Stage Master

During the course of immune responses B cells diversify their immunoglobulin (Ig) genes by somatic hypermutation (SHM) and class switch recombination (CSR). These reactions establish highly specific and adapted humoral responses by increasing antibody affinity and by allowing the expression of a different antibody isotype with unique immunological functions. SHM diversifies the variable region Immunoglobulin heavy (IgH) and light (IgL) chain genes, producing families of related clones bearing mutated receptors that are positively selected on the basis of antigen binding affinity. CSR diversifies the B cell repertoire by combining a single heavy chain variable region with a different constant region, switching the antibody isotype expressed (from IgM to IgG, IgE or IgA) while retaining the antigen specificity of the receptor.

These reactions are initiated by Activation Induced Cytidine Deaminase (AID), an enzyme that deaminates cytosines in DNA. AID-induced lesions are recognized by the DNA repair machinery and are processed in different ways to trigger mutations or double stranded DNA break intermediates during CSR. AID has the potential to induce significant collateral DNA damage and has been implicated in the generation of B cell malignancies. At present in is not understood how AID is specifically targeted to immunoglobulin genes or how collateral genomic damage is restricted.

The project's goal is to characterize the functional role in CSR of a selected gene that we have identified through a genome-wide CRISPR/Cas9 screen coupled to a BioID screen looking for AID partner proteins. The specific objectives are: 1) Generate knockout clones using CRISPR/Cas9 in CH12 cells, a B cell line that undergoes CSR from IgM to IgA very efficiently in vitro. 2) Determine whether CSR is affected following gene inactivation and pinpoint the mechanistic role of the selected gene.