Integrated analysis of chromatin conformation by Hi-C and electron tomography

2021 Fiscal Year Research-status Report

• Project/Area Number: 21K06029
• Research Institution: Institute of Physical and Chemical Research
• 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)
• 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)
• Project Period (FY): 2021-04-01 – 2024-03-31
• Keywords: Tomography / Electron microscope / Chromatin 3D structure / In-situ hybridization / Non-coding RNA / XIST

Outline of Annual Research Achievements

We have performed the ChromEMT protocol in our laboratory using MCF-10A cells, which were used instead of THP-1 cells for convenience for now while establishing the ChromEMT protocol. This involves fixation with glutaraldehyde, staining with DRAQ5 and confirmation with light microscopy, performing the photo-oxidation reaction to induce diaminobenzidine polymerization near chromatin, and staining the diaminobenzidine polymers with osmium tetroxide. After that, the cells were embedded in resin, sectioned into thin slices, and imaged on a transmission electron microscope. Electron microscope tomography by taking images after tilting the sample through a succession of angles in the electron microscope has also been performed. The micrographs confirm that the chromatin was successfully stained with osmium tetroxide, making chromatin visible as dark spots in the electron microscope images.

Next, we performed in-situ hybridization in MCF-10A cells together with the ChromEMT protocol, using primary in-situ hybridization probes against XIST transcripts, and secondary probes carrying fluorescent labels to visualize the position of XIST transcripts in the light microscope. Imaging using the light microscope confirmed that in-situ hybridization of XIST in MCF-10A cells was successful. Whereas the background signal in the light microscope was fairly strong, this may be due to glutaraldehyde; as this will not be observable in the electron microscope, the background signal is unlikely to create a significant impediment to our project.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

We have established the ChromEMT protocol in our laboratory, and the electron microscope images confirm that the protocol performs as expected. While for reasons of convenience we are using MCF-10A cells for establishing the protocol instead of THP-1 cells, changing to THP-1 cells is straightforward. In our research proposal, we had planned to perform antibody labeling of histone modifications to identify promoters and enhancers in the first year, and in-situ hybridization of specific RNAs in the second year. Instead, we have decided to perform in-situ hybridization in the first year to ensure that we can identify specific genes in the electron microscope micrographs, as this is the more critical point for making practical use of chromatin electron microscope tomography in the future. Overall, the amount of progress is consistent with our original research plan in the project proposal.

Strategy for Future Research Activity

There are no significant changes to our research plan. We will continue with biotinylation of the primary in-situ hybridization probes, which will allow us to use streptavidin conjugated to fluorescein as a secondary probe. After confirming that we can observe the fluorescent labels indicating the position of XIST in the light microscope, we will repeat the experiment with Fluoronanogold secondary probes, which consist of streptavidin conjugated to both a fluorescent moiety and a gold bead and can therefore be observed in the light microscope as will as in the electron microscope. After confirming that these secondary probes work, we are ready to take our first high-resolution electron microscope tomography images and to reconstruct the 3D structure of chromatin, with the position of XIST therein, using the AMIRA software for image analysis. We will then proceed with antibodies against histone modifications to identify the position of promoters and enhancers, and again take high-resolution electron microscope tomography images. At that point, we will have established in our laboratory all experimental procedures proposed in our research plan. We will then apply the same protocol to THP-1 cells to generate the 3D chromatin structure data, with the position of selected messenger RNAs, promoters, and enhancers, and integrate the 3D chromatin structure with Hi-C chromatin conformation and other sequencing data in THP-1.

Causes of Carryover

Originally, our plan was to perform antibodies against histone modifications to identify promoters and enhancers in the first fiscal year, and to perform in-situ hybridization against specific RNAs in the second fiscal year. To ensure that we can identify specific genes in the electron microscope images, instead we performed in-situ hybridization in the first fiscal year, and will perform identification of promoters and enhancers using antibodies in the second fiscal year. As antibodies are expensive and constitute a major part of the total cost of this project, compared to in-situ hybridization probes, we ended up spending less budget in the first fiscal year, and we will spend more budget (on antibodies) in the second fiscal year.