Defining Gene Regulatory Elements Essential to Cancer Cell Viability

项目概要/摘要： 组蛋白3赖氨酸4（H3 K4 me 1）的单甲基化是与转录调控密切相关的染色质标记。 增强子和其他基因间调控元件;然而，这种组蛋白的功能意义 修改还有待证明。我们的实验室以前已经确定Trr和MLL 3/4是主要的H3 K4- 在果蝇和哺乳动物中分别有一种单甲基化酶。最近的全基因组关联研究 鉴定了trr人类直系同源物MLL 3和MLL 4，作为在多种人类中频繁突变的基因， 癌症，沿着其他COMPASS亚基。以前的工作已经确定，Trr蛋白是必需的， 调节增强子功能;然而，尚未测试Trr依赖性H3 K4 me 1在增强子中的作用 直接.为了解决这个问题，我们利用了一个胚胎隐性致死Trr-空等位基因trr[1]， 测试Trr依赖性甲基化酶活性的必要性。我们已经发现TRR[1]的致命性可以通过 表达其SET结构域含有催化失活（C2398 A）或催化失活（C2398 A）的Trr转基因， 过度活跃（Y2383 F）点突变。正如预期的那样，蛋白质印迹证实了H3 K4 me 1的显著减少 在催化死亡的突变体中，而催化过度活跃的突变显示出显著增加， H3K4me2/3。我们的ChIP-seq研究证实这些变化专门发生在增强子元件上。一边 从这些分子表型来看，这两个突变蝇系没有表现出明显的发育、生殖或 与对照trr-WT拯救线相比的行为差异。这些意见提出了三个 重要的问题：1）Trr依赖的H3 K4甲基化在果蝇发育中的作用，2） Trr在调节增强子介导的过程中的非酶功能是什么，以及3）非酶功能如何 MLL 3/4中发现的催化突变破坏增强子功能并导致人类癌症发病机制？通过 利用果蝇，其中MLL 3/4仅由一种酶trr代表，我将揭示 关于这些蛋白质如何调节增强子活性的基本细节， MLL 3/4的突变导致癌症形成。

Project Summary/Abstract: Mono-methylation of histone 3 lysine 4 (H3K4me1) is a chromatin mark closely associated with transcriptional enhancers and other intergenic regulatory elements; however, the functional significance of this histone modification has yet to be demonstrated. Our lab has previously identified Trr and MLL3/4 as the major H3K4- monomethylases in Drosophila and mammals, respectively. Recent genome-wide association studies identified the trr human orthologues, MLL3 and MLL4, as genes frequently mutated in a wide variety of human cancers, along with other COMPASS subunits. Previous work has established that Trr protein is necessary for regulating enhancer function; however, the role of Trr-dependent H3K4me1 at enhancers has not been tested directly. To address this, we have taken advantage of an embryonic recessive lethal Trr-NULL allele, trr[1], to test the necessity of Trr-dependent methylase activity. We have found the trr[1] lethality can be rescued by expressing a Trr transgene whose SET domain contains either a catalytic-inactive (C2398A) or a catalytic- hyperactive (Y2383F) point mutation. As expected, western blots confirm substantial reductions of H3K4me1 in the catalytic-dead mutant, while the catalytic-hyperactive mutation shows significant increases in H3K4me2/3. Our ChIP-seq studies verify these changes are occurring specifically at enhancer elements. Aside from these molecular phenotypes, the two mutant fly lines show no obvious developmental, reproductive, or behavioral differences in comparison with a control trr-WT rescue line. These observations raise three important questions: 1) what is the function of Trr-dependent H3K4-methylation in Drosophila development, 2) what are the non-enzymatic functions of Trr in regulating enhancer-mediated processes, and 3) how do non- catalytic mutations found in MLL3/4 disrupt enhancer function and lead to human cancer pathogenesis? By exploiting Drosophila melanogaster, in which MLL3/4 are represented by just one enzyme, trr, I will uncover fundamental details regarding how these proteins function to regulate enhancer activity, and how deleterious mutations to MLL3/4 result in cancer formation.