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 were previously able to show mutational enhancer elements (MEEs) mediate the targeting of of SHM/GCV to Ig gene loci, and identified two such MEEs in the chicken IGL locus. During the last fiscal year we studied the role of two evolutionarily conserved sequence motifs that have been identified within the 3'MEE. While one of them had no effect on mutation frequencies, scrambling the 2nd site led to a reproducible 25% decrease in mutations without a concomitant reduction in transcription. Studies to identify the corresponding trans-acting factor are ongoing. Furthermore, we initiated studies to determine whether MEEs act by (in)directly recruiting/activating AID or by altering faithful DNA repair. 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 |
