Mechanisms Of Genome Stability And Dna Recombination In B Cells Revealed By An Innovative Single-Locus Proteomics Approach Using The Crispr/Cas9 System.
Reference : PhD Bernardo REINA-SAN-MARTIN
Offer publication : April 6, 2016
Diversification of the B cell repertoire during immune responses is initiated by the targeting of Activation Induced Cytidine Deaminase (AID) to immunoglobulin (Ig) loci and by the subsequent generation of DNA breaks. The current model for AID targeting proposes that AID is recruited to promoter-proximal regions that are enriched in genes bearing chromatin modifications associated with active transcription and stalled/paused RNA polymerase II.
Although this model explains several features of somatic hypermutation (SHM) and class switch recombination (CSR), neither AID recruitment alone, nor transcription per se, appear to be sufficient for optimal AID activity. Indeed, a large subset of actively transcribed genes in activated B cells is not targeted by AID and not all loci targeted by AID are mutated.
In addition, although AID can be recruited to numerous non-Ig loci, these are mutated at much lower frequencies when compared to Ig genes. At present it is not understood how this differential outcome is controlled. The working hypothesis is that post-AID recruitment events are responsible for controlling the outcome of AID recruitment. To gain insight into these
events we propose to identify the proteins that are recruited to Ig and non-Ig loci after AID recruitment by making use of an innovative system we have developed in which we have coupled the CRSIPR/Cas9 and BioID technologies. The system is based on CH12 cells, a cell line that is capable of undergoing very efficient AID-dependent CSR in vitro, and that expresses the dead Cas9 (dCas9) fused to the promiscuous E. coli biotin ligase mutant (BirA*). This enzyme features proximity-dependent biotinylation of proteins that are near-neighbors of the targeting protein (dCas9). Expression of the BirA*-dCas9 fusion protein allows for the efficient isolation of chromatin-bound biotinylated proteins by using streptavidin-coupled beads and their
subsequent identification by mass spectrometry. The functional role of in CSR and DNA repair of selected proteins identified will be studied by generating knockout cellular models using the CRISPR/Cas9 system.
- 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