组蛋白修饰对酿酒酵母复制起始相关蛋白基因表达的调控

The Regulation of Histone Modifications on DNA Replication initiation Proteins of Saccharomyces cerevisiae

真核生物的DNA复制是一个复杂且有序的过程,其中DNA复制起始参与调控众多生物学活动,对生物正常的生长发育具有重要作用。组蛋白修饰在调节DNA复制过程中具有重要作用。为了检测组蛋白特定位点的修饰对DNA复制起始的影响,本研究以酿酒酵母组蛋白H3/H4定点突变菌株为研究对象,采用倍比稀释表型分析法检测了组蛋白11种H3/H4甲基化、乙酰化突变菌株的生长情况,显示,以野生型菌株BY4741为对照,组蛋白H3/H4乙酰化突变菌株H3K64A、H3K56A、H3K14Q、H4K5R、H4K16Q以及组蛋白H4甲基化突变菌株H4K20Q表现为生长缺陷,其他菌株与BY4741生长情况基本一致;通过GeNorm和NormFinder软件分析得出本实验最适内参基因为β-actin;采用Real-time PCR检测了菌株细胞内复制起始相关蛋白基因mcm2、mcm10、cdc45的表达情况,表明,组蛋白H3/H4甲基化突变菌株H3K9A、H3R72K、H4K59A、H4K20Q胞内mcm2、mcm10、cdc45基因表达均显著下调(p<0.01),而H4R3A胞内mcm2、mcm10表达显著上调(p<0.05)、cdc45显著下调(p<0.01),组蛋白H3/H4乙酰化突变菌株H3K14Q、H4K5R、H4K8A、H4K16Q胞内mcm2表达显著下调(p<0.01),而在H3K64A、H3K56A胞内无显著变化,mcm10在6种组蛋白H3/H4乙酰化突变菌株内表达均下调(p<0.01),cdc45基因在H4K5R胞内表达无显著变化,在其余5种乙酰化突变菌株中表达均显著下调(p<0.01),为进一步阐明组蛋白特定位点修饰调控DNA复制起始的深入研究提供理论帮助。

DNA replication is a complicated and ordered progress in eukaryotic cells,which is regulated by histone modifications and important for growth and development.In order to study the influence of histone modifications on DNA replication initiation,the growth of the Saccharomyces cerevisiae histone H3/H4 acetylated and methylated mutants was detected,and the results showed that H3 K64 A,H3 K56 A,H3 K14 Q,H4 K5 R,H4 K16 Q,H4 K20 Q appeared growth defects and the others were consistent with WT.β-actin was the optimal reference gene analyzed by Ge Norm and NormFinder software.The gene expression of mcm2,mcm10,cdc45 encoding DNA replication initiation proteins was tested by Real-time PCR,the results indicated that all three genes were downregulated in histone H3/H4 methylated mutants H3 K9 A,H3 R72 K,H4 K59 A,H4 K20 Q(p<0.01)while mcm2,mcm10 was upregulated(p<0.05)and cdc45 was downregulated(p<0.01)in H4 R3 A.For histone H3/H4 acetylated mutants,mcm2 was downregulated in H3 K14 Q,H4 K5 R,H4 K8 A,H4 K16 Q(p<0.01),which had no significant changes in H3 K64 A and H3 K56 A,mcm10 was downregulated in above 6 strains(p<0.01),and cdc45 did not change significantly in H4 K5 R which was downregulated in the others(p<0.01).It provided a theoretical basis for histone modifications regulating on DNA replication initiation.