体外组装含有组蛋白变体H2A.Z及H3.3的核小体  

Assembly Nucleosome Containing Histone Variant H2A. Z and H3. 3 in vitro

作　　者：赵宏宇[1,2] 刘媛[2] 张凤慧[2] 邢永强[2] 蔡禄[2] 

机构地区：[1]内蒙古大学物理科学与技术学院,呼和浩特010021 [2]内蒙古科技大学数理与生物工程学院,包头014010

出　　处：《中国生物工程杂志》2016年第3期53-60,共8页China Biotechnology

基　　金：国家自然科学基金(61271448);内蒙古自然科学基金(2013MS0514;2014MS0312)资助项目

摘　　要：核小体是构成真核生物染色质的基本结构单位,组蛋白变体H2A.Z及H3.3对染色质结构及基因转录过程发挥着重要的调控作用。体内研究核小体及染色质结构受到诸多因素限制,体外重构含有H2A.Z及H3.3的核小体结构是研究与组蛋白变体相关基因表达调控的重要方法之一。实验表达纯化了6种组蛋白,在复性的过程中装配了含有H2A.Z和H3.3的组蛋白八聚体。基于DNA序列10bp周期性及序列模体设计了3条易于形成核小体的DNA序列,通过PCR大量扩增的方法,回收了标记Cy3荧光分子的目的 DNA序列。采用盐透析法体外组装了含有H2A.Z和H3.3的核小体结构,利用荧光标记、EB染色及考马斯亮蓝染色检测了含有组蛋白变体的核小体形成效率及形成过程的吉布斯自由能变化。结果发现,设计的3条DNA序列可以有效地组装形成含有组蛋白电梯的核小体结构,而且随着组蛋白八聚体与DNA比例的增加,核小体的形成效率显著提高;采用Cy3荧光标记可以灵敏且定量地计算组装过程的吉布斯自由能。该方法的建立对研究组蛋白变体相关的结构生物学及转录调控等具有一定的意义。The nucleosomes constitute the basic structural unit of eukaryotic chromatin. The variant H2 A. Z and H3. 3,both of which are highly conserved evolutionarily,have been proposed to play crucial and specific roles in the regulation of chromatin dynamics and transcription. In vivo eukaryotic nucleosome formation will be subject to interference from a variety of factors,and in vitro assembling nucleosome structure by DNA and histone variants H2 A. Z and H3. 3 is one of basic method to study the nucleosome positioning and chromatin structure.The H2 A,H2B,H3,H4,H2 A. Z and H3. 3 histones have been expressed and assembled to form octamer containing histone variants. Based on 10 base periodicity and sequence motif on nucleosome DNA,CS1,CS2 and CS3,which could have high affinity to histones,was designed to assembly the nucleosome structure. Then the DNA sequences labeling Cy3 are obtained through using PCR and gel extraction method. After a salt dilution method employing to assembly nucleosome,reconstituted nucleosome was analyzed by Cy3 labeling,EB staining and Coomassie blue staining. The results show that the nucleosome structure can be effectively reconstituted in vitro and the formation efficiency of nucleosome was positively related to the ratio of octamer to DNA. The quantitative result by Cy3 labeling can be used to compute the Gibbs free energy of the reconstituted assay. The work laid the foundation for further study of epigenetic and structural biology associated with histone variant H2 A. Z and H3. 3.

关 键 词：组蛋白变体 核小体定位 染色质结构 荧光标记 

分 类 号：Q816[生物学—生物工程]