重复序列GAA及R5Y5的核小体定位理论与实验研究

发布时间：2018-05-02 02:44

本文选题：核小体定位 + 三核苷酸重复序列　；参考：《内蒙古大学》2016年博士论文

【摘要】：真核生物基因组以高度压缩的染色体形式存在,核小体是构成染色质的基本结构单元,基因组上核小体的相对位置称为核小体定位。核小体定位对包括DNA复制、修复、重组及可变剪接等在内的众多生物学过程起着调控作用。比如,核小体的形成影响反式作用因子与核小体DNA的结合,为调控蛋白的结合提供了可选择性；构成核小体的组蛋白尾部发生化学修饰可以改变染色质结构,从而影响其与调控因子的结合。所以,核小体作为一种真核生物染色体基本结构单元,履行着结构与功能的双重作用。核小体定位受到DNA序列内在特性及诸如组蛋白变体、组蛋白修饰、转录因子及RNA聚合酶竞争等外在因素的影响,其中DNA序列特性是影响核小体定位最为重要的因素之一。基因组中特定位点三核苷酸重复序列(如GAA、CAG等)与40余种人类神经系统的遗传疾病相关,这类疾病的致病机理可能与重复序列局部染色质结构有关。研究发现Friedreich共济失调(FRDA)患者体内FXN基因第一内含子中GAA重复序列周围表观遗传修饰异常,而体外实验证实GAA22重复序列形成核小体能力较弱。在正常人体内,GAA重复序列的核小体定位特征及其对染色质局部结构的影响仍然是一个悬而未决的问题,该问题对于在染色质结构水平上理解FRDA疾病的致病机理具有重要的理论意义。近30余年,人们利用各种方法寻找可能隐含在核小体DNA中的序列模体。以色列科学家Trofonov教授通过分析线虫、果蝇、人和酵母等模式生物的核小体定位数据,分别建立了N-gram延伸模型、弯曲矩阵模型和堆积能模型。三个模型从不同的角度对核小体占据的数据进行分析后提出了核小体定位的RRRRRYYYYY(R5Y5)序列模体,其中R是嘌呤,Y是嘧啶。R5Y5序列模体提出后,在国际上引起很大争议,到目前为止,依然缺乏有效的实验证明R5Y5序列模体对核小体定位的影响。论文结合理论统计分析和体外实验方法,研究了含GAA重复序列和R5Y5模体序列核小体定位的规律,期望理解DNA序列对核小体定位影响的机制。主要研究内容如下：(1)首先,统计了人基因组中18种三核苷酸重复序列核小体定位特征,结果发现富含AA或TT的三核苷酸重复序列形成核小体的能力非常弱。其次,重点关注与疾病相关的GAA重复序列的核小体定位特征,发现基因组中紧邻GAA重复序列上游分布着大量的A-tracts,这些A-tracts的存在会影响局部染色质结构,GAA重复片段本身可以导致局部的核小体缺乏,而上游A-tracts会进一步加剧这种缺乏程度。体内含有GAA重复序列特定位点的核小体定位图谱同样证实GAA重复序列是一个弱的核小体定位元件。(2)从大肠杆菌中表达纯化了6种重组组蛋白(H2A、H2B、H3、H4、 H2A.Z和H3.3),装配了常规组蛋白八聚体、含变体H2A.Z及H3.3的组蛋白八聚体,这三种组蛋白八聚体可用于体外组装核小体及染色质实验。(3)通过体外重组染色质实验,发现了GAA重复序列可导致局部核小体占据率下降。构建了含有GAA7、GAA27、GAA42片段的3种重组质粒及含有CAG7、CAG27、CAG44和601序列共4种实验对照质粒。在质粒上组装了染色质结构,通过微球菌核酸酶酶切实验、电镜和蔗糖梯度密度离心等方法发现：相比于601序列,GAA42重复序列插入到质粒中不利于质粒形成染色质结构。进一步通过分析超速离心定量检测发现,含有GAA重复序列的染色质结构比含有CAG重复序列或601序列的染色质结构松弛、核小体占据水平偏低,说明GAA重复序列插入到质粒中可能降低了重组质粒体外形成染色质结构的能力。(4)通过体外重组核小体的实验,发现R5Y5是一种有利于常规核小体形成的序列模体。基于R5Y5模体及TA 10.5-bp周期性规律,设计了CS1-CS6共6条序列,利用体外组装核小体的实验技术,研究了这些序列形成核小体的效率。结果发现：在常规核小体形成过程中,DNA序列中R5Y5和TA 10.5-bp周期特性都极其显著地促进核小体的形成,相比较而言,TA 10.5 bp周期性的影响更为显著。在含有H2A.Z变体的核小体体外组装过程中,TA 10.5-bp周期性显著地影响核小体的形成,但是不能判断DNA序列中R5Y5模体对组装含有H2A.Z变体的核小体的影响程度。在6条DNA序列上进行体外组装含有H3.3变体的核小体,TA 10.5-bp周期性对核小体的形成具有显著的影响,实验结果不能判断R5Y5模体对核小体形成的影响程度,而R5Y5模体和TA 10.5-bp周期规律之间对含有H3.3核小体组装的影响没有显著性差异。
[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.

【学位授予单位】：内蒙古大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q343.23

【参考文献】