本文選題：核小體定位 + 三核苷酸重復(fù)序列　； 參考：《內(nèi)蒙古大學(xué)》2016年博士論文

【摘要】：真核生物基因組以高度壓縮的染色體形式存在,核小體是構(gòu)成染色質(zhì)的基本結(jié)構(gòu)單元,基因組上核小體的相對(duì)位置稱為核小體定位。核小體定位對(duì)包括DNA復(fù)制、修復(fù)、重組及可變剪接等在內(nèi)的眾多生物學(xué)過程起著調(diào)控作用。比如,核小體的形成影響反式作用因子與核小體DNA的結(jié)合,為調(diào)控蛋白的結(jié)合提供了可選擇性；構(gòu)成核小體的組蛋白尾部發(fā)生化學(xué)修飾可以改變?nèi)旧|(zhì)結(jié)構(gòu),從而影響其與調(diào)控因子的結(jié)合。所以,核小體作為一種真核生物染色體基本結(jié)構(gòu)單元,履行著結(jié)構(gòu)與功能的雙重作用。核小體定位受到DNA序列內(nèi)在特性及諸如組蛋白變體、組蛋白修飾、轉(zhuǎn)錄因子及RNA聚合酶競爭等外在因素的影響,其中DNA序列特性是影響核小體定位最為重要的因素之一�；蚪M中特定位點(diǎn)三核苷酸重復(fù)序列(如GAA、CAG等)與40余種人類神經(jīng)系統(tǒng)的遺傳疾病相關(guān),這類疾病的致病機(jī)理可能與重復(fù)序列局部染色質(zhì)結(jié)構(gòu)有關(guān)。研究發(fā)現(xiàn)Friedreich共濟(jì)失調(diào)(FRDA)患者體內(nèi)FXN基因第一內(nèi)含子中GAA重復(fù)序列周圍表觀遺傳修飾異常,而體外實(shí)驗(yàn)證實(shí)GAA22重復(fù)序列形成核小體能力較弱。在正常人體內(nèi),GAA重復(fù)序列的核小體定位特征及其對(duì)染色質(zhì)局部結(jié)構(gòu)的影響仍然是一個(gè)懸而未決的問題,該問題對(duì)于在染色質(zhì)結(jié)構(gòu)水平上理解FRDA疾病的致病機(jī)理具有重要的理論意義。近30余年,人們利用各種方法尋找可能隱含在核小體DNA中的序列模體。以色列科學(xué)家Trofonov教授通過分析線蟲、果蠅、人和酵母等模式生物的核小體定位數(shù)據(jù),分別建立了N-gram延伸模型、彎曲矩陣模型和堆積能模型。三個(gè)模型從不同的角度對(duì)核小體占據(jù)的數(shù)據(jù)進(jìn)行分析后提出了核小體定位的RRRRRYYYYY(R5Y5)序列模體,其中R是嘌呤,Y是嘧啶。R5Y5序列模體提出后,在國際上引起很大爭議,到目前為止,依然缺乏有效的實(shí)驗(yàn)證明R5Y5序列模體對(duì)核小體定位的影響。論文結(jié)合理論統(tǒng)計(jì)分析和體外實(shí)驗(yàn)方法,研究了含GAA重復(fù)序列和R5Y5模體序列核小體定位的規(guī)律,期望理解DNA序列對(duì)核小體定位影響的機(jī)制。主要研究內(nèi)容如下：(1)首先,統(tǒng)計(jì)了人基因組中18種三核苷酸重復(fù)序列核小體定位特征,結(jié)果發(fā)現(xiàn)富含AA或TT的三核苷酸重復(fù)序列形成核小體的能力非常弱。其次,重點(diǎn)關(guān)注與疾病相關(guān)的GAA重復(fù)序列的核小體定位特征,發(fā)現(xiàn)基因組中緊鄰GAA重復(fù)序列上游分布著大量的A-tracts,這些A-tracts的存在會(huì)影響局部染色質(zhì)結(jié)構(gòu),GAA重復(fù)片段本身可以導(dǎo)致局部的核小體缺乏,而上游A-tracts會(huì)進(jìn)一步加劇這種缺乏程度。體內(nèi)含有GAA重復(fù)序列特定位點(diǎn)的核小體定位圖譜同樣證實(shí)GAA重復(fù)序列是一個(gè)弱的核小體定位元件。(2)從大腸桿菌中表達(dá)純化了6種重組組蛋白(H2A、H2B、H3、H4、 H2A.Z和H3.3),裝配了常規(guī)組蛋白八聚體、含變體H2A.Z及H3.3的組蛋白八聚體,這三種組蛋白八聚體可用于體外組裝核小體及染色質(zhì)實(shí)驗(yàn)。(3)通過體外重組染色質(zhì)實(shí)驗(yàn),發(fā)現(xiàn)了GAA重復(fù)序列可導(dǎo)致局部核小體占據(jù)率下降。構(gòu)建了含有GAA7、GAA27、GAA42片段的3種重組質(zhì)粒及含有CAG7、CAG27、CAG44和601序列共4種實(shí)驗(yàn)對(duì)照質(zhì)粒。在質(zhì)粒上組裝了染色質(zhì)結(jié)構(gòu),通過微球菌核酸酶酶切實(shí)驗(yàn)、電鏡和蔗糖梯度密度離心等方法發(fā)現(xiàn)：相比于601序列,GAA42重復(fù)序列插入到質(zhì)粒中不利于質(zhì)粒形成染色質(zhì)結(jié)構(gòu)。進(jìn)一步通過分析超速離心定量檢測發(fā)現(xiàn),含有GAA重復(fù)序列的染色質(zhì)結(jié)構(gòu)比含有CAG重復(fù)序列或601序列的染色質(zhì)結(jié)構(gòu)松弛、核小體占據(jù)水平偏低,說明GAA重復(fù)序列插入到質(zhì)粒中可能降低了重組質(zhì)粒體外形成染色質(zhì)結(jié)構(gòu)的能力。(4)通過體外重組核小體的實(shí)驗(yàn),發(fā)現(xiàn)R5Y5是一種有利于常規(guī)核小體形成的序列模體�；赗5Y5模體及TA 10.5-bp周期性規(guī)律,設(shè)計(jì)了CS1-CS6共6條序列,利用體外組裝核小體的實(shí)驗(yàn)技術(shù),研究了這些序列形成核小體的效率。結(jié)果發(fā)現(xiàn)：在常規(guī)核小體形成過程中,DNA序列中R5Y5和TA 10.5-bp周期特性都極其顯著地促進(jìn)核小體的形成,相比較而言,TA 10.5 bp周期性的影響更為顯著。在含有H2A.Z變體的核小體體外組裝過程中,TA 10.5-bp周期性顯著地影響核小體的形成,但是不能判斷DNA序列中R5Y5模體對(duì)組裝含有H2A.Z變體的核小體的影響程度。在6條DNA序列上進(jìn)行體外組裝含有H3.3變體的核小體,TA 10.5-bp周期性對(duì)核小體的形成具有顯著的影響,實(shí)驗(yàn)結(jié)果不能判斷R5Y5模體對(duì)核小體形成的影響程度,而R5Y5模體和TA 10.5-bp周期規(guī)律之間對(duì)含有H3.3核小體組裝的影響沒有顯著性差異。
