The Assembly And Dissociation Dynamics Of Histone Variant H2A.Z-containing Nucleosome In Vitro

As the basic structural unit of eukaryotic chromatin,nucleosomes in vivo are highly dynamic in many biological processes,such as DNA replication,repair,recombination,or transcription.In eukaryotes,histone variant H2 A.Z is one of the highly conserved variants in evolution level,and it can regulate chromatin structure and thus affect gene transcription.However,its detailed regulatory mechanism is still unclear.Histone variant H2 A.Z and its epigenetic modifications play an important role in regulating chromatin dynamic structure and function.At present,the dynamic mechanism of nucleosome assembly and dissociation containing histone variant H2 A.Z has not been clearly explained.The study of nucleosome assembly and dissociation dynamics containing H2 A.Z can promote the understanding of the effect of nucleosome localization on gene expression regulation.In view of the possible competitive relationship between H2 A.Z and H2 A in nucleosome assembly,the competitive kinetics between histone variant H2 A.Z and histone H2 A was investigated based on the dynamics of nucleosome assembly and dissociation.The results of this study are expected to provide a theoretical basis for further understanding of nucleosome assembly dynamics of histone variant H2 A.Z.Main research contents in this paper are as follows:1.Based on the canonical nucleosome assembly dynamic model proposed by our research group,it was determined by experiments whether nucleosomes containing histone variant H2 A.Z were suitable for this dynamic model.The relationship between nucleosome assembly efficiency and histone concentration dependence,DNA sequence specificity and assembly reaction time was investigated in this experiment.The experimental results showed that the nucleosome assembly efficiency of H2 A.Z was positively correlated with histone concentration in assembly reaction system and assembly reaction time,which was consistent with the correlation in the dynamic model.The reaction rate constant can be used to evaluate the affinity of DNA sequence to histone variant H2 A.Z.These results indicate that the nucleosome assembly process of H2 A.Z in vitro are also subject to the principle of chemical reaction kinetics.2.Based on the nucleosome assembly dynamic model,the competitive kinetic model of histone variant H2 A.Z and canonical histone H2 A in the same reaction system nucleosome assembly was proposed.The nucleosome containing H2 A.Z and H2 A assembly experiments were performed under the assembly ratio of total histone to DNA of 0.8,1.0 and 1.2,and then Native-PAGE and Western Blot was used to detect the assembly efficiency.It was found that the canonical histone H2 A was more competitive to assemble nucleosome than histone variant H2 A.Z,and this competition ability of H2 A is unrelated to the ration of histone variant H2 A.Z and histone H2 A in the assembly reaction system.3.In order to compare the stability of nucleosomes containing histone variant H2 A.Z and canonical nucleosomes under the action of salt ions,F(?)rster resonance energy transfer method was used to detect the effect of sodium chloride,potassium chloride,manganese chloride,calcium chloride,magnesium chloride and nickel chloride on the dissociation of nucleosomes.In this experiment,the DNA sequence of Widom 601 was labeled with Cy3 and Cy5,and the dissociation of nucleosomes was evaluated by the change of fluorescence signal value.F(?)rster resonance energy transfer experiment results showed that nucleosomes containing histone variant H2 A.Z dissociation rate was slower than canonical nucleosomes under the action of sodium chloride,potassium chloride,manganese chloride,calcium chloride,magnesium chloride and nickel chloride.And calcium chloride,manganese chloride,magnesium chloride and nickel chloride had more obvious effects on nucleosome dissociation.By incubating assembled nucleosome samples at 75℃,it was found that the dissociation rate of nucleosomes containing histone variant H2 A.Z was slower than that of conventional nucleosomes.Furthermore,the analysis by fluorescence thermal shift experiment indicated that the increase rate of fluorescence signal value of canonical nucleosomes showed a slowing trend at 70℃.The fluorescence signal value of nucleosomes containing the histone variant H2 A.Z increased slowly at 75°C,and the maximum value of fluorescence signal value is about 87℃.The dissociation rate of canonical nucleosomes are faster than that of histone variant H2 A.Z.The results showed that the structure of the nucleosome containing histone variant H2 A.Z was more stable than that of the conventional nucleosome.