Endogenous Gene Segments Research Articles

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

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).

Sequence-dependent dynamics of synthetic and endogenous RSSs in V(D)J recombination.

Developing lymphocytes of jawed vertebrates cleave and combine distinct gene segments to assemble antigen–receptor genes. This process called V(D)J recombination that involves the RAG recombinase binding and cutting recombination signal sequences (RSSs) composed of conserved heptamer and nonamer sequences flanking less well-conserved 12- or 23-bp spacers. Little quantitative information is known about the contributions of individual RSS positions over the course of the RAG–RSS interaction. We employ a single-molecule method known as tethered particle motion to track the formation, lifetime and cleavage of individual RAG–12RSS–23RSS paired complexes (PCs) for numerous synthetic and endogenous 12RSSs. We reveal that single-bp changes, including in the 12RSS spacer, can significantly and selectively alter PC formation or the probability of RAG-mediated cleavage in the PC. We find that some rarely used endogenous gene segments can be mapped directly to poor RAG binding on their adjacent 12RSSs. Finally, we find that while abrogating RSS nicking with Ca2+ leads to substantially shorter PC lifetimes, analysis of the complete lifetime distributions of any 12RSS even on this reduced system reveals that the process of exiting the PC involves unidentified molecular details whose involvement in RAG–RSS dynamics are crucial to quantitatively capture kinetics in V(D)J recombination.