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. Regulation of X chromosome gene expression in the nematode C. elegans is an excellent model to decipher the molecular mechanisms that regulate chromatin structure, nuclear architecture and gene expression. In this organism, a protein complex called condensin binds both X chromosomes of XX hermaphrodites to downregulate gene expression two-fold thereby equalizing X-linked gene expression between the sexes. Condensin is best known for its function in mitotic chromosome compaction, but C. elegans dosage compensation provides us with an opportunity to uncover its interphase roles. Condensin affects X chromosome structure on multiple levels, including posttranslational modifications of histone, alterations in chromosome topology and compaction, and tethering the chromosome to the nuclear periphery. These functions require cooperation with other cellular machinery, such as histone modifiers and proteins of the nuclear lamina. Recent evidence indicates that condensin also cooperates with the nuclear RNAi machinery to remodel the X chromosome and repress its genes. The proposed research will examine the interactions between condensin, nuclear RNAi, histone modifiers, and the nuclear lamina, and define how these processes cooperate to establish and then maintain repression of the X chromosomes. State-of-the art superresolution microscopy methods will be combined with genomic methods and bioinformatics to identify chromosome structure changes that are causally involved in gene repression. Using precise genome editing techniques, the relative contributions of condensin binding and the other repressive mechanisms will be uncovered. Condensin mutations have recently been reported in patients with microcephaly and various cancers. Therefore, findings from this project may become directly relevant to human health.