| Project/Area Number |
21K06029
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Multi-year Fund |
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| Section | 一般 |
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| Review Section |
Basic Section 43010:Molecular biology-related
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
DE・HOON MICHIEL 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 (70525617)
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| Co-Investigator(Kenkyū-buntansha) |
蓑田 亜希子 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 (40721569)
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| Project Period (FY) |
2021-04-01 – 2024-03-31
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| Project Status |
Granted (Fiscal Year 2022)
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| Budget Amount *help |
¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2023: ¥520,000 (Direct Cost: ¥400,000、Indirect Cost: ¥120,000)
Fiscal Year 2022: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2021: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
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| Keywords | Tomography / Electron microscope / Chromatin structure / In-situ hybridization / Non-coding RNA / XIST / Chromatin 3D structure / Chromatin conformation / Electron microscopy / THP-1 Differentiation / Imaging / Hi-C |
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| Outline of Research at the Start |
Physical interactions between genes and genomic control regions underlie gene regulation that determines cell type and cell state. We use electron microscopy to visualize those interactions at specific control regions at nanometer-scale resolution to understand the mechanism of gene regulation.
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| Outline of Annual Research Achievements |
During this year, we have performed further ChromEMT experiments following by electron microscope (EM) imaging by 3D tomography. This year, in collaboration with Okinawa Institute of Science and Technology, we obtained high-resolution images. The images confirmed that the ChromEMT protocol is working correctly, though the contrast observed in the images still needs some improvement.
Our next aim is to label specific RNA molecules for identification in the EM images. We use a primary unmodified probe against a specific RNA, a secondary probe with an attached biotin moiety and a fluorescent label for confirmation by light microscopy, a tertiary probe consisting of an anti-biotin antibody with a nanogold bead attached, and a quaternary probe consisting of an antibodywith an attached fluorophore at a different color. As only the primary probe (which is relatively cheap) is specific to the RNA, this setup allows us to minimize costs when targeting different RNAs. We are using XIST as a target for protocol development. Overlapping signals from the secondary and quaternary probe in the light microscope confirmed that our probes are working. An earlier attempt using streptavidin failed, as it was unable to enter the nucleus. In contrast, the 1.4 nm nanogold bead on the tertiary probe can enter the nucleus. For visualization in EM, we use gold enhancement to increase the size of the gold bead. While EM tomography confirmed that the probes are able to enter the nucleus, we currently still have a strong background signal with spurious binding of the tertiary probe inside the nucleus.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
We have now successfully acquired high-resolution images in electron microscope tomography, including the use of fiducial beads to be able to reconstruct the 3D structure of chromatin from the images. On the other hand, the contrast of chromatin in the images is lower than in the published protocol. Further optimization of the protocol for our particular cell types may still be needed, which will require some additional time..
We have been successful in overcoming obstacles in the development of RNA hybridization probes for electron microscopy. Based on the evidence from light microscopy, it seems that these probes are working correctly. However, we absolutely need to reduce the background signal to be able to confirm that we are targeting XIST. Reducing the background signal may take some time. It may be as simple as increasing the number of washing steps, or using a different blocking method in the protocol.
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| Strategy for Future Research Activity |
The essential point is to ensure that our RNA hybridization probes are binding to the target RNA at an acceptable background level. If these two issues can be resolved quickly, we can move on to applying this protocol to a different cell type, such as THP-1 as in our original research proposal. In any case, just the introduction of in-situ RNA hybridization methods in chromatin electron microscope tomography would be a very important result of this project and will be appreciated as such by the scientific community.
In addition to labeling specific RNA molecules, we will also attempt to label promoters and enhancers using antibodies against the corresponding histone modifications.
Until now, we have performed the basic image data processing using the IMOD software package. In this fiscal year, we will use the Sculptor and ChimeraX software packages for downstream analysis of the 3D chromatin structure, in particular the positioning of nucleosome particles in it by fitting the known crystallographic structure of the nucleosome at atomic resolution to the EM image.
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