The Establishment Of Theoretical Model And Experimental Study On Nucleosome Assembly In Vitro

Nucleosomes are the basic structural units of eukaryotic chromatin and are formed from approximately 147 bp of double helix DNA wound around histone octamers.Histone octamer is composed of four canonical histones of two molecules of H2A,H2B,H3 and H4.During the dynamic changes of nucleosomes in vivo,histones can undergo post-translational modification or be replaced by histone variants,which modulates the epigenetic regulation of the genome.The assembly and depolymerization process of nucleosome structure can directly regulate the contact ability between trans-acting factors and functional elements on chromatin,and affect many biological processes with DNA as template,such as transcription,replication,recombination and repair.The dynamic study of assembly and depolymerization of nucleosomes has important biological significance for nucleosome localization.The assembly and depolymerization of nucleosomes in vivo are highly dynamic and are subjected to the regulation of many factors.At present,nucleosome assembly in vitro is an effective method to research the assembly and depolymerization of nucleosomes.The assembly and depolymerization of nucleosomes were investigated by constructing kinetic models and experiments in vitro.These results was helpful for deciphering the regulation mechanism of gene expression and chromatin dynamics from the perspective of nucleosome sliding and nucleosome localization in vivo,etc.,and provided an important theoretical guideline for understanding nucleosome function in many biological processes.The main research contents can be summarized as follows.1.A kinetic model of nucleosome assembly was proposed based on the kinetic principle of chemical reaction and was verified by experiment.According to the kinetic model,the effects of assembly time,histone concentration and assembly rate constant on the assembly efficiency of nucleosomes were experimentally verified.The experimental results revealed that the nucleosome assembly efficiency is positively correlated with assembly time,histone concentration and assembly rate constant.This result is consistent with the relationship in the model.Experiments verified the relationship between DNA concentration and nucleosome assembly efficiency,which were conducted to determine the optimum concentration of histone octamer in the system.When the DNA concentration is much smaller than the histone octamer concentration,the assembly efficiency increases with the increase of the DNA concentration.When the DNA concentration is greater than the histone octamer concentration,the nucleosome DNA proportion decreases with increasing DNA concentration in the system.2.The effects of temperature,assembly time and salt ion concentration on the structure of nucleosomes were investigated.Firstly,high temperature was used to stimulate nucleosome depolymerization,and native PAGE and FRET techniques were used to detect the depolymerization of nucleosomes during different process time of temperature stimulation.The results of electrophoresis showed that the process of nucleosome depolymerization under heat stress carried out in different time-course stages.In the early period of the depolymerization process,the DNA content of nucleosomes had no significant change.However,the FRET results showed that although the histones and DNA are not separated in the first stage of nucleosome depolymerization,the nucleosome structure has become loose.Then,FRET technology was used to detect the effects of six salt ions on the structure of the nucleosomes.The similar nucleosome structure change was observed in different ion environments.The structure of the nucleosomes became gradually loosened as the ion concentration increased.Taking sodium potassium ion as an example,the analysis of fluorescence energy transfer efficiency with the rate of change of salt ion concentration revealed that the depolymerization process of nucleosome structure with the change of salt ion concentration is carried out in different stages.Especially,in the sodium ion environment,the optimum sodium ion concentration during the depolymerization process is consistent with the optimum ion concentration required the in vitro assembly process by salt dialysis.The influence of ion concentration on nucleosome structure is more obvious in some 2-valent salt ion environments.3.The changes of nucleosome state in the process of temperature-stimulated nucleosome depolymerization were detected by FTS experiment.The results revealed that the separation of DNA and histone octamer may be carried out step by step in the process of nucleosomal depolymerization.The depolymerization process may be mainly divided into two steps.First of all,two H2A/H2B dimers are detached from the DNA.And then,the?H3/H4?₂ tetramer is separated from the DNA.This is consistent with the sequential assembly mechanism of the nucleosome assembly process.