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

2023 Fiscal Year Research-status Report

• Project/Area Number: 22KF0405
• Allocation Type: Multi-year Fund
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: 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)
• 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
• Project Period (FY): 2023-03-08 – 2025-03-31
• Keywords: transcription / electron microscopy / epigenomics

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).

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.

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.

Causes of Carryover

ChromEMT is a new and complicated methodology. Modifying the existing protocol to accommodate targeted labeling required optimization at multiple steps including testing different labeling, permeabilization, gold enhancement, and photo oxidation.
In FY2023, we took a careful, steady approach to solving these challenges. Although our timelines for experiments and spending were delayed, we successfully developed a working protocol optimized for signal-to-noise. We are now ready for full scale sample preparation and data acquisition. The carryover budget is needed to cover associated costs.