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

真核细胞的发育过程是由称为增强子的DNA元件控制的。增强子的分子性质尚不清楚，尽管目前的证据表明它们与其他DNA元素不同，因为它们在染色体内具有独特的结构。在细胞核中负责包装DNA的组蛋白的酶修饰可引起结构差异。这个项目的重点是研究修饰增强子元件的酶，以及它们的作用如何帮助在发育过程中正确的时间和位置打开关键基因。这项工作将采用多学科方法，将遗传、生化和高通量生物信息学工具结合起来，以果蝇为模式生物。因为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，所以研究结果应该具有深远的影响，从酵母到人类。该项目将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、遗传学和高级显微镜成像，从而产生广泛的教育影响。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，准备口头和书面摘要并发表他们的工作，这些技能为各种科学事业提供了强大的技术知识基础。后生动物COMPASS相关的辅激活因子复合物催化赖氨酸4 （H3K4）上组蛋白H3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 compass样复合体负责H3K4的单甲基化，并与正常发育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在两个保守的簇中含有植物同源结构域Zn指，它们与核小体中的染色体压实共同进化了15亿年。尽管在转录控制中具有保守性和重要性，但目前尚不清楚compass样复合物如何在发育过程中启动和维持增强子活性。这个项目的重点是保守的PHD指结构域在染色质识别和依赖于精确增强子控制的关键细胞信号通路中的作用。利用多用途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集群PHD结构域有助于基本的表观遗传组蛋白读取器功能，这些功能驱动基因增强子的正确启动和调控；(2)果蝇compass样共激活子复合体调节增强子利用的时间，以整合关键的发育信号。该项目的一个目的是探索保守PHD集群中手指结构域的组合功能，这些功能域对于适当的增强子调节至关重要。PHD指结构域和靶向诱变的结构建模研究，结合体外和体内染色质关联的测量，将定义Cmi和哺乳动物PHD指结构域的组蛋白识别和结合特性，并进一步阐明compass样复合物控制增强子的机制。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，帮助将特定增强子表观遗传标记与compass样复杂功能联系起来，并为染色质调控的保守机制提供重要的新见解。

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.