PROJECT SUMMARY Background: Class switch recombination (CSR) and somatic hypermutation (SHM) are two B lymphocyte specific processes that mediate antibody diversification. Activation Induced Cytidine Deaminase (AID) is essential for initiating both of these processes by deaminating cytidine residues in immunoglobulin (Ig) loci DNA. Despite its specific and indispensible function in the Ig loci, AID has been demonstrated to also deaminate non-Ig locus genes, with the concomitant negative outcome of catalyzing various oncogenic translocations that manifest in tumorigenesis. Recent work has indicated that AID's DNA deamination activity requires its association with transcriptionally stalled RNA polymerase II and the RNA exosome complex. RNA exosome is a cellular non-coding RNA (ncRNA) processing and/or degradation macromolecular complex. How ncRNA and RNA exosome facilitate AID's DNA deamination activity is a question we plan to address. Objective/Hypothesis: We will determine how RNA exosome activity on ncRNAs support AID activity at the IgH locus and stabilizes 3-dimensional organization of the B cell genome. Specific aims: AIM 1: To understand the chromatin associated function of noncoding RNAs and RNA exosome in controlling AID activity. AIM 2: To understand the role of CTCF binding element (cbe) ncRNAs in TADIgH organization. AIM 3: To understand the role of novel RNA exosome cofactors in regulating AID activity. Study Design: Using mouse models that are deficient in RNA exosome-mediated RNA degradation, we have identified regions in the B cell genome that express exosome sensitive ncRNAs and also are mutated by AID. We will evaluate the mechanism of formation of single-strand DNA structures following localized chromatin remodeling at these identified AID target DNA sequences. We have discovered a new family of exosome sensitive ncRNAs that are associated with CTCF-binding elements (CBE), defined as cbeRNAs. We will evaluate how cbeRNA processing regulates the 3 dimensional topologically associating domain structure of IgH and influences SHM and CSR efficiencies. Finally, using B cells from TAP-tagged Exosome expressing mouse model, we have purified and identified chromatin associated RNA exosome co-factors. We will evaluate the role of RNA exosome co-factor(s) in stimulating AID/RNA exosome complex function. Disease Relevance: The proposed studies will lead to a better understanding of the mechanisms initiating AID dependent oncogenesis in B lymphocytes (particularly in the contexts of DLBCL and multiple myeloma) as well as have direct implications in understanding B lymphocyte-based immunodeficiency syndromes like Hyper-IgM syndrome type 2. RNA exosome is implicated in many diseases including numerous neurodegenerative disorders and multiple myeloma and findings from this grant will lead to a better understanding of the etiologies of the disorders and development of therapies.