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 generated several DT40 cell lines in which we deleted the two known regulatory elements within the Ig light chain (IgL) locus, a putative matrix attachment region and the enhancer, individually and in combination. Neither of these elements is absolutely essential for SHM and GCV, but we identified an additional transcriptional control element downstream of the known enhancer that seems to be critical for transcription of the IgL locus. The role of this element in SHM and GCV is the topic of ongoing studies. We also continued our search for the DNA element that prevents the spreading of mutagenic activity onto neighboring genes, and identified an 800bp element that might contain a such a protective boundary element. To gain insight in the underlying molecular mechanisms of both aspects, we started to analyze changes of the chromatin state of the locus that occur when defined cis-acting elements within the locus are missing. These studies will reveal further insight into how histone modifications and DNA methylation are involved in the regulation and control of transcription and SHM/GCV. 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 |
