Gene expression analysis in the presence or absence of N-terminal tag (9myc) of Swd2 in Saccharomyces cerevisiae

project abstract : H3K4 methylation is a well-conserved histone modification from yeast to human. Because H3K4 methylase Set1 and its complex, COMPASS (Complex of proteins associated with Set1), are conserved from yeast to humans, budding yeast has been studied as an acceptable model organism. Since COMPASS components affect Set1 protein stability and H3K4 methylation activity variously, it is important to study how Set1 is regulated by complex components. However, deletion mutant of Swd2 component of COMPASS is not viable, although overexpression of Sen1 fragment enables the construction of Swd2 deletion mutant. This study found that positioning epitope tag to the N-terminal of Swd2 did not decrease interaction between Swd2 and Set1, but reduced the stability of both proteins, Swd2 and Set1, and global H3K4 methylation. Also, we observed that overexpression of N-terminal tagged Swd2 caused increased Set1 protein level and bulk H3K4 methylation. Therefore, Set1 protein can maintain its protein level only when enough Swd2 exist to cover the protein amount of Set1. Also, by comparing RNA sequencing analysis of N-terminal tagged Swd2 and Swd2 deletion mutant with Sen1 fragment overexpression, we isolated genes regulated by Swd2. In conclusion, we suggest that the abundance of Swd2 is important to regulate the protein stability of Set1 and the regulation of gene expression.项目摘要：H3K4甲基化是一类从酵母到人类均高度保守的组蛋白修饰。由于H3K4甲基转移酶Set1及其复合物COMPASS（Complex of proteins associated with Set1）在从酵母到人类中均保守，因此出芽酵母（budding yeast）被作为公认的模式生物用于相关研究。鉴于COMPASS的各组分可不同程度地影响Set1蛋白稳定性与H3K4甲基化活性，探究复合物组分对Set1的调控机制具有重要学术价值。然而，COMPASS的Swd2组分的敲除突变体无法存活，而过表达Sen1片段可成功构建Swd2敲除突变体。本研究发现，在Swd2的N端引入表位标签（epitope tag）并不会降低Swd2与Set1之间的相互作用，但会降低Swd2和Set1两种蛋白的稳定性，以及全局H3K4甲基化水平。此外，本研究团队观察到，过表达N端带有表位标签的Swd2会导致Set1蛋白水平升高，整体H3K4甲基化水平提升。由此可见，仅当存在足够数量的Swd2以匹配Set1的蛋白总量时，Set1才能维持自身的蛋白稳定性。此外，通过对比分析N端标签修饰Swd2菌株与过表达Sen1片段的Swd2敲除突变体的RNA测序（RNA sequencing）结果，本研究筛选得到了受Swd2调控的基因。综上，本研究提出Swd2的丰度对于调控Set1的蛋白稳定性以及基因表达调控具有关键作用。