As they develop, B cells undergo a formidable series of genetic alterations seen nowhere else in the organism. Confined to the immunoglobulin (Ig) loci, these mutations confer to B lymphocytes the ability to drastically increase the diversity of the antibodies they produce (V(D)J recombination), to generate different effector function options (CSR), and to synthesize immunoglobulins with increased affinity for the pathogens they aim to neutralize (SHM). CSR and SHM cannot proceed without the catalytic action of AID. AID deaminates cytidine residues of its DNA substrates. This deamination results in the presence of a uridine residues, eliciting a variety of cellular responses to the dU:dG mismatch. These cellular responses help entrench not only the mutations that are initiated by AID in the variable regions of Ig heavy and light chain loci (in the case of SHM), but also the AID-dependent double-strand breaks (DSB) in the constant region of the Ig heavy chain locus (for CSR). Many of the proteins involved in the cellular responses to AID deamination of cytosine residues are usually found in the context of replication, during which they help the cell rid itself of the varius mistakes that can arise in this setting. Our understanding of the mechanisms that co-opt these factors for the purposes of antibody diversification is still lacking however. In this respect, we propose in our first aim that proliferating cell nuclear antigen (PCNA), a DNA clamp also usually found in the context of replication, helps AID recruit these other factors, thereby helping entrench mutations and DSB at sites of AID activity. To test this, we will evaluate AID-dependent diversification events (e.g. CSR) in primary cells that express mutant AID which has lost the ability to interact with PCNA. AID is a single-stranded DNA deaminase that nonetheless accesses both the template and the non-template strands of its substrates in vivo. We know that the RNA exosome, a multi-subunit complex involved in RNA processing and degradation, plays a crucial role in stimulating AID activity at its substrates, and in enabling AID to access both th non-template and the template strands of its Ig substrates. Yet, the mechanisms responsible for AID being targeted to its physiologic substrates are still unknown. Given the important role played by the RNA exosome in supporting AID activity, we want to explore how the RNA exosome influences AID targeting activity to its physiologic substrates in B cells: this will be ou second aim. AID-caused deamination events are concentrated in Ig loci. Accordingly, these deaminations result in Ig-locus restricted recombinations (CSR) and mutations (SHM). Dysregulation of AID mutagenic activity can therefore have catastrophic consequences on genomic stability. Indeed, signature translocations of hematological malignancies have been linked to AID (e.g., c-myc/IgH in Burkitt's lymphoma). Using mouse primary B cells deficient in RNA exosome, we will evaluate genomic instability by molecular cytogenetic techniques and translocation quantitative assays.

Public Health Relevance

AID dysregulation has been implicated in immune deficiency and lymphomas, the most common blood malignancy in the developed world. However, the regulation of AID mutagenic activity is poorly known. There is evidence that PCNA and the RNA exosome are important for the proper progression of AID-initiated events. Full understanding of the roles played by these two factors would constitute crucial progress in the quest for novel therapies against diseases of the immune system.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1-F07-E (20))
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Adger-Johnson, Diane S
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Columbia University (N.Y.)
Internal Medicine/Medicine
Schools of Medicine
New York
United States
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Pefanis, Evangelos; Wang, Jiguang; Rothschild, Gerson et al. (2015) RNA exosome-regulated long non-coding RNA transcription controls super-enhancer activity. Cell 161:774-89
Sun, Jianbo; Keim, Celia D; Wang, Jiguang et al. (2013) E3-ubiquitin ligase Nedd4 determines the fate of AID-associated RNA polymerase II in B cells. Genes Dev 27:1821-33
Keim, Celia; Kazadi, David; Rothschild, Gerson et al. (2013) Regulation of AID, the B-cell genome mutator. Genes Dev 27:1-17