核小体与转录因子RFX7的相互作用分析

The interactions of nucleosome and transcription factor RFX7

关键词:  核小体  转录调控  冷冻电镜技术

Key words:  nucleosome  transcription regulation  cryo-electron microscopy

摘要:

目的利用冷冻电镜技术解析核小体与RFX7结合复合物的冷冻结构，阐明核小体与RFX7相互作用的具体方式。 方法利用pET28a表达载体和人源RFX7 eDBD（extended DNA binding domain）基因构建重组蛋白表达质粒pET28a-RFX7-eDBD，在原核蛋白表达感受态BL21（DE3）中表达蛋白，亲和层析获得蛋白，体积排阻色谱进一步分离纯化RFX7 eDBD蛋白。构建含有多拷贝人源RFX7特异性识别核小体DNA序列的质粒，转入大肠杆菌大量扩增DNA序列用于核小体组装。纯化4种组蛋白，组成组蛋白八聚体，利用盐透析法在体外装配核小体。通过EMSA凝胶迁移实验检测核小体与RFX7 eDBD的结合情况。负染电镜观察复合物蛋白状态并优化样品；冷冻电镜收集数据，冷冻电镜数据处理软件CryoSparc解析复合物的冷冻电镜结构，Chimera分析模型。 结果亲和层析成功纯化人源RFX7 eDBD蛋白以及4种组蛋白，并通过体积排阻色谱获得高纯度的RFX7 eDBD蛋白和组装好的组蛋白八聚体；聚乙二醇(PEG)6000沉淀法纯化DNA，利用盐透析法成功在体外组装核小体；EMSA凝胶迁移实验证实RFX7可以结合核小体，通过负染电镜、冷冻电镜及单颗粒重构技术解析了复合体的空间结构，初步确认了核小体与RFX7之间的相互作用。 结论RFX7具有核小体结合能力，利用冷冻电镜技术可以初步获得核小体与RFX7 eDBD蛋白复合体的空间结构模型。

Abstract:

ObjectiveTo investigate the interaction of nucleosomes and transcriptive factor RFX7. MethodsRecombinant protein expression plasmid pET28a-RFX7-eDBD was constructed from pET28a expression vector and human RFX7 eDBD(extended DNA binding domain), and the protein was expressed in Escherichia coli BL21(DE3); followed by affinity chromatography, size exclusion chromatography was used to further isolate and purify the RFX7 eDBD protein. Plasmids containing multiple copies of human RFX7-specific recognition nucleosome DNA sequences were constructed and transferred into E.coli to get large quantities of DNA sequences for nucleosome assembly. Four histones were purified to assemble histone octamers. And then, nucleosomes were assembled by salt dialysis. The binding of nucleosomes to RFX7 eDBD was detected by electrophoretic mobility shift assay(EMSA). Negative staining electron microscopy was used to observe the state of complexes and optimize the samples. Cryo-electron microscopy(Cryo-EM) was used to collect data. The structure of the complexes were resolved by Cryo-EM data processing software CryoSparc. Then, Chimera was used to analyze the model. ResultsHuman RFX7 eDBD protein, four histones, RFX7 eDBD protein and assembled histone octamer were successfully purified by affinity chromatography and size exclusion chromatography. The DNA was purified by polyethylene glycol(PEG 6000) precipitation, and nucleosomes were successfully assembled by salt dialysis. EMSA confirmed that RFX7 was able to bind nucleosomes. And the structure of the complex was resolved by negative-staining electron microscopy, cryo-electron microscopy and single-particle reconstruction technique, which preliminarily confirmed the interactions between the nucleosomes and RFX7. ConclusionRFX7 has nucleosome-binding ability. Additionally, the preliminary structure models of the nucleosome and RFX7 eDBD protein complex can be obtained using cryo-electron microscopy.