PROJECT SUMMARY Chromatin structure and organization are critical to establishing developmentally appropriate gene expression programs for each cell type, and aberrations in these processes lead to developmental abnormalities and disease. Our research program’s long-term goal is to define how a specialized molecular machine called condensin can restructure chromosomes to regulate genes in interphase as well as to promote chromosome segregation in mitosis. Condensin mutations have been reported in patients with microcephaly and various cancers. Therefore, findings from this project will have direct relevance to human health. Condensin complexes are conserved from yeast to human, and while their roles in mitosis are relatively well defined, their function in interphase gene regulation is not well understood. Regulation of X chromosome-wide gene expression in C. elegans provides us with an opportunity to uncover condensin’s interphase roles. In this organism, in a process called sex chromosome dosage compensation, a specialized condensin complex binds both X chromosomes of XX hermaphrodites to downregulate gene expression by half thereby equalizing X-linked gene expression between the sexes. Condensin-mediated gene repression involves altering X chromosome structure on multiple levels, including posttranslational modifications of histones, looping of the chromatin fiber, chromosome compaction, and nuclear organization. The first major project will determine how mutations in conserved domains affect condensin’s interphase roles compared to its mitotic roles. The effect of the phosphorylation, which is known to modulate condensin’s activity in mitosis, will also be investigated. The second project centers on the roles of histone modifiers which cooperate with condensin in the process of dosage compensation. These histone modifiers perform additional functions in the germline. The project will characterize the dual functionality of this cellular machinery to reveal how the same activity can be co-opted to fulfill different biological functions in different tissues. The third project examines how developmental regulators can coordinate sex chromosome dosage compensation with developmental transitions. Defining the regulation of key dosage compensation proteins at two key transitions, loss of the pluripotent state and then terminal differentiation, will reveal the mechanistic link between these processes. Precise genome editing techniques and tissue and developmental stage-specific protein depletion techniques will be employed to reveal the mechanism of action and regulation of condensin in gene regulation. State-of-the art microscopy techniques will be combined with genomic methods and bioinformatics to identify chromosome and chromatin structure changes that are linked to gene repression.