| 研究課題/領域番号 |
15K15154
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| 研究機関 | 大阪大学 |
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研究代表者 |
Jun Huang 大阪大学, 免疫学フロンティア研究センター, 特任研究員(常勤) (00751207)
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| 研究期間 (年度) |
2015-04-01 – 2018-03-31
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| キーワード | Self tolerance / Autoimmunity / Epigenetic regulation / Treg development |
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| 研究実績の概要 |
We identified that a gene (thereafter called X), one component of a protein complex implicated in multiple epigenetic mechanisms, plays a crucial role in thymic regulatory T cell (tTreg) development. By performing function assays both in vitro and vivo and analyzing the mice with Treg lineage-specific X depletion, we demonstrated that the role of X in Tregs is developmentally restrictive, as X deletion in differentiated Tregs has no effects on Tregs function nor their stability. By further characterization of tTreg development in the absence of X, we demonstrated that X is crucial for thymic CDSP cells to differentiate into Treg precursors, but dispensable for later steps of tTreg development.
In order to understand the underlying mechanism of our discovery, we carried out several experiments and had gained several crucial insights: 1) X contributes to Treg development in a cell-intrinsic manner rather than through affecting other T cells, as demonstrated by bone marrow co-transfer experiment. 2) By bisulfite sequencing, we demonstrated that tTregs developed in the absence of X display enhanced Foxp3 CNS2 demethylation. Thus X does not contribute to tTreg development via promoting demethylation of Treg signature genes.3) we got several lines of evidences from global transcriptome analysis and flow cytometric analysis suggesting that X might contribute to tTreg (precursors) development through modulate T-Cell Receptor (TCR) Signaling.
Results so far set the stage for further comprehensive studies to fully elucidate mechanistic role of X during Treg development.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
We had narrowed down candidate genes for further functional interrogation and obtained CRISPR/dCas9 lentiviral activation particles targeting those genes, which are ready for bone marrow cells transduction.
The order and delivery of experimental mice were slightly delayed.
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| 今後の研究の推進方策 |
In particular, we observed that several genes significantly downregulated in the absence of X, but it is still not clear whether these genes functionally mediate the downstream effects of X. Next, we will apply lentivirus-based CRISPR/Cas9 system, including CRISPR/dCas9 lentiviral activation system which we did not envisage its application previously, to functionally interrogate the role of these genes in the process of Treg development.
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| 次年度使用額が生じた理由 |
The order and delivery of laboratory mice were slightly delayed.
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| 次年度使用額の使用計画 |
Incurring amount will be mainly used for the purchase of laboratory mice.
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