【作者】 别丽华；

【导师】 袁巧霞；

【作者基本信息】 华中农业大学 ， 生物信息技术与工程， 2018， 博士

【摘要】 转录是基因表达的第一步,由于蛋白-蛋白、蛋白-DNA之间的相互作用,以及一些复杂大分子复合物的形成,导致真核生物转录水平的调控是一个多级的复杂过程。转录因子TF(Transcription factor)作为一种序列特异性的DNA结合蛋白,能通过结合到顺式元件中的启动子和增强子中,从而控制遗传信息从DNA到mRNA的转录速率。众多实验研究表明,DNA甲基化(DNA methylation)能动态调控这一结合过程,进而调控基因的打开或关闭,保证它们的正确表达。同时,DNA甲基化水平也与人类发育和肿瘤疾病密切相关。因此,研究DNA甲基化在TF-DNA相互作用过程中的识别和调控机理具有重要意义,也使这一领域成为表观遗传学和表观基因组学的研究热点。随着深度测序等技术的快速发展,当前在不同生物过程和种族中已经累积了大量的DNA甲基化数据,但这些数据仍不能解释DNA甲基化水平动态变化的机制,表型领域中DNA甲基化和生理结果之间的因果关系仍然是个挑战性问题。要理解其中的分子机制,需要采用分子模拟的方法。当前的计算机技术和力场精度使得分子动力学已经可以实现对生物分子大体系进行长时间的模拟采样,利用分子模拟技术来探讨甲基化对蛋白-核酸相互作用的动态调控机制也成为了一种可行的研究方案。因此,本课题选择了分子动力学的模拟方法来研究甲基化对蛋白-核酸相互作用的调控机制。经过文献调研和晶体结构数据搜索,论文最终选取了bZIP家族转录因子作为研究体系本论文的研究思路为,通过比较不同位置单甲基化位点和对称双甲基化位点对转录因子与核酸相互作用的影响,研究5mC甲基化的调控机制。为此,首先对所选体系进行了长时间、多轨迹的动力学模拟(总模拟时间达到微秒量级),然后分别从结合自由能、熵焓互补、氢键、RMSF、DNA结构参数、蛋白质相对构象和空间位阻效应等各个角度进行了分析和系统比较,主要进行了以下几方面的研究工作:1、以C/EBP?转录因子结合DNA为例,研究了单位点甲基化(5mCpG)对转录因子与DNA相互作用的影响机制。2、以Jun/Jun同源二聚体与DNA结合物为例,研究了不同位置单位点、双位点甲基化对转录因子与DNA相互作用的影响机制。3、在上述两方面研究中,由于发现分子动力学方法对自由能的评估精度太低,需要采用量子力学方法开展相关研究。因此,采用从头算动力学方法以氧气激活过程为例开展了探索性的方法测试。更多还原

【Abstract】 Transcription is the first step in gene expression.The regulation of eukaryotic transcription levels is a multi-stage and complicated process,due to the interactions of protein-protein and protein-DNA,and the formation of complex macromolecular complexes.Transcription factors,as a sequence-specific DNA-binding protein,can control the transcription rate of genetic information from DNA to mRNA by binding to promoters and enhancers in cis-elements.Experimental studies have shown that DNA methylation can dynamically regulate this binding process,thereby regulating gene active or silencing and ensuring their correct expression.Recent studies have also found that DNA methylation levels are closely related to human development and tumor diseases.Therefore,it is of great significance to study the mechanism of DNA methylation recognition and regulation in the process of TF-DNA interaction,and it has become a research hotspot in epigenetics and epigenomics.With the rapid development of technologies especially deep sequencing,there has accumulated a large amount of DNA methylation data in different biological processes and races,but these data still cannot explain the dynamic mechanism of DNA methylation.The relationship between DNA methylation and physiological outcomes remains a challenging issue.the molecular simulation method is needed to solve this problem.Using molecular simulation techniques to explore the mechanism of dynamic regulation of protein-nucleic acid interactions has also become a viable research protocol.Therefore,this study selected the molecular dynamics simulation method to study the regulation mechanism of methylation on protein-DNA interaction.After literature research and crystal structure data search,we selected the bZIP family transcription factor as the research system.The research idea of this thesis is to study the regulation mechanism of 5mC methylation by comparing the effects of different single methylation sites and symmetric dimethylation sites on the interaction between transcription factors and nucleic acids.To this end,we first performed long-term and multiple parallel dynamics simulations on methylated and unmethylated structures(the total simulation time reached the order of microseconds),and then the binding free energy,entropy and enthalpy equilibrium,hydrogen bond,RMSF,DNA structure parameters,protein relative conformation and steric hindrance effects were analyzed and systematically compared.The following research progresses were made:Firstly,taking C/EBP protein binding to consistent DNA sequence containing a single methylated cytosine as an example,the mechanism of the influence of single-site methylation on the interaction between transcription factor and DNA was studied.Secondly,taking Jun/Jun homodimers-DNA complex as examples,the effects of single-site and double-site methylation on the interaction between transcription factors and DNA were studied.Thirdly,in the above two aspects of research,we found that the accuracy of the evaluation of binding free energy by molecular dynamics method is not enough,and it is necessary to use the ab initio quantum dynamics method to carry out related research.Therefore,we use the ab initio dynamics method to conduct an exploratory method test using the oxygen activation process as an example.更多还原