ABSTRACT
Objective To study the effects of missense mutation of H4 Lysine 20,expore the molecular mechanisms,and find some specific interaction proteins,such as H4K20me3 demethyhransferase.Methods Stable HeLaS3 cell line expressed H4 or H4 mutations that fuses a Flag-HA tag to carboxyl(C)-terminal was established by virus infection.Mutations include lysine mutates to methionine and alanine.Harveste cells expressed purpose protein.Staining with propidium iodide (PI) and analyze cell cycle analysis by fluorescence activating cell sorter (FACS).Prepare mono nuclesome (mono-N) of three cells samples and purify associated protein complex by immunoprecipitation assay.Distingwish protein differences by Siliver staining and cut different protein bands for mass spectrometry analysis.Results HeLa S3 overexpressed H4K20M exhibit abnormal phenotype when transfected virus 72h,mainly show abnormal cell and nuclei volume increases,stop growing and no longer split.FACS shows cells overexpressed H4K20M were blocked in G2/M period.Mass spectrometry data shows the expression of PRBP4/7 increases in cells overexpressed H4K20M.Conclusion Histone H4 lysine 20 mutation to methionine block cell cycle,this block is likely to be caused by protein PRBP4/7.