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

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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.