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. Using a systematic deletion approach we determined that neither the only known enhancer, nor a matrix attachment region are critical for transcription and SHM/GCV in this cell line. We did, however, identify an as of yet uncharacterized 4kb DNA fragment in the IgL locus that contains both a transcriptional enhancer and cis-acting targeting elements for SHM and GCV. We named this part of the locus 3 regulatory region (3'RR). The identification of the minimal cis-acting DNA elements required for targeting AID-mediated Ig gene diversification and its functional characterization is the topic of ongoing studies. In addition, we are trying to move the targeting element to unrelated gene loci to determine whether it is sufficient to transfer mutability to non-Ig genes. In addition, we started to study the role of SIRT6 in SHM/GCV, as this protein is thought to control base excision repair (BER), the DNA repair pathway that is critical for both processes. We generated a number of reagents for these studies, including SIRT6 deficient DT40 cell lines and polyclonal antisera directed against chicken SIRT6. In contrast to our hypothesis, preliminary results suggest that SIRT6 is not essential for SHM/GCV, and that it might not even be critical for BER in 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.
| Kothapalli, Nagarama; Norton, Darrell D; Fugmann, Sebastian D (2008) Cutting edge: a cis-acting DNA element targets AID-mediated sequence diversification to the chicken Ig light chain gene locus. J Immunol 180:2019-23 |
| Gopal, Anjali R; Fugmann, Sebastian D (2008) AID-mediated diversification within the IgL locus of chicken DT40 cells is restricted to the transcribed IgL gene. Mol Immunol 45:2062-8 |
| Yang, Shu Yuan; Fugmann, Sebastian D; Gramlich, Hillary S et al. (2007) Activation-induced cytidine deaminase-mediated sequence diversification is transiently targeted to newly integrated DNA substrates. J Biol Chem 282:25308-13 |
| Fugmann, Sebastian D; Messier, Cynthia; Novack, Laura A et al. (2006) An ancient evolutionary origin of the Rag1/2 gene locus. Proc Natl Acad Sci U S A 103:3728-33 |
