The recognition of pathogens by the adaptive immune system relies on a diverse antigen receptor repertoire on the surface of B and T lymphocytes. The genes encoding these receptors are assembled from individual gene segments by a process named V(D)J recombination. The key factors in this process are the products of the recombination activating genes 1 and 2. Studies during last fiscal year led to the identification of homologs of RAG1 and RAG2, named spRAG1L and spRAG2L respectively, in the genome of the purple sea urchin, strongylocentrotus purpuratus. This is unexpected as echinoderms lack a diversified antigen receptor repertoire and are devoid an adaptive immune system. Now we have evidence from pull-down experiments that spRAG1L and spRAG2L form a specific stable complex that closely resembles the vertebrate RAG1/RAG2 complex. We started to generate polyclonal antisera against both proteins to be able to study their expression pattern in developing and adult sea urchins. Our studies have major implications for the current model of how adaptive immunity evolved in jawed vertebrates, and will help to illuminate conserved features of how V(D)J recombination is tightly controlled to avoid potentially dangerous modifications of the genome. ? During an immune response, the immunoglobulins (Ig) genes are further diversified to improve the specificity of the encoded antibodies by two closely related mechanisms: somatic hypermutation (SHM) and immunoglobulin gene conversion (GCV). Both processes rely on the activation induced cytidine deaminase, AID. During this fiscal year we continued our studies to characterize the mechanisms that restrict AID-dependent sequence diversification specifically to the Ig genes. We generated several cell lines in which we deleted individual regulatory elements, and are in the process of analyzing their phenotype. We also extended our studies investigating the boundary elements that prevent spreading of SHM from the Ig genes to neighboring genes. One approach is the analysis of the chromatin modifications in the IgL locus in B-cell lines that constitutively undergo GCV and SHM. An analysis of these cells will shed a light on the mechanisms that protect the rest of the genome from potentially deleterious AID-mediated modifications.
| 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 |
