| 研究実績の概要 |
As described in the summary for FY2017, we previously obtained very interesting and promising results regarding the integration of transcriptomic (CAGE-seq and RNA-seq) and epigenomic (DNA methylation and histone modification) data, especially regarding the REP522 DNA repeat family. In FY2018, in collaboration with Dr. Kazuhide Watanabe and Dr. Harukazu Suzuki group from RIKEN IMS we performed a series of epigenetic perturbation in normal epithelial cells (MCF10A). We tested four conditions, where the cells were treated with: 1) DAC (DNA demethylating agent), 2) TSA (Histone Deacetylase inhibitor) , 3) DAC and TSA combination, and 4) DMSO control. The cells were then profiled by CAGE 5’start RNA sequencing to allow for promoter level gene expression analysis. Additionally, we performed DNA methylation profiling using Illumina EPIC DNA methylation array that covers ~830 thousand methylation sites across the genome. The integration of promoter level gene expression data and genome-wide DNA methylation profiles allows us to study how changes the epigenome are reflected in transcription and to better understand how disrupted epigenome of cancer cells impacts the gene regulation. The integrative analyses of the data sets are ongoing.
As described above, the experiment was performed in normal cells, in FY2019, we plan to perform the perturbation experiment in MCF7 cancer cell line, which will allow to study if cancer and normal cells respond differently to epigenetic drugs.
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| 今後の研究の推進方策 |
In the FY2019, following activities are planned:
1) performing the perturbation experiment with a demethylating agent in a cancer cell line.
2) performing single-cell gene expression profiling after the