Investigating catalytic and non-catalytic roles for Trr/COMPASS in regulating enhancer function during Drosophila development

研究 Trr/COMPASS 在果蝇发育过程中调节增强子功能的催化和非催化作用

• 批准号: 9259512
• 负责人: Ryan Adam Rickels
• 金额: $ 2.78万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-01 至 2017-09-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9259512
• 关键词: Address; Affect; Alleles; Behavioral; Binding Sites; Biochemical; Biochemical Genetics; Biochemistry; Biological Process; Cells; ChIP-seq; Chromatin; Communication; Complement; Data; Deletion Mutation; Deposition; Development; Developmental Gene; Drosophila genus; Drosophila melanogaster; Embryo; Enhancers; Enzymes; Epigenetic Process; Family; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Enhancer Element; Genetic Transcription; Genetic study; Histones; Homeobox Genes; Homeostasis; Human; Hyperactive behavior; Investigation; Laboratories; Lead; Light; Lysine; Malignant Neoplasms; Mammalian Cell; Mammals; Mediating; Methylation; Methyltransferase; Molecular; Mono-S; Mutate; Mutation; Organism; Pathogenesis; Pattern; Play; Point Mutation; Positioning Attribute; Proteins; Reagent; Recruitment Activity; Regulation; Regulatory Element; Role; SET Domain; Sequence Homology; System; Testing; Transcriptional Activation; Transgenes; Work; Yeasts; cancer genome; fly; genome wide association study; genome-wide; histone methylation; histone methyltransferase; histone modification; member; molecular phenotype; mutant; promoter; protein function; protein protein interaction; reproductive; targeted treatment; therapeutic target; tool; transcription factor

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, immunoblots 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 activity and transcriptional activation, and 3) how do 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.

项目摘要/摘要： 组蛋白3赖氨酸4（H3K4ME1）的单甲基化是与转录型密切相关的染色质标记 增强剂和其他基因间调节元素；但是，该组蛋白的功能意义 修改尚未证明。我们的实验室以前已将TRR和MLL3/4确定为主要的H3K4-- 果蝇和哺乳动物中的单甲基酶。最近的全基因组关联研究确定了 TRR人类直系同源物mll3和mll4，因为基因经常在各种各样的人类癌症中突变， 以及其他指南针亚基。以前的工作已经确定TRR蛋白对于 调节增强子功能；但是，尚未测试TRR依赖性H3K4ME1在增强器中的作用 直接地。为了解决这个问题，我们利用了胚胎隐性致命的TRR等位基因TRR [1]， 测试TRR依赖性甲基化酶活性的必要性。我们发现TRR [1]的致命性可以由 表达TRR转基因的设置域含有催化无效（C2398A）或催化 - 多动（Y2383F）点突变。不出所料，免疫印迹证实了H3K4ME1的大幅降低 催化已故的突变体，而催化混合性突变在H3K4ME2/3中显示出显着增加。 我们的CHIP-seq研究验证这些变化是在增强子元素上特别发生的。除了这些 分子表型，两个突变蝇线没有明显的发育，生殖或行为 与对照trr-wt救援线相比的差异。这些观察提出了三个重要的 问题：1）依赖TRR的H3K4-甲基化在果蝇发展中的功能是什么，2）什么是 TRR在调节增强子活性和转录激活中的非酶函数，以及3）如何如何 在MLL3/4中发现的突变破坏了增强子功能并导致人类癌症发病机理？通过利用 果蝇Melanogaster，其中Mll3/4仅由一种酶，TRR表示，我将发现基本 有关这些蛋白质如何调节增强剂活性以及如何有害突变的详细信息 MLL3/4导致癌症形成。