Biochemical reconstitution of antigen receptor gene regulation

National Institute On Aging

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Abstract

During FY12 we accomplished the following: 1. Studies of the role of H3K4me3 in the initiation of V(D)J recombination were hampered by varying effects of exogenously added H3K4me3 peptide for RAG1/2-induced DNA cleavage. Working with our collaborator, Dr. Stephen Desiderio (JHUSOM), we determined that this was due to endogenous peptides co-purifying with full-length RAG2 protein. By appropriately altering the purification procedure, Dr. Desiderios laboratory produced RAG 1/2 proteins that were more sensitive to H3K4me3. We are currently using these preparations to re-visit the allosteric role of H3K4me3 in the RAG cleavage reaction using specifically modified mononucleosomes. 2. We initiated and have almost completed a new set of biochemical experiments in collaboration with Dr. Patricia Gearhart (NIA/IRP). We carried out in vitro transcription studies using nuclear extracts from a human B lymphoma cell line and various different VH genes sub-cloned into plasmids. Using kinetic and pulse/chase studies we found that RNA polymerase II stalled within each VH gene segment studied; in contrast, no stalling was observed within the CD19 gene. By exchanging promoter and coding sequences between VH and CD19, we determined that VH coding sequences were responsible for this phenomenon. We are now adding back purified activation induced cytidine deaminase (AID) protein to see if D-loops caused by stalled polymerase creates substrates for AID activity.