Chromatin recruitment of Rad6 determines H3K4me3 in Saccharomyces cerevisiae

Project abstract : The trimethylation of histone H3 lysine 4 (H3K4me3) is a crucial factor in defining the promoter regions of active genes in all eukaryotes ranging from Saccharomyces cerevisiae (yeast) to humans. In budding yeast, this trimethylation process facilitated by the Set1 complex results in H3K4me3 requiring a prior mono-ubiquitination at the histone H2BK123 residue (H2Bub) by E2 enzyme Rad6 and E3 enzyme Bre1. A previous in vitro study suggested that ubiquitinated H2B directly facilitates H3K4me3. However, even low levels of global H2Bub is sufficient for the required H3K4me3 in yeast cells, thereby indicating that other factors resulting in the H2Bub-dependent H3K4me3 remain unknown. This study revealed the high level of correlation of H3K4me3 with chromatin recruitment of Rad6 at the genome-wide level. Rad6 is confirmd to interact and co-localize with Swd2/Cps35, a key factor for the H2Bub-dependent H3K4me3 in genes with high levels of H3K4me3 and intronic genes rather than non-intronic genes. This study therefore provides a mechanistic insight of the H2Bub-Rad6- Swd2/Cps35-H3K4me3 axis and its potential role in RNA biogenesis. To define the global binding sites of Rad6 and to identify whether chromatin recruitment of Rad6 is affected by catalytic activity or phosphorylation status of Rad6, chromatin immunoprecipitation (ChIP) sequencing against 9myc antibody is performed with yeast strains endogenously tagged with 9myc at Rad6 locus in WT, Rad6 C88A catalytically dead mutant, Rad6S120A phosphorylation site mutant background. To confirm if H2B ubiquitination contributes to chromatin recruitment of Rad6, ChIP with 9myc antibody is performed to endogenously expressed Rad6-9myc at H2B WT and H2B K123R strain. 9myc ChIPseq is carried out with no tag WT strain as control. To define the chromatin binding sites of Swd2 and to compare with chromatin binding of Rad6 and H3K4me3, ChIP against 3HA tag of endogenously tagged Swd2-3HA WT strain is performed.

项目摘要：组蛋白H3赖氨酸4三甲基化（histone H3 lysine 4 trimethylation, H3K4me3）是定义所有真核生物（从酿酒酵母Saccharomyces cerevisiae到人类）中活跃基因启动子区域的关键调控因子。在酿酒酵母中，由Set1复合物（Set1 complex）介导的该三甲基化过程，即H3K4me3的形成，需要先由E2酶Rad6和E3酶Bre1在组蛋白H2BK123位点完成单泛素化修饰（H2Bub）。既往一项体外研究提示，泛素化的H2B可直接促进H3K4me3的生成。然而，即便酵母细胞中全局水平的H2Bub含量极低，也足以满足H3K4me3形成的需求，这表明介导H2B泛素依赖性H3K4me3的其他潜在因子仍未被阐明。本研究在全基因组层面揭示了H3K4me3与Rad6的染色质招募水平存在高度相关性。实验证实，在H3K4me3高表达基因及内含子基因中，Rad6可与Swd2/Cps35相互作用并共定位——Swd2/Cps35是H2B泛素依赖性H3K4me3形成的关键因子，而在无内含子基因中则无此现象。本研究由此解析了H2Bub-Rad6-Swd2/Cps35-H3K4me3调控轴的机制，并阐明其在RNA生物发生中的潜在作用。为明确Rad6的全基因组结合位点，并探究Rad6的染色质招募是否受其催化活性或磷酸化状态影响，本研究针对在Rad6基因座内源携带9myc标签的酵母菌株，分别在野生型（WT）、Rad6 C88A催化失活突变体、Rad6S120A磷酸化位点突变体背景下，开展了针对9myc抗体的染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）实验。为验证H2B泛素化是否影响Rad6的染色质招募，本研究分别以H2B野生型及H2B K123R菌株中内源表达的Rad6-9myc为靶标，使用9myc抗体开展ChIP实验，并以无标签的野生型菌株作为对照进行9myc ChIP-seq。为明确Swd2的染色质结合位点，并将其与Rad6及H3K4me3的染色质结合情况进行比较，本研究针对内源携带3HA标签的Swd2-3HA野生型菌株，开展了针对3HA标签的ChIP实验。