During this fiscal year we continued our studies to characterize the mechanisms that recruit and restrict the activation induced cytidine deaminase (AID) and its accompanying error-prone DNA repair machinery specifically to the immunoglobulin (Ig) genes. Our model system remains the DT40 cell line, a chicken B-cell line constantly undergoing somatic hypermutation (SHM) and Ig gene conversion (GCV), and showing the unique feature of being modifiable by standard gene targeting strategies. We previously identified a 1.5 kb DNA fragment within the 3′regulatory region of the IgL locus containing both a transcriptional enhancer and cis-acting targeting elements for SHM and GCV. Transient transfection based luciferase assay helped to identify a """"""""complete"""""""" enhancer region that is sufficient for driving normal levels of IgL transcription, but unable to promote SHM/GCV. This demonstrated for the first time that transcriptional enhancer elements and mutational enhancer elements are distinct from each other. In addition, we were able to localize evolutionary conserved elements critical for SHM/GCV within 450bp. Lastly, we have started our analysis of DT40 cells deficient in 25 individual putative AID interacting factors, and analyzed the phenotype of RNF126-/- deficient cells. Interestingly RNF126 is dispensable for GCV (and SHM) in DT40 cells, but clearly plays an important role in class switch recombination in murine B cells. Overall, our studies will provide a framework to explain the multiple levels at which the targeted introduction of mutations into Ig genes is controlled to protect the rest of the genome from potentially deleterious and cancer promoting alterations.
| Sen, Ranjan; Fugmann, Sebastian D (2012) Transcription, splicing, and release: are we there yet? Cell 150:241-3 |
| Kothapalli, Naga Rama; Fugmann, Sebastian D (2011) Targeting of AID-mediated sequence diversification to immunoglobulin genes. Curr Opin Immunol 23:184-9 |
| Kothapalli, Naga Rama; Norton, Darrell D; Fugmann, Sebastian D (2011) Classical Mus musculus Igýý enhancers support transcription but not high level somatic hypermutation from a V-lambda promoter in chicken DT40 cells. PLoS One 6:e18955 |
| Kothapalli, Nagarama; Fugmann, Sebastian D (2011) Characterizing somatic hypermutation and gene conversion in the chicken DT40 cell system. Methods Mol Biol 748:255-71 |
| Kothapalli, Naga Rama; Collura, Kaitlin M; Norton, Darrell D et al. (2011) Separation of mutational and transcriptional enhancers in Ig genes. J Immunol 187:3247-55 |
| Delker, Rebecca K; Fugmann, Sebastian D; Papavasiliou, F Nina (2009) A coming-of-age story: activation-induced cytidine deaminase turns 10. Nat Immunol 10:1147-53 |
