2023 Fiscal Year Annual Research Report
Integrated analysis of chromatin conformation by Hi-C and electron tomography
| Project/Area Number |
21K06029
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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蓑田 亜希子 国立研究開発法人理化学研究所, 生命医科学研究センター, 客員主管研究員 (40721569) [Withdrawn]
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| Project Period (FY) |
2021-04-01 – 2024-03-31
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| Keywords | electron microscopy / tomography / 3D reconstruction / promoters / enhancers / histone modifications / in-situ hybridization |
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| Outline of Annual Research Achievements |
The aim of this project is to use electron microscopy (EM) to generate a 3D image of gene regulation. We successfully stained DNA using heavy metals to enhance its contrast in the EM. To reduce costs, for in-situ hybridization, instead of a biotinylated primary probe we used a primary probe specific to an RNA together with a biotinylated generic secondary probe. Also, instead of streptavidin, we used anti-biotin antibodies conjugated to nanogold beads, as they entered the nucleus more easily. We also used smaller nanogold beads to allow their entry into the nucleus, and applied gold enhancement to increase their size after entry into the nucleus for easy observation in the EM images. We confirmed that the protocul labels specific RNAs by applying it to XIST, for which we confirmed the nuclear localization to the inactive X chromosome by transmission electron microscopy.
As planned, we also labeled specific histone modifications to identify promoters, enhancers, and inactive chromatin, and optimized these procedures to reduce the background signal to acceptable levels. Next month, we will perform tomography by taking images of the generated samples at varying angles in an EM, and reconstructing the 3D model of gene regulatory systems from these images. We will then analyze and annotate these images, and summarize the results in a manuscript for publication. In a future project, we plan to combine electron microscopy with light microscopy to label specific genes by fluorescence in-situ hybridization and identify the genes associated with promoters seen in the EM image.
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