| Project/Area Number |
22KF0405
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| Project/Area Number (Other) |
22F22072 (2022)
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| Research Category |
Grant-in-Aid for JSPS Fellows
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| Allocation Type | Multi-year Fund (2023)
Single-year Grants (2022) |
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| Section | 外国 |
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| Review Section |
Basic Section 43050:Genome biology-related
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
石川 文彦 (2023) 国立研究開発法人理化学研究所, 生命医科学研究センター, チームリーダー (30403918)
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| Co-Investigator(Kenkyū-buntansha) |
LIANG MINGGAO 国立研究開発法人理化学研究所, 生命医科学研究センター, 外国人特別研究員
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| Host Researcher |
石川 文彦 (2022) 国立研究開発法人理化学研究所, 生命医科学研究センター, チームリーダー (30403918)
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| Foreign Research Fellow |
LIANG MINGGAO 国立研究開発法人理化学研究所, 生命医科学研究センター, 外国人特別研究員
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| Project Period (FY) |
2023-03-08 – 2025-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2024: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 2023: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2022: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | transcription / electron microscopy / epigenomics / non-coding / electronmicroscopy |
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| Outline of Research at the Start |
To understand gene dysregulation in acute myeloid leukemia, we complement existing sequencing approaches with chromatin electron microscopy to directly visualize chromatin architecture at high resolution, using RNA probes to landmark specific genomic sites in the EM images.
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| Outline of Annual Research Achievements |
We successfully established the chromEMT protocol at RIKEN for cultured adherent cells (MCF10A), including EM-fixation, photo-oxidation and staining of DNA with DRAQ5 + OsO4, resin embedding, and ultrathin sectioning. We next developed a modified chromEMT protocol to incorporate immunolabeling of specific genomic landmarks with fluorescent nanogold probes for visualization by light microscopy and EM.
We have acquired TEM images of labeled samples that demonstrate successful labeling of single targets, including lncRNA (XIST), histone modifications (H3K27Ac), and RNA polymerases (RNAPII-S2 and POL1). In addition, we have preliminary data (fluorescent microscopy) supporting on-target labeling of additional targets (H3K27me3, H3K4me3, RNAPII-S5).
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
We have developed a modified chromEMT protocol to label genomic landmarks for EM imaging. Preliminary data from conventional TEM imaging confirm quality, specificity, and good signal-to-noise of labeling. We are now preparing samples with labeling for regulatory genome landmarks (H3K27me3, H3K27Ac, and RNAPII-S2) for tilt-axis EM imaging and to construct 3D tomograms. These will form the primary dataset for our study, after which we will be ready for analysis and manuscript writing.
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| Strategy for Future Research Activity |
We have revised the original goal of imaging of patient-specific regulatory genome mutations due to time constraints. We will instead aim to complete the project with comprehensive labeling of different classes (active, repressed, and transcribed) of regulatory elements in the MCF10A system where our assay is established and working. Final sample preparation will be completed by the end of May. Tilt-axis imaging will be done in early June at OIST. 3D tomography, chromatin structure modeling, and manuscript writing will be performed thereafter.
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