The interactions of nucleosome and transcription factor RFX7
Objective To investigate the interaction of nucleosomes and transcriptive factor RFX7.Methods Recombinant 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.Results Human 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.Conclusion RFX7 has nucleosome-binding ability.Additionally,the preliminary structure models of the nucleosome and RFX7 eDBD protein complex can be obtained using cryo-electron microscopy.
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