Mechanisms of epigenetic gene regulation by the Drosophila COMPASS-like complex

真核细胞的发育过程由被称为增强子的DNA元件控制。增强子的分子本质还不是很清楚，尽管目前的证据表明，它们与其他DNA元件不同，因为它们在染色体中具有不同的结构。结构上的差异可能源于负责将DNA包装在细胞核中的组蛋白的酶修饰。这个项目的重点是研究修改增强子元件的酶，以及它们的作用如何帮助在发育过程中的正确时间和位置打开关键基因。这项工作将使用多学科方法进行，将遗传、生化和果蝇的高通量生物信息学工具结合起来，作为模式生物。由于控制基因在发育过程中如何、何时、何地开启和关闭的机制在整个进化过程中都是保守的，因此结果应该会产生深远的影响，从酵母到人类。该项目将产生广泛的教育影响，为学生提供多个领域的培训，包括蛋白质结构建模、生物化学、生物信息学、遗传学和高级显微成像。学生还将学习如何发展科学假设，独立进行实验和解释结果，准备口头和书面总结并发表他们的工作，这些技能为不同的科学生涯提供强大的技术知识基础。后生动物指南针相关的共激活复合体催化组蛋白H3在赖氨酸4(H3K4)上的甲基化，这是与控制真核基因转录相关的表观遗传标记。果蝇中的CMI/TRR罗盘状复合体负责H3K4的单甲基化，并与对正常发育至关重要的转录因子合作调节增强子活性。TRR提供组蛋白甲基转移酶活性，而CMI包含两个保守的簇中的植物同源结构域锌指，这两个簇与核小体中的染色体紧密结合在一起进化了15亿年。尽管在转录控制中具有保守性和重要性，但目前还不清楚指南针状复合体如何在发育过程中启动和维持增强子活性。本项目的重点是保守的PhD指状域在染色质识别中的作用以及依赖于精确增强子控制的关键细胞信号通路。利用多功能的果蝇遗传模型系统，该项目测试了广泛的假设：(1)在CMI和同源脊椎动物蛋白中发现的簇状PHD结构域有助于必要的表观遗传组蛋白阅读器功能，从而驱动适当的启动和调节基因增强子，以及(2)果蝇罗盘状共激活物复合体调节增强子利用的时间，以整合关键的发育信号。该项目的一个目标是探索保守的PHD簇中的指状域的组合功能，这些功能对于适当的增强子调控是必不可少的。对PhD指节结构域和定向突变的结构模拟研究，结合体外和体内染色质结合的测量，将确定CMI和哺乳动物PhD指节结构域的组蛋白识别和结合特性，并进一步阐明指南针状复合体控制增强子的机制。第二个目标是结合发育、遗传和分子分析(ChIP-SEQ、RNA-SEQ、染色质捕获)，将扩大我们对体内靶基因调控的理解，帮助将特定的增强子表观遗传标记与COMPASS类复杂功能关联起来，并提供关于染色质调节的保守机制的重要新见解。

Developmental processes in eukaryotic cells are controlled by DNA elements called enhancers. The molecular nature of enhancers is not well understood, although current evidence suggests they differ from other DNA elements by having a distinct structure within the chromosomes. Structural differences can arise from enzymatic modification of the histone proteins responsible for packaging the DNA in the nucleus. This project focuses on studying the enzymes that modify enhancer elements and how their action helps turn on critical genes at the right time and location during development. The work will be carried out using a multi-disciplinary approach that combines genetic, biochemical and high-throughput bioinformatics tools in the fruit fly, Drosophila melanogaster, as the model organism. Because the mechanisms governing how, when and where genes are turned on and off during development are conserved across evolution, the results should have far-reaching impact, from yeast to humans. The project will have broad educational impact by providing students with training in multiple areas, including protein structure modeling, biochemistry, bioinformatics, genetics and advanced microscopy imaging. Students also learn how to develop scientific hypotheses, independently carry out experiments and interpret results, prepare oral and written summaries and publish their work, skills that provide a strong technical knowledge base for diverse scientific careers.The metazoan COMPASS related coactivator complexes catalyze the methylation of histone H3 on Lysine 4 (H3K4), epigenetic marks associated with controlling eukaryotic gene transcription. The Cmi/Trr COMPASS-like complex in Drosophila is responsible for monomethylating H3K4 and regulates enhancer activity in cooperation with transcription factors important for normal development. Trr provides histone methyltransferase activity, while Cmi contains plant homeodomain Zn fingers in two conserved clusters that co-evolved over 1.5 billion years with chromosome compaction in nucleosomes. Despite the conservation and importance in transcription control, it is not well understood how the COMPASS-like complexes are able to prime and maintain enhancer activities during development. This project focuses on the role of the conserved PHD finger domains in chromatin recognition and key cellular signaling pathways that depend on precise enhancer control. Taking advantage of the versatile Drosophila genetic model system, this project tests broad hypotheses (1) that the clustered PHD domains found in Cmi and homologous vertebrate proteins contribute essential epigenetic histone reader functions that drive the proper priming and regulation of gene enhancers and (2) the Drosophila COMPASS-like coactivator complex regulates the timing of enhancer utilization to integrate key developmental signals. One aim of this project explores the combined functions of the finger domains in the conserved PHD cluster that are essential for proper enhancer regulation. Structural modeling studies of the PHD finger domains and targeted mutagenesis, combined with in vitro and in vivo measurements of chromatin association, will define the histone recognition and binding properties of Cmi and mammalian PHD finger domains and further elucidate the mechanisms of enhancer control by the COMPASS-like complexes. A second aim incorporating developmental, genetic and molecular analyses (ChIP-seq, RNA-seq, chromatin capture) will expand our understanding of target gene regulation in vivo, help correlate specific enhancer epigenetic marks with COMPASS-like complex functions, and provide significant new insights regarding conserved mechanisms of chromatin regulation.