NAP1蛋白可以结合组蛋白H2A-H28,我们的结果支持了NAP1作为H2A-H2B分子伴侣的模式在植物中也是保守的。对NAP1的结合蛋白的分析表明烟草NAP1蛋白可以和微管蛋白及有丝分裂细胞周期蛋白Nicta;CYCB1;1结合,暗示其在微管动力学调控上的潜在功能。利用绿色荧光蛋白作为标记,我进一步研究了水稻和烟草NAP1家族成员的亚细胞定位。核定位输出抑制剂LMB处理以及核定位输出信号突变实验表明Orysa;NAP1;1和Nicta;NAP1;1可以在细胞质和细胞核中穿梭。比较有趣的是,其它三个烟草NAP1蛋白以及Orysa;NAP1;2只定位于细胞质中,而且并不具有核、质穿梭能力。此外, Orysa;NAP1;3缺少典型的核定位信号,但却在细胞核和细胞质中均有分布。我们还发现只有Orysa;NAP1;3可以在体外被酪蛋白激酶2(CK2,Casein kinase 2)磷酸化。然而,通过点突变分析,我们却发现Orysa;NAP1;3的磷酸化并不影响该蛋白的亚细胞定位。以上结果已发表于Plant Physiology杂志。 由于拟南芥作为模式植物在遗传分析上具有巨大的优势,我们转而利用拟南芥系统对NAP1家族蛋白进行功能研究。拟南芥编码4个NAP1基因,我们发现其中一个基因(AtNAP1;4)特异表达于雄蕊以及根延长区的部分,而其它三个NAP1基因则全局表达。更进一步,我们发现拟南芥NAP1蛋白可以形成同源和异源二聚体,同时他们也可以结合植物的组蛋白H2A和H28。在我们实验室的标准生长条件下,三个全局表达的NAP1基因的缺失的两个三突变(Atnap1;1-1Atnap1;2-1Atnap1;3-1和Atnap1;1-1Atnap1;2-1Atnap1;3-2)未表现出任何明显表型。而通过各种DNA损伤试剂的处理,我们发现这两个三突变表现出中等强度的紫外(UV-C)敏感以及体外修复DNA损伤的能力降低。两个三突变的转录谱和染色质免疫共沉淀分析表明拟南芥NAP1蛋白可以直接结合到一些核苷酸剪切修复(NER,Nucleotide excision repair)途径基因的启动子上,并调节这些基因的表达。这些结果提示拟南芥NAP1家族成员蛋白保守地行使组蛋白H2A/H28分子伴侣的功能,并且参与调节核苷酸剪切修复途径。 有趣的是,三突变的转录谱还表明,除了核苷酸剪切修复途径基因外,某些激素和压力应答相关基因的表达也发生了改变,提示我们去研究NAP1蛋白在植物对激素和压力应答方面的功能。我们发现三突变Atnap1;1-1Atnap1;2-1Atnap1;3-1表现出对脱落酸(ABA,Abscisic acid)的轻微超敏感。相反,Atnap1;1-1Atnap1;2-1Atnap1;3-2则在苗的生长和气孔活动方面比较强烈地表现出对脱落酸的不敏感,并导致该三突变植株盐耐受性的降低。此外,所有含有Atnap1;3-2等位的突变体以及Atnap1;3-2杂合子都表现对脱落酸的脱敏性。更细致的研究表明在所有含有Atnap1;3-2等位的突变体以及Atnap1;3-2杂合子中均存在一个AtNAP1;3的截短蛋白(碳末端34个氨基酸缺失,命名为AtNAP1;3T)。过量表达这个截短蛋白导致植物对脱落酸的脱敏,并导致一些ABA信号转导途径基因的表达降低,证实该AtNAP1;3T可能通过调节这些基因的表达显性影响拟南芥对脱落酸的反应。 综上所述,我的博士论文工作比较深入地阐述了植物NAP1家族成员的分子、细胞和生物学功能,表明植物NAP1蛋白参与基因表达调控以及对非生物逆境的反应。 关键词:拟南芥,组蛋白分子伴侣,染色质,表观遗传学,DNA修复,非生物逆境 中图分类号:Q7

