| 研究実績の概要 |
In order to produce high affinity antibody, Immunoglobulin (Ig) gene locus of mature B cells undergo Somatic Hyper Mutation (SHM) and Class Switch Recombination (CSR) upon antigen challenge. Expression of Activation-Induced Cytidine Deaminase (AID) is essential to initiate SHM and CSR by a complex DNA break and recombination mechanism, which is poorly understood. During this time aberrant genomic damage also takes place in the chromosomal loci other than IgH, causing oncogenic chromosomal translocations. We aim to know how AID exerts such a function and how a non-IgH locus become the target of AID induced DNA damage.
(A) AID-AID interaction analysis previously revealed that AID forms monomer as well as dimer, and interacts with specific RNA binding proteins (RBPs) for DNA break and recombination, respectively. One of the recombination specific RBP co-factor (hnRNPL) was subjected to investigation in depth to understand the molecular basis of its interaction with AID and its function.
(B) The second goal was the target locus specific recombination regulator identification. Initial characterization of the two putative factors suggest that they are involved in DNA repair regulation in CSR. Interestingly, one of the factors appears to be affecting the NHEJ via regulating the chromatin dynamics, whereas other factor seems to beaffecting the NHEJ by regulating the DNA end processing.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
As we obtained interesting data on a RBP co-factor that contributes to recombination function of AID, it is important to know the RNA substrates that specifically associate to the protein. We hypothesize that the individual AID-RNP complex associated RNA species play an important role in regulating the locus specific recombination. We are conducting various RNA IPs for high throughput sequence analysis.
In the second part, due to the high background, we could not get the expected result using locus specific TALE binder as initially planned. Therefore, we changed the strategy to different DNA binding modules such as Gal4-DBD and LexA-DBD. Multi copy Gal4 and LexA motifs have been inserted upstream of donor and acceptor switch regions, respectively, in the IgH locus. Site-specific protein pull down will be examined using the pure clones that are currently under selection.
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| 今後の研究の推進方策 |
AID has two functions in antibody gene diversification, DNA break and recombination. The two functions are regulated at various level, and distinctly. AID’s own structural transition (monomer/dimer) associates with distinct co-factors. We think different RNA species are bound to specific AID-RNP complex, which needs to be understood. We made progress on isolating and analyzing the function of such RNA binding co-factors, but we need considerable effort on understanding the specific RNA substrates bound and their contribution in recombination.
Currently, chromatin regulation behind CSR versus aberrant genomic rearrangement in B cells are not well understood. We identified 2 new chromatin factors whose knockdown strongly blocked IgH/cMyc translocation, suggesting yet unknown regulatory pathway that promotes AID induced genomic instability in B cell. Interestingly, these factors affected SHM differentially, which suggests SHM, CSR and translocation pathways can be regulated uniquely by various cellular and locus specific regulators. Therefore, it is important to investigate further to find out the exact mechanism involved in the regulation of beneficial versus harmful genomic instability in B cell.
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