[Abstract]:The genome of eukaryotes exists in the form of highly compressed chromosomes. Nucleosomes are the basic structural units that constitute chromatin. The relative position of the nucleosomes in the genome is called the nucleosome location. Nucleosome positioning plays a regulatory role in many biological processes, including DNA replication, repair, recombination and variable splicing. For example, nucleosomes Its formation affects the binding of the trans acting factor and the nucleosome DNA, which provides selectivity for the binding of the regulatory proteins; the chemical modification of the histone tail of the nucleosome can change the chromatin structure and affect its combination with the regulator. Therefore, the nucleosome acts as a basic structural unit of the eukaryotic chromosomes, which is performed as a basic structural unit of the eukaryotic chromosomes. The localization of nucleosome is influenced by the intrinsic characteristics of the DNA sequence and the factors such as histone variants, histone modification, transcription factors and RNA polymerase competition. The DNA sequence characteristics are one of the most important factors affecting the localization of nucleosomes. GAA, CAG, etc. are associated with more than 40 genetic diseases of the human nervous system. The pathogenesis of these diseases may be related to the repeated sequence of local chromatin structure. The study found that the epigenetic modification of the GAA repeat in the first intron of the Friedreich ataxia (FRDA) patients was abnormal, and in vitro experiments confirmed that the GAA22 repetition was repeated. The nucleosome ability of sequence formation is weak. In normal human body, the nucleosome localization characteristics of GAA repeat sequence and its effect on the local structure of chromatin are still an unresolved problem. This problem has important theoretical significance for understanding the pathogenesis mechanism of FRDA disease at the level of chromatin structure. In the last 30 years, people have used various kinds of methods. Methods to find sequence models that may be hidden in the nucleosome DNA. Professor Trofonov, an Israeli scientist, established the N-gram extension model, the bending matrix model and the accumulative energy model by analyzing the nucleosome location data of the nematode, Drosophila, human and yeast model organisms. Three models were used for the data of the nucleosome from different angles. After the analysis, the RRRRRYYYYY (R5Y5) sequence of nucleosome positioning is proposed, in which R is a purine and the Y is a pyrimidine.R5Y5 sequence model, which has caused great controversy in the world. So far, there is still a lack of effective experiments to prove the effect of R5Y5 sequence model body on the localization of nucleosome. The rules of nucleosome localization containing GAA repeat sequences and R5Y5 modules are studied in order to understand the mechanism of the effect of DNA sequences on nucleosome localization. The main contents are as follows: (1) first, the nucleosome localization characteristics of 18 trinucleotide repeat sequences in the human genome are counted, and the results of the nucleosome repeat sequences rich in AA or TT are found to form the nucleus. The ability of the body is very weak. Secondly, focus on the nucleosome localization characteristics of the GAA repeat sequence related to the disease. It is found that a large number of A-tracts in the upstream of the GAA repeat sequence in the genome will affect the local chromatin structure, and the GAA repeat fragment can lead to the local