This is an application for an exploratory grant to build on our preliminary results suggesting that the C terminus of activation-induced cytidine deaminase (AID) is important for the recombination step during antibody class switch recombination (CSR). It has been known for several years that the C terminal 10 amino acids of AID are very important for CSR, although they do not appear to have any role during somatic hypermutation (SHM) of antibody genes, a process also dependent upon AID. Also, the AID C terminus is important for preventing chromosomal translocations between the IgH and c-myc loci. We have obtained novel results indicating that in splenic B cells induced to undergo CSR, AID binds to Ig switch (S) regions cooperatively with other enzymes involved in introducing DNA breaks into S regions, specifically UNG and Msh2-Msh6, and that this binding is dependent upon the AID C terminus. Using retroviral over-expression in aid-/- mouse splenic B cells of AID, we detect AID binding to S? and S?? in chromatin immunoprecipitation (ChIP) assays, whereas C terminal deleted AID (?AID) does not ChIP with S region DNA. Likewise, both UNG and Msh2-Msh6 are also detected by ChIP at S? in aid-/- cells expressing ?AID, but they are not detected in cells expressing AID, suggesting that the binding of these repair proteins depends on the AID C terminus, and the binding of AID and these proteins might be cooperative, i.e. co-dependent. Consistent with the hypothesis that these proteins bind cooperatively to S? DNA, in ung-/-aid-/- B cells or in msh2-/-aid-/- B cells, transduced full lengt AID cannot be detected at S? by ChIP. These results suggest that in order for AID and UNG and Msh2-Msh6 to bind sufficiently stably to be detected by ChIP at S regions, they must bind DNA cooperatively with each other, and this cooperative binding depends on the AID C terminus. We propose to test the hypotheses (1) that the AID C terminus recruits UNG and Msh2-Msh6 to S regions via an intermediary protein, and (2) that the C terminus of AID is important for recruiting UNG and Msh2-Msh6 to S regions during the G1 phase of the cell cycle, and (3) that the C terminus of AID is important for steps subsequent to formation of DNA breaks that direct CSR towards non-homologous end-joining (NHEJ). In order to test these hypotheses we propose 3 Specific Aims: 1) To investigate the mechanism of interaction between AID and UNG and Msh2-Msh6. 2) To determine if the recruitment of UNG and Msh2-Msh6 by AID is important for creating DSBs in S regions during G1 phase and also for their repair during G1 phase. In normal splenic B cells, AID-dependent S? DSBs are restricted to the G1 phase. However, in other cell types, UNG and Msh2- Msh6 are recruited by the DNA replication complex to DNA during S phase. 3) To determine if the AID C terminus is important for recruiting enzymes involved in NHEJ to S regions and thereby directing CSR toward NHEJ.
This project investigates the function of the enzyme (AID) that initiates antibody class switching, which is required for generation of an effective antibody immune response. This enzyme initiates formation of breaks in DNA which when everything goes well leads to an effective immune response. However, AID can cause collateral damage, which leads to chromosomal deletions and translocations and to B cell lymphomas, the most common type of cancer. We will investigate how AID is regulated to introduce DNA breaks that lead to proper antibody class switching.
|Kadungure, Tatenda; Ucher, Anna J; Linehan, Erin K et al. (2015) Individual substitution mutations in the AID C terminus that ablate IgH class switch recombination. PLoS One 10:e0134397|
|Stavnezer, Janet; Schrader, Carol E (2014) IgH chain class switch recombination: mechanism and regulation. J Immunol 193:5370-8|
|Khair, Lyne; Guikema, Jeroen E J; Linehan, Erin K et al. (2014) ATM increases activation-induced cytidine deaminase activity at downstream S regions during class-switch recombination. J Immunol 192:4887-96|
|Ucher, Anna J; Ranjit, Sanjay; Kadungure, Tatenda et al. (2014) Mismatch repair proteins and AID activity are required for the dominant negative function of C-terminally deleted AID in class switching. J Immunol 193:1440-50|
|Vuong, Bao Q; Herrick-Reynolds, Kayleigh; Vaidyanathan, Bharat et al. (2013) A DNA break- and phosphorylation-dependent positive feedback loop promotes immunoglobulin class-switch recombination. Nat Immunol 14:1183-9|