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

基本信息

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
项目类别：
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

项目摘要

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密切相关的染色质标记增强子和其他基因间调节元件；然而，该组蛋白的功能意义修改尚未得到证实。我们的实验室之前已确定 Trr 和 MLL3/4 是主要的 H3K4- 分别存在于果蝇和哺乳动物中的单甲基化酶。最近的全基因组关联研究发现 trr 人类直系同源物 MLL3 和 MLL4，作为在多种人类癌症中频繁突变的基因，以及其他 COMPASS 子单元。先前的工作已证实 Trr 蛋白是调节增强子功能；然而，Trr 依赖性 H3K4me1 在增强子中的作用尚未经过测试直接地。为了解决这个问题，我们利用胚胎隐性致死 Trr-NULL 等位基因 trr[1] 来测试Trr依赖性甲基化酶活性的必要性。我们发现 trr[1] 的杀伤力可以通过以下方式挽救表达 Trr 转基因，其 SET 结构域包含催化失活 (C2398A) 或催化失活 (C2398A) 过度活跃（Y2383F）点突变。正如预期的那样，免疫印迹证实 H3K4me1 显着减少催化死亡突变体，而催化高活性突变体显示 H3K4me2/3 显着增加。我们的 ChIP-seq 研究证实这些变化专门发生在增强子元件上。除了这些之外分子表型，这两个突变蝇系没有表现出明显的发育、生殖或行为与对照 trr-WT 救援系相比的差异。这些观察提出了三个重要的问题：1）Trr 依赖性 H3K4 甲基化在果蝇发育中的功能是什么，2）什么是 Trr 在调节增强子活性和转录激活中的非酶功能，以及 3) 如何 MLL3/4 中发现的突变会破坏增强子功能并导致人类癌症发病机制？通过利用黑腹果蝇，其中 MLL3/4 仅由一种酶 trr 代表，我将揭示基本原理有关这些蛋白质如何发挥调节增强子活性以及有害突变如何发挥作用的详细信息 MLL3/4 导致癌症形成。