統合ゲノム解析と超微細胞構造イメージングによる癌ゲノム構造変化の理解

2022 Fiscal Year Annual Research Report

• Project/Area Number: 22F22072
• Allocation Type: Single-year Grants
• Research Institution: Institute of Physical and Chemical Research
• Host Researcher: 石川 文彦 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)
• 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
• Project Period (FY): 2022-07-27 – 2025-03-31
• Keywords: transcription / non-coding / electronmicroscopy

Outline of Annual Research Achievements

We developed a novel methodology to directly visualize chromatin and gene regulatory dynamics in situ. Our strategy involves 2 components:
1) the chromEMT protocol, which combines targeted staining of DNA with OsO4, tilt-axis electron microscopy imaging, and tomography to generate ultra-resolution 3D models of chromatin in-situ.
2) immunogold labeling of regulatory genome landmarks (polymerases, histone modifications, and specific RNAs) for visualization by EM.

Combining the two, our approach aims to directly visualize regulatory genome features (enhancers, promoters, transcriptional units) in the context of ultra-resolution chromatin imaging in-situ.

Current Status of Research Progress

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

Reason

We have spent the past 2 years developing a working protocol and overcoming two key challenges:
1) Establishing chromEMT at RIKEN, including sample prep, staining, sectioning, and imaging
2) Developing an in-situ labeling strategy compatible with chromEMT. For the latter, we used immuno-fluoronanogold particles that can easily enter fixed nuclei, bind specific targets, and can be chemically enhanced for visualization by EM.

We have successfully labeled various targets, including lncRNA (XIST), polymerases (POL1, RNAPII), and histone modifications (H3K27Ac) and have optimized our method for sensitivity and specificity. These are supported by preliminary 2D EM imaging and fluorescent imaging data. We are now ready to proceed with 3D tilt-axis imaging and tomography.

Strategy for Future Research Activity

Using the established protocol, we will proceed with tilt-axis imaging of labeled samples for tomography and 3D modeling.

Experiments will be performed in the MCF10A cell line where our assay is currently established and optimized. Labeling will be done for XIST, RNAPII-S5, and histone modifications to cover a variety of regulatory element classes. Imaging will be performed at OIST, where specialized TEM is available for tilt-axis imaging.

Sample preparation and imaging for the final dataset will be completed in 2 months time, and computational analysis will proceed immediately after. We will write a manuscript to report achievements for the program.

Remarks

We were able to make considerable progress over the past 2 years and develop a working protocol. Our findings have been well received at internal symposia and have sparked new collaborations.