Project Summary Homologous chromosome pairing, the physical matching up of homologous chromosomes, is a fundamental aspect of chromosome mechanics. In most organisms, pairing must occur only during meiosis to allow for recombination. Accidental pairing at other times can lead to numerous problems, including aneuploidy and many types of cancer. Understanding the mechanisms that control such somatic pairing will provide insight into fundamental aspects of chromosome biology. To study homologous chromosome pairing in somatic cells, we require a system where we can perform controlled manipulations of the state of pairing across the genome. Unlike most other organisms, Drosophila flies pair all of their somatic chromosomes all of the time; however, the offspring of one Drosophila melanogaster fly and one Drosophila simulans fly lose pairing in a small number of replicable regions of the genome. This phenomenon is an opening that we can exploit to understand somatic pairing broadly. Drosophila's wealth of genetic tools, presence of somatic pairing in normal flies, and aberrant pairing in hybrid flies provide a powerful system to understand the mechanistic basis of pairing. I plan to measure pairing in normal individuals and interspecies hybrids by using the Hi-C sequencing technique. This approach will provide the first genome-wide description of properly and aberrantly paired genome regions in interspecies hybrids. I will then use the D. melanogaster genome annotation to identify genome features such as genes and repeated DNA motifs that are overrepresented in non-pairing regions. DNA features that are consistently found where pairing is lost will provide insight into the factors that govern pairing. I will confirm these features by up- or down-regulating pairing-related genes, changing the rate of pairing across the chromosomes and replicating the results of the interspecies hybridization. Because pairing is known to influence gene expression through the phenomenon of transvection, I will use allele-specific expression to measure each gene's cis- and trans- regulation with and without pairing, demonstrating the effect of pairing on gene expression. Together, these experiments will characterize the mechanisms that underlie the fundamental process of chromosome pairing. This new knowledge will inform our understanding of pairing as it relates to cell function, disease, and evolution.