PROJECT SUMMARY Eukaryotic nuclei are crowded compartments that not only compact but also organize meters of DNA. This feat is achieved by wrapping DNA around histone proteins to form nucleosomes, the basic units of chromatin. Histone levels in the cell are precisely balanced: a lack of histone expression causes cell cycle arrest, whereas histone overexpression leads to genomic instability. Although histone gene regulation is crucial for proper cell viability and function, mechanisms that regulate histone gene expression are poorly defined. Histone genes are often clustered in metazoan genomes. In the genome of the excellent model system Drosophila melanogaster, a single cluster encompasses all the canonical histone genes. A suite of transcription and processing factors occupy the histone gene locus and form a conserved structure known as the histone locus body (HLB). Mutations in known HLB members lead to defects in HLB formation and/or histone gene expression, often causing animal lethality or infertility. We do not know all the factors that occupy the histone locus and contribute to histone gene regulation, and there are large gaps in our understanding of how HLB factors specifically target the histone gene locus. The overall goal of this proposal is to define the molecular interactions at the histone locus that specify histone genes for unique regulation. Recent studies identified the Drosophila protein CLAMP, which binds specifically to GA-repeat sequences within the histone3/histone4 promoter, opens chromatin across the histone locus, and promotes expression of all histone genes. However, it is unclear if CLAMP is required at the histone locus prior to the localization of HLB-specific factors, which would implicate CLAMP in specifying the locus for HLB formation. In Aim 1, I will define the DNA-protein interactions that lead to HLB formation and histone gene expression. I will take advantage of an established transgenic histone locus system to (A) probe the positional requirements of the CLAMP-GA-repeat interaction and (B) determine the placement of CLAMP in the developmental hierarchy of HLB formation. It is also critical that we define HLB composition, as this is an important step towards defining mechanisms of HLB formation and histone gene regulation. In Aim 2, I will discover novel HLB factors using both an unbiased proteomic screen and a candidate approach that involves mining existing -omics datasets. This candidate approach will also be the basis of a Course-Based Undergraduate Research Experience (CURE) module that I will develop and utilize in both my future independent laboratory and classroom. Collectively, these experiments will define mechanisms of HLB formation and histone gene expression while expanding my experimental repertoire and generating new directions for my future laboratory and classroom.