| 研究課題/領域番号 |
24590352
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| 研究機関 | 京都大学 |
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研究代表者 |
BEGUM NasimAra 京都大学, 医学(系)研究科(研究院), 准教授 (80362507)
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| キーワード | AID / TOP1 / CSR / SHM / H3K4me3 / FACT / SPT6 / Genomic instability |
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| 研究概要 |
AID induces genomic instability at IgH locus during antibody gene diversification. It also induces aberrant genomic rearrangement responsible for oncogenic chromosomal translocations. Therefore, it is important to understand the underlying mechanism of AID induced genomic break and repair.
Study of histone chaperones, FACT and SPT6, revealed the importance of chromatin regulation in AID induced DNA break. Unlike FACT, SPT6 regulated the chromatin of both AID locus and the AID-target loci. Interestingly, H3K36me3 and H3K4me3 chromatin marks controlled the two regulations by SPT6, respectively. Highly mutated target regions also showed a strong correlation with FACT, histone variant H3.3 and H3K4me3 enrichment. We could publish these observations, and also successfully characterized the H3K4me3 enriched Top1-DNA cleavage complex isolated from AID activated cells.
Chromatin proteins and histone epigenetic marks are also important at the repair step of the recombination. We have isolated a novel adapter protein that supports CSR specific repair complex formation on the chromatin through acetylated histone bridging. The complex regulated the recruitment of UNG, a base excision repair enzyme whose deficiency leads to severe CSR block. UNG appears to regulate the recruitment of several key repair factors involved in CSR synapse formation and the DNA break end joining. We have recently published this novel phenomenon of UNG.
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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
Among the identified CSR associated novel chromatin regulators, two of them appeared to be working at the repair-recombination phase of CSR. Interestingly, we found that this repair complex contained UNG, a repair enzyme whose deficiency severely blocked CSR. As we have been exploring a non-canonical mode of action of UNG, this unexpected coincidence helped to understand the functional relevance and the mechanism of action of UNG and the novel chromatin protein identified.
We also made considerable progress on understanding AID induced DNA-cleavage complex formation. While FACT is required to generate H3K4me3 dependent DNA cleavage complex formation with Top1, another associated chromatin remodeling protein plays an important role in Top1 loading in the transcribed S-region. This dual mode of Top1 regulation in AID activated B cells is a very promising finding, especially in the context of DNA break events associated with CSR, SHM, and aberrant chromosomal rearrangements.
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| 今後の研究の推進方策 |
(1) Chromatin factors and features involved in AID induced DNA damage: We will complete the characterization of the H3K4me3-Top1-DNA cleavage complex in respect of function. Although MS analysis identified several chromatin factors, which are likely involved in AID induced DNA break and recombination processes, we plan to do all types of functional analyses for the two chromatin factors which may explain the functional significance of the H3K4me3 complex.
(2) Chromatin factors and features involved in AID induced recombination: We will mainly focus on the identified bromodomain chromatin adapter protein. To complete the work we need to do (i) extensive ChIP analysis of various CSR repair factors after knocking down the adapter protein or by inhibiting its interaction with the acetylated histones, and (ii) demonstration of the general DNA repair efficiency of the adapter protein such as radiation induced global DNA damage or meganuclease induced site-specific DNA cleavage.
(3) Development of DNA cleavage regulation assay and generation SPT6-CKO mice: We have generated S-region specific TALE binder proteins to examine their ability to interfere with the S-region repeat structure and DNA cleavage. As ChIP assay for the TALE-binders turned out to be very inefficient, we could not validate their binding specificity. Now, we are planning to examine the CSR regulation property by an alternative functional assay. We are still in the process of B cell specific SPT6-CKO mice generation for in vivo chromatin regulation study.
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