Establishing novel and unique technology to uncover how Activation-Induced Deaminase specifically targets immunoglobulin regions

Abstract

Abstract Activation-induced deaminase (AID) is the critical enzyme required for somatic hypermutation (SHM) and class switch recombination (CSR). Upon exposure to antigen, B cells become activated and express AID, initiating the process of affinity maturation. While we understand how AID is targeted to the switch region to initiate CSR, relatively little is known about why AID is targeted to the variable region to initiate SHM. The difficulty lies in the requirement to study SHM in vivo in a small population of germinal center B cells. To address this, we developed a knock-in mouse model called JH1, which utilizes a VDJ containing endogenous gene segments in the Variable (V) gene locus where upstream and downstream sequence isintact and intervening DNA is deleted. This unique model’s V region represents what is observed in endogenous B cells generated from the bone marrow. I have used and validated CUT&RUN on our low cell inputs, expanding possibilities for probing critical targets in precise populations of interest. Our lab and others have shown that SPT5 (a subunit of the RNA Polymerase II modulator, DSIF) is required for AID targeting and thus SHM. Unlike a typical gene, promoter-proximal pausing occurs up to 2kb downstream of the V promoter and is necessary for AID activity. We hypothesize distinctive regulation of RNA Polymerase II activity is what allows AID targeting and SHM. I am currently performing analysis of my generated CUT&RUN datasets for well-known transcriptional regulatory proteins to determine if there is a unique signature for SHM. Understanding how AID is targeted to the V region will help us to understand the elusive, yet indispensable process of SHM, which may be useful to fine-tune humoral responses via vaccines and other treatments. This work was supported entirely through the Intramural Research Program at the National Institutes of Health (NIH), National Institute on Aging (NIA).