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 imunoglobulin (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 4kb DNA fragment (now named 3′RR) in the IgL locus containing both a transcriptional enhancer and cis-acting targeting elements for SHM and GCV. Using a systematic deletion approach we continued our efforts to identify the minimal cis-acting DNA element required for targeting. We are now focusing on a 1.5kb region within the 3′RR that is necessary for GCV and SHM of the IgL gene in DT40 cells. In addition, we are trying to identify the respective elements in the mouse Ig loci using a complementation approach. As the binding sites for transcriptional enhancers and targeting factors are likely to be overlapping/intermingled, we are currently also dissecting the transcriptional control elements in the chicken IgL locus using luciferase-based reporter constructs. We identified at least one previously unknown enhancer element that is important (but not essential) for transcription of the IgL gene in DT40 cells. A complementary approach towards understanding the mechanism of AID targeting is the characterization of AID-interacting proteins. AID does not contain a sequence-specific DNA binding domain and hence depends on adaptors proteins that act in recruiting AID to the Ig loci via the cis-acting targeting elements described above. We are in the process of generating DT40 cells deficient in 25 individual candidate factors and are planning to analyze their phenotype in the next fiscal years. 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 |
