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

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The genetic code of every living organism is contained within chromosomes, referred to collectively as the genome. In all eukaryotes, the genome is compacted into the nucleus through the formation of condensed molecules known as chromatin. The smallest individual units of compaction are known as nucleosomes that are comprised of 147 base pairs of DNA wrapped around an octamer of small basic histone proteins. In order for regulatory factors and enzymes to access the genetic code and properly regulate processes essential for normal development and cell survival, the nucleosomes need to be moved aside or directly modified on specific amino acid residues in a process termed chromatin remodeling. The enzymes and other proteins that carry out chromatin remodeling are ancient and remarkably conserved, with increasing complexity from single celled organisms up through vertebrates. The overall impact of this project will be a better understanding of how chromatin remodeling controls expression of the information that is encoded within the genome. In addition, high school, undergraduate and graduate students will participate in mentored research. Women and minorities will be highly represented. Multidisciplinary training and education are vital to prepare for diverse science careers and this project employs the unique tools currently available for Drosophila studies. Students will be trained in molecular and developmental genetics, biochemistry, molecular biology and bioinformatics, structural and developmental biology. The project will enhance interactions among faculty scientists within the institution and with students at multiple undergraduate institutions to explore the rapidly emerging field of bioinformatics. Undergraduate students will be tasked with learning new informatics tools and approaches using experimental data derived from this project. In order to define the histone recognition and binding properties of Cmi and related mammalian domains and elucidate the potential mechanism of COMPASS-like complex targeting to gene enhancers, collaborative structural studies that include X-ray crystallography and nuclear magnetic resonance of the histone recognition domains and targeted mutagenesis combined with in vitro measurements of binding affinities and in vivo chromatin association will be employed. The project also uses next-generation high throughput chromatin and RNA analyses (ChIP-Seq, RNA-Seq) and target gene studies. ChIP-Seq using chromatin from cmi mutant animals will enable correlation of epigenetic marks with Cmi function and align the RNA-Seq gene expression data with chromatin binding to address key developmental functions. Tissue-specific targeted removal of both cmi and trr and overexpression of cmi using unique genetic tools developed for these studies will allow for direct testing of COMPASS-like functions on target genes. Leading edge chromatin technologies that examine physical connections between distant gene regions will elucidate novel regulatory roles for the Drosophila COMPASS-like complex in facilitating enhancer-promoter communication necessary for proper gene control. These studies will help to reveal essential and foundational properties of chromatin remodeling and modifying complexes and provide critical insight into the mechanisms of developmental gene regulation.