Mechanisms Of Aid Regulation During Immunoglobulin Class Switch Recombination.
Reference : PhD Bernardo REINA-SAN-MARTIN
Offer publication : April 6, 2016
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 identify proteins that associate with AID, which potentially regulate its activity, and study their role in CSR. To do this we willuse the CH12 cell ine, a mouse lymphoma B in which CSR can be induced very efficiently in vitro and expressing AID at physiological levels. To identify AID partners, we will use the system CRISPR/Cas9 system to knockin the sequence encoding the biotin ligase mutant BirA* into AID's exon 1.
BirA* is capable of bioninylating proteins located at close proximity. The expression of BirA*-AID will allow us to purify biotinylated proteins (located in the vicinity of AID) using magnetic beads coupled to streptavidin and identify them by mass spectrometry. We propose to study the functional role in
isotype switching of the most promising candidates identified by generating knockouts via the CRISPR/Cas9 system. We will concentrate our efforts in studying the different stages of recombination (transcription switch regions, expression of AID, recruiting AID IgH locus, training and repair of double
strand breaks in DNA) by functional genomics approaches including ChIP-Seq-Seq and 4C, among others.
- WISHED SKILLS : Experience in molecular biology, biochemistry and tissue culture.
- EXPERTISES WHICH WILL BE ACQUIRED DURING THE TRAINING : Solid training in immunology, molecular biology, biochemistry and DNA repair.
Application Deadline : Dec. 31, 2016