2012 Theses Doctoral

Post Translational Regulation of AID Targeting to Both Strands of a Transcribed DNA Substrate

Keim, Celia D.

Activation induced Cytidine Deaminase (AID) contributes to the generation of antibody affinity by participating in two reactions, class switch recombination (CSR) and somatic hypermutation (SHM). Both reactions occur after VDJ recombination, subsequent to antigen exposure. During CSR, a deletion and recombination event occur to alter the effector function from IgM to either IgG, IgE, or IgA. SHM then occurs, which introduces point mutations at a high frequency into the variable regions of both the immunoglobulin heavy and light chains. These point mutations increase the antibody binding affinity for antigen, and antibodies with greatest affinity for antigen will be positively selected and further expanded during an immune response. The ability of AID to act as a mutator gene underscores the importance of understanding its regulation throughout the genome. Action of AID on genes outside of the Ig loci can lead to genomic instability. Hyperactivity of AID has been shown to cause chromosomal translocations and other oncogenic malignancies. Loss of AID can lead to immunodeficiencies. Therefore, it is imperative to understand how AID identifies and interacts with target sequences and mutates both strands of the DNA. Previous studies have identified DNA secondary structure such as R loops, transcription factors, miRNA, and phosphorylation as events important for determining AID's ability to access its substrate sequences. However, none of these studies demonstrated how AID mutates both strands of DNA, reminiscent to its in vivo mode of action.The focus of this thesis is to identify how AID mutates both strands of the DNA duplex, and how target genes are identified. To this end, we have discovered that AID functionally interacts with the cellular non-coding RNA degradation complex, RNA exosome. We observe that the RNA exosome stimulates AID activity on both strands of DNA in in vitro reconstituted reactions. The RNA exosome/AID complex binds to switch (S) sequences in a manner that is both transcription- and AID-dependent. Knockdown of exosome core component ExoSc3 results in defects in CSR. Additionally, this work focuses on the role of the neddylation (Nedd8) of AID in recruitment to its target sequences. Neddylation, a 10kDa modifier, is a small ubiquitin like modifier which functions in a variety of cellular processes. We have used a combination of proteomics, computational approaches, and candidate screening to identify and validate the role of E1, E2 and E3 in CSR. We have identified NEDD4 as the AID-specific E3 Neddylation ligase and demonstrated its requirement for CSR in mouse B cells. Using mass spectrometry, we have identified AID neddylation sites from in vitro neddylated AID proteins. We observe that mutation of these AID-neddylation sites affects AID/RNA exosome interaction and CSR efficiency in B cells. These observations point towards a role of NEDD4 in recruiting AID/RNA exosome complex to the immunoglobulin locus. Additionally, we confirm the role of NEDD4 as an E3 ubiquitin ligase of RNA polymerase. In both cell lines and primary cells, we observe an increase of germline transcripts and S region resident RNA polymerase in the absence of NEDD4. We propose NEDD4 ubiquitination can promote the degradation of stalled RNA polymerase complexes at the Ig S region, facilitating exosome access to germline transcripts and AID access to the template strand.