英文摘要

Eukaryotic genomes are packaged into chromatin, a regularly repeated structure whose fundamental building block is the nucleosome. Each nucleosome core particle is composed of a histone octamer consisting of two molecules each of the core histones H2A, H2B, H3, and H4, around which approximately 146-147 bp of DNA is wrapped. The assembly of nucleosome is the first step of chromatin assembly which is important for the chromatin structure affecting a broad ranges of biological events including DNA replication, repair, recombination, transcription, cell differentiation, proliferation, and organism development, and is fulfilled with the help of histone chaperones, which are important for the organization and dynamics of chromatin templates, and are involved in the storage, translocation to the nucleus and exchange ofhistor.es and their deposition onto the DNA for replication-dependent chromatin assembly. Nucleosome Assembly Protein 1 (NAP1) represents the primary chaperone of H2A and H2B and is highly conserved from yeast to human. Through H2A-H2B dimer removal or exchange, or causing nucleosome sliding, NAP1 can change chromatin structure, and thus affects chromatin metabolism, which provides a mechanism for controlling cell differentiation and development properly. Consistent with this view, genetic studies reveal important functions of NAP1 in Drosophila and mouse, in which deletion of a NAP1 gene leads to embryonic lethality. NAP1 is conserved in plants. However, few studies have been done on its property and biological function. My thesis work aimed to characterize NAP1 to reveal its molecular and cellular activities and its biological functions in higher plants. The first part of my thesis focused on the functional analyses of NAP1 family proteins in rice (Oryza sativa) and tobacco (Nicotiana tabacum). The previous work in our labs described the isolation of three cDNAs encoding NAP1 family proteins from the monocotyledon rice (Oryza sativa) and four from the dicotyledon tobacco (Nicotiana tabacum) (Dong et al., 2003. Planta 216, 561 - 570). Intracellular localization analyses showed that some NAP1 family proteins localize in the nucleus whereas others are cytoplasm-localized. I further investigated the details about intracellular localization of rice and tobacco NAP1 family proteins firstly by identification of interacting partners and by examination of the localization of green fluorescent protein-tagged proteins. Through treatment of tobacco cells with leptomycin B and mutagenesis of nuclear export signal, we demonstrated that Orysa;NAP1;1and Nicta;NAP1;1 shuttle between the cytoplasm and the nucleus. Together with the demonstration that tobacco NAP1 proteins bind histone H2A and H2B, our results support the current model and provide additional evidence that the function of NAP1 as histone chaperones appears to be conserved in plants. In addition, we showed that tobacco NAP1 proteins interact with tubulin and the mitotic cyclin Nicta;CYCBl;l, suggesting a role for NAP1 in microtubule dynamics. Interestingly, in spite of their high homology with the above NAP1 proteins, the other three tobacco proteins and Orysa;NAP1;2 did not show nucleocytoplasmic shuttling and were localized only in the cytoplasm. Moreover, Orysa;NAP1;3 that lacks a typical nuclear localization signal sequence is localized in both the cytoplasm and the nucleus. Finally, we showed that only Orysa;NAP1 ;3 could be phosphorylated by casein kinase 2α in vitro. However, this phosphorylation was not responsible for nuclear import of Orysa;NAP1;3 as being demonstrated through mutagenesis studies. Together, our results provide an important step toward elucidating the molecular mechanism of function of the NAP1 family proteins in plants. These results described above were published in the journal "Plant Journal". Because of the difficulties of functional analyses of NAP1 proteins in rice and tobacco, and great advantages of the model plant Arabidopsis in genetic analysis, we switched to Arabidopsis for further studies. The second part of my thesis focused on the functional analyses of NAP1 family proteins in Arabidopsis. The Arabidopsis genome encodes 4 NAP1 genes. We found that one of them, AtNAP1;4, is specifically expressed in stamen and a small part of the elongation zone of the root, while the other three NAP1-subgroup genes are universally expressed and their encoded proteins are mostly localized in the cytoplasm. Further, we showed that Arabidopsis NAP1 proteins form homo- and heterodimeric complexes, and bind plant histone H2A and H2B. Although triple mutants of the three universally expressed NAP1 genes, Atnap1;1-1Atnap1;2-1Atnap1;3-1 and Atnap1; 1-1 Alnap1;2-1Atnap1;3-2, show a normal growth phenotype under our laboratory standard growth conditions, both triple mutants exhibit a moderate UV-C sensitivity as well as reduced efficiency of in vitro repair of UV-damaged DNA. Transcription profilings and ChIP analyses of the mutants showed that AtNAP1 proteins directly targeted to the promoter regions of some nucleotide excision repair (NER) pathway genes and activated expression of these genes. These results suggest that NAP1 family proteins in Arabidopsis act as H2A/H2B chaperones and are involved in modulating NER pathway. Interestingly, transcription profilings of triple mutants also showed that, in addition to some NER pathway genes, several phytohormone and stress responsive genes were misregulated, stimulating us to study the function of NAP1 proteins in the plant responses to diverse phytohormones and stresses. We reported that loss-of-function mutations in AtNAP1;1, 2, and 3 resulted in a slight abscisic acid (ABA)-sensitive phenotype. By the constrast, the triple mutant Atnap1;1-1 Atnap1;2-1Atnap1;3-2 exhibited pleiotropic and strong ABA-insensitive phenotypes in seedling growth and stomatal movement and led to decreased tolerance of the mutant seedlings to salt stress. Furthermore, the ABA-insensitive phenotypes of Atnap 1; 1 -1 Atnap1 ;2-1 Atnap1; 3-2 were observed in all Atnap1 ;3-2 mutant allele-containing mutants and Atnap1;3-2 heterozygote plants. Detailed study revealed that a stable truncated AtNAP1 ;3 protein (named as AtNAP1 ;3T) with C-terminal 34 aa of the full-length protein lost was present in all Atnap1; 3-2 mutant allele-containing mutants and Atnap1;3-2 heterozygote, and overexpressing this AtNAP1;3T protein in wild-type plants conferred the plant with ABA hyposensitivity, and caused the downregulation of some genes which are involved in ABA signaling. Taken together, our results demonstrated the dominant role of this truncated AtNAP1 ;3 protein on Arabidopsis responses to ABA and revealed the significant role of the C-terminal acidic region in NAPPs structure and function, providing informations about AtNAP1 ;3T protein which might be useful for future biotechonological applications. In conclusion, my thesis work has gained no-previously obtained knowledge about the molecular, cellular and functional properties of NAP1 family proteins in higher plants. My results indicate that AtNAP1 proteins can function as modulators between environmental stress and chromatin structure and function. Key words:Arabidopsis, Histone chaperone, Chromatin, Epigenetics, DNA repair, Abiotic stress Classfiction code: Q7

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