组蛋白H4第20位赖氨酸突变阻滞细胞周期

Mutation of Histone H4 Lysine 20 Blocks Cell Cycle

目的研究组蛋白H4第20位赖氨酸突变对细胞可能存在的影响并探究其分子机制,期望寻找作用于该位点的特异性蛋白,比如H4K20me3去甲基化酶。方法建立能够表达羧基(C)末端带Flag-HA标签的组蛋白H4及其突变体H4K20M(赖氨酸突变为蛋氨酸),H4K20A(赖氨酸突变为丙氨酸)的HeLa S3细胞系。通过病毒感染法获得能够表达目的蛋白的细胞,碘化丙啶(PI)染色后进行流式细胞(FACS)周期分析。制备单个核小体(mono nucleosome,mono-N),通过免疫共沉淀的方法得到相互作用蛋白复合物成分,银染观察蛋白组分的差异,将差异蛋白条带切割后进行质谱分析。结果在病毒感染大约72h过表达K20M的HeLa S3细胞表型异常,主要表现为细胞体积和细胞核体积明显增大,并停止生长,不再分裂。FACS结果显示过表达H4K20M的细胞被阻滞在G_2/M期,质谱结果表明在过表达K20M的细胞中RBBP4/7的表达量增高。结论组蛋白H4第20位赖氨酸突变为蛋氨酸会阻滞细胞周期,而这种阻滞很可能由RBBP4/7所引起。

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 demethyltransferase. 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.