| 研究実績の概要 |
In response to antigenic challenge, activated B cells change the Immunoglobulin (Ig) gene locus to produce high affinity antibody. “Somatic Hyper Mutation (SHM)” and Ig “Class Switch Recombination (CSR)” are two key mechanisms involved. Activation-Induced Cytidine Deaminase (AID) initiates both SHM and CSR by a yet unclear DNA break-recombination mechanism, which mainly targets “Variable” and “Switch” regions of Ig locus. This event also causes aberrant genomic rearrangement between Ig and non-Ig locus such as IgH/cMyc translocation. Thus we aim to know how AID exerts its functions and how a genomic locus become the target of DNA damage upon AID activation in B cells.
a) In order to understand AID’s action and regulation, we performed AID-AID interaction analysis by Bimolecular Fluorescence Complementation Assay (BiFC). AID forms both monomer and dimer, and interacts with distinct group of RNA binding proteins. Analysis of SHM and CSR specific AID mutants suggests monomeric AID is probably involved in DNA break, whereas dimeric AID possibly form CSR specific complex.
b) In order to understand the target locus and associated chromatin regulators, we planned to to perform candidate gene screening by siRNA. Among the genes so far screened, knockdown of two of those showed CSR defect, 80% and 50%, respectively. However, in both the cases, there was a strong block of IgH/cMyc translocation, suggesting “Cis” and “Trans” recombination or genomic rearrangements induced by AID are differentially regulated.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
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理由
As explained above, we made reasonable progress and obtained promising primary data. However one of the big challenges remains is to pull down the target locus specific protein complexes involved in AID induced DNA break and recombination.
In the first approach, we are going to apply epitope tagged site-specific binder protein TALE (Transcription activator-like effectors). We have completed construction of two TALE binder proteins, which are expected to target upstream and down stream of donor “Switch” regions, respectively. It needs to be tested whether these molecules can pull down locus-specific proteins.
A second strategy of site-specific protein pull down is to apply recently developed iChIP or enChIP method. In order to achieve such a goal unique sequence motif(s) needs to be inserted at the vicinity of the target locus subjected to AID induced DNA damage. We selected two loci, one in the donor Sμ and the other in the cMyc locus. Currently, we are constructing donor constructs for homologous recombination (HR) and the target region specific CRISPR/Cas9 constructs, which will introduce nicks to initiate the HR. Successful HR, followed by single cell isolation will generate the desired B cell lines to perform enChIP in AID targeted IgH and non-IgH loci.
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| 今後の研究の推進方策 |
We identified two novel candidate genes that affected CSR. The encoded proteins were not previously known important for CSR or related to recombination. Therefore, it will be very exciting if we could characterize them at the context of antibody gene diversification. We plan to examine the effect of the identified factors on AID induced SHM and DNA break. And also verify whether they play a role in post break DNA repair/S-S synapse phase. Their relationship with SHM and CSR will be investigated in detail once we know whether they are working at break or repair step.
Currently, we do not have the full-view of the AID induced DNA/break-recombination complex that forms at IgH (variable or Switch regions) or any non-Ig locus. Characterization of such a complex and/or the individual components of the complex are of prime importance. Therefore, both TALE and enChIP strategies will be applied, followed by MS analysis of the target associated proteins.
Moreover, AID’s DNA break and recombination functions are differentially regulated. AID’s own monomeric versus dimeric structure as well as associated distinct co-factor are playing important role. How the RNA binding co-factors of AID influenced SHM and CSR differentially will be addressed in future. We think different RNA species are bound/presented to AID monomer and dimer via individual RNA binding co-factor, which needs to be clarified to understand the functional regulation of AID.
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