Regulation of histone methylation

在真核生物中，DNA被包装成染色质，染色质是一种核蛋白结构，由DNA、组蛋白和非组蛋白组成。染色质对基因表达的保真度很重要，其结构可以通过在组蛋白上添加翻译后修饰(PTM)来调节。一种这样的修饰，组蛋白H3在赖氨酸36处的甲基化，通过RNA聚合酶II的通过而被定位于活性基因，而这种PTM可以说是所有真核生物中最公认的高产转录的标志。然而，尽管对H3K36甲基化建立和功能背后的机制进行了十多年的研究，但令人惊讶的是，关于这种修饰是如何在基因抑制后被移除的知之甚少。在这项申请中提出的研究将揭示在基因抑制过程中涉及去除H3K36甲基化的酶和途径。我们将使用酿酒酵母作为模式生物，因为参与调控H3K36甲基化的基因在酵母和其他真核生物之间具有很好的保守性，并且这种模式生物具有遗传易感性。使用的技术包括染色质免疫沉淀(ChIP)、定量聚合酶链式反应(QPCR)、深度测序的文库准备、生物信息学、蛋白质纯化、酶动力学和酵母遗传学，因此，我的NSERC资助的学生将学习到一套在多个生命科学研究领域备受追捧的技能。

In eukaryotes DNA is packaged into chromatin, a nucleoprotein structure consisting of DNA, histones, and non-histone proteins. Chromatin is important for the fidelity of gene expression and its structure can be regulated by the addition of post-translational modifications (PTMs) to histones. One such modification, methylation of histone H3 at lysine 36, is targeted to active genes by the passage of RNA polymerase II, and this PTM is arguably the most recognized hallmark of productive transcription in all eukaryotes. However, despite over a decade worth of research into the mechanisms behind the establishment and function of H3K36 methylation, there is surprisingly little known about how this modification is removed following gene repression. The research proposed in this application will reveal the enzymes and pathways involved in removal of H3K36 methylation during gene repression. We will use S. cerevisiae as a model organism due to the excellent conservation of genes involved in regulation of H3K36 methylation between yeast and other eukaryotes, and the genetic tractability of this model organism. The techniques used include ChIP (chromatin immunoprecipitation), qPCR (quantitative polymerase chain reaction), library preparation for deep sequencing, bioinformatics, protein purification, enzyme kinetics, and yeast genetics, and thus students supported by my NSERC grant will learn a diverse set of skills that are highly sought after in multiple fields of life science research.