Unraveling the mechanism of DOT1A-mediated replication regulation in Trypanosoma brucei

组蛋白翻译后修饰调控许多重要的生物过程，如复制、DNA修复和细胞周期控制。我们以单细胞真核寄生虫布氏锥虫为模型系统，研究染色质机制对复制调控和发育分化的调控作用。我们特别感兴趣的是Dot1赖氨酸甲基转移酶家族成员对组蛋白H3甲基化的功能。DOT1(端粒沉默的干扰因子，也被称为KMT4)最初是在酵母中发现的，是在基因筛查中发现的，这些基因的过度表达导致端粒沉默的缺陷。在之前的资助阶段，我们想要揭示染色质结构在布鲁氏锥虫细胞周期调节和发育分化中的作用。在这些项目的过程中，我们开始关注两个锥虫Dot1同系物DOT1A和DOT1B的功能，它们分别负责H3K76的二甲基化和三甲基化。令人惊讶的是，我们发现DOT1A介导的H3K76单甲基化或双甲基化调节复制启动。这是一种在真核生物中复制的新的调控机制，在我们发表结果后不久，人类细胞也描述了这一机制。基于这个成功的初步项目，我们现在的目标是揭开这个细胞过程背后的机械事件。我们计划确定介导H3K76甲基化依赖的复制调控的分子机制，以了解更多关于这一基本生物学过程的信息。

Post-translational histone modifications regulate many important biological processes like replication, DNA repair and cell cycle control. We use the unicellular, eukaryotic parasite Trypanosoma brucei as a model system to study how replication regulation and developmental differentiation are controlled by chromatin-based mechanisms. We are specifically interested in the function of histone H3 methylation by a member of the Dot1 family of lysine methyltransferases. Dot1 (disruptor of telomeric silencing, also called KMT4) was initially discovered in yeast in a genetic screen for genes whose over-expression caused defects in telomeric silencing. In the previous funding period, we wanted to unravel the function of chromatin structure during cell cycle regulation and developmental differentiation in T. brucei. In the course of these projects, we began to focus on the function of two trypanosome Dot1 homologues DOT1A and DOT1B, which are responsible for di-methylation and tri-methylation of H3K76, respectively. Surprisingly, we discovered that DOT1A-mediated H3K76 mono- or di-methylation regulates replication initiation. This is a novel regulatory mechanism for replication in eukaryotes, which was also described in human cells shortly after we published our results. Based on this successful initial project, we now aim to unravel the mechanistic events behind this cellular process. We plan to identify the molecular machinery, which mediates H3K76 methylation-dependent regulation of replication to learn more about this basic biological process.