Mechanisms of targeting in antibody diversification

Abstract

In mature B cells, the immunoglobulin heavy chain (IgH) locus is targeted for a variety of DNA transactions that allow the production of antibodies that can bind a wide variety of antigens such as bacteria and viruses, and allow for their disposal. These two capacities are brought about by somatic hypermutation (SHM), which introduces point mutations in the variable region, and class switch recombination (CSR) which replaces the IgM constant region with downstream isotypes such as IgG, IgA or IgE. An enzyme called activation induced cytidine deaminase (AID) initiates both processes by deamination of cytosines into uracils at the V region, or at the switch region, a segment of DNA that is upstream of every constant region and participates in the actual DNA recombination event.;How the various regions of the IgH gene are correctly targeted for these two highly mutagenic processes has been a longstanding question in immunology. The dysregulation or mistargeting of these mutagenic processes is thought to be responsible for mutations in oncogenes and chromosomal translocations in the IgH locus that result in B cell lymphomas.;One hypothesis is that histone modifications play a role in demarcating which regions of the IgH gene is targeted. The first part of my thesis project was to examine the chromatin modifications on switch regions during CSR using ex vivo splenic B cell cultures as a model system, and to determine if there were special histone modifications that predicted which switch regions would undergo CSR. I found two histone modifications that correlated with actively recombining switch regions: H3 acetyl K9 and H3 tri-methyl K9. The appearance of H3 tri-methyl K9 is a surprise because it is canonically thought to be present at silent genes and heterochromatin. Nevertheless, we believe that these modifications play a role in targeting of CSR based on the time course of appearance, the regions on the IgH gene they associate with, and their independence of AID's presence.;Since AID is an essential factor to both SHM and CSR, the ability to adequately track its location in the cell and on DNA is important. The second part of my thesis project was to generate antibodies against AID that are useful in applications such as IP and chromatin IP, and to determine AID's association with various regions of the IgH gene during SHM.