nucleosome deficiency and the upstream A-tr. Acts will further exacerbate this lack. The nucleosome mapping of the GAA repeat loci also confirms that the GAA repeat sequence is a weak nucleosome positioning element. (2) 6 recombinant histones (H2A, H2B, H3, H4, H2A.Z and H3.3) were expressed and purified from Escherichia coli, and the conventional histone eight polymer, containing variant H2A, was assembled. The histone eight polymer of.Z and H3.3, these three histone eight polymers can be used to assemble nucleosome and chromatin experiments in vitro. (3) through in vitro recombinant chromatin test, it was found that the GAA repeat sequence could lead to the decrease of local nucleosome occupancy. 3 recombinant plasmids containing GAA7, GAA27, GAA42 segments and CAG7, CAG27, CAG44 and 601 sequence were constructed. A total of 4 kinds of experimental control plasmids were included. The chromatin structure was assembled on the plasmid. It was found by Micrococcus nuclease enzyme digestion, electron microscopy and sucrose gradient density centrifugation. Compared with the 601 sequence, the GAA42 repeat sequence was inserted into the plasmid and was not beneficial to the plasmids to form chromatin structure. The chromatin structure containing the GAA repeat sequence is more relaxed than the chromatin structure containing CAG repeats or 601 sequences, and the nucleosome occupies a low level. It indicates that the insertion of GAA repeats into plasmids may reduce the ability of the recombinant plasmid to form chromatin structure in vitro. (4) through the experiment of recombinant nucleosome in vitro, it is found that R5Y5 is a beneficial one. A sequence model body formed by conventional nucleosomes. Based on the periodic laws of R5Y5 modules and TA 10.5-bp, a total of 6 CS1-CS6 sequences are designed. The efficiency of the nucleosome formation is studied by the experimental technique of assembling nucleosomes in vitro. The results show that the periodic characteristics of R5Y5 and TA 10.5-bp in the DNA sequence are all polar during the formation of the conventional nucleosome. It significantly promotes the formation of nucleosomes. In comparison, the periodic effect of TA 10.5 BP is more significant. In the process of in vitro assembly of nucleosomes containing H2A.Z variants, TA 10.5-bp periodically affects the formation of nucleosomes, but it is not possible to judge the extent to which the R5Y5 modules in the DNA sequence have a degree of influence on the nucleosomes containing H2A.Z variants in the DNA sequence. The nucleosomes containing H3.3 variants were assembled on the DNA sequence in vitro, and the periodicity of TA 10.5-bp had a significant influence on the formation of nucleosomes. The experimental results could not determine the effect of R5Y5 modules on the formation of nucleosomes, but there was no significant difference in the effect of the R5Y5 module and the TA 10.5-bp periodic law on the assembly of H3.3 nucleosomes.

【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q343.23

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 柴榮;趙宏宇;蔡祿;;與人遺傳病相關(guān)的DNA重復(fù)序列的體外核小體定位特性[J];基礎(chǔ)醫(yī)學(xué)與臨床;2013年03期

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本文編號(hào)：1832047