PROJECT SUMMARY The goal of this proposal is to investigate the role of ubiquitin-dependent protein degradation in initiating and maintaining X-chromosome inactivation (XCI), which produces the inactive X chromosome (Xi) to equalize the dose of X-linked genes between male and female mammals. XCI is necessary for proper development and depends on the long non-coding (lnc) RNA Xist, which implements silencing early in development. During initiation of XCI, Xist spreads in cis from its site of transcription on the X-chromosome to cover the entire chromosome, recruits repressive chromatin regulators, induces gene silencing, leads to the depletion of active chromatin and transcriptional regulators, and mediates chromosome-wide compaction. The molecular mechanisms underlying all these changes on the Xi during initiation of XCI and how they contribute to XCI maintenance are still poorly understood. Interestingly, studies of Xist deletion support a model where Xist promotes removal of active transcriptional and chromatin regulators from the Xi and continued removal is important to prevent reactivation of genes on the Xi. The mechanism by which Xist carries out this function is not understood. We recently discovered that a subunit of the anaphase-promoting complex/cyclosome (APC/C) accumulates on the Xi and that this localization depends on Xist. APC/C is a multi-subunit ubiquitin ligase that catalyzes the attachment of ubiquitin to target proteins to trigger degradation by the proteasome. APC/C functions in various cellular processes including regulating progression through different phases of the cell cycle and also has cell cycle-independent functions. Based on our preliminary findings, we hypothesize that Xist recruits APC/C to the Xi where it ubiquitinates regulators of active chromatin and transcriptional regulators, such as chromatin modifying enzymes or transcription factors, to promote their degradation by the ubiquitin- proteasome system. Here, we will test this model by defining the localization of APC/C and components of the ubiquitin-proteasome system on the Xi, and by examining how blocking APC/C and the ubiquitin-proteasome system affects the Xi. This work is a multi-PI application combining the expertise of two labs: Kathrin Plath in XCI and genomics approaches, and Lars Dreier in cell biological studies of the ubiquitin-proteasome system, allowing us to effectively integrate cell biological and genomic approaches. Our complementary strengths, together with promising preliminary data and expert support, position us well to explore a role for ubiquitin- dependent protein degradation on the Xi. The proposed experiments will provide a starting point for defining critical proteins that need to be continuously degraded to prevent a transcriptionally permissive environment on the Xi. Our work will also provide novel insights into the regulation of chromatin states by lncRNAs and may link the function of two fundamental processes occurring in the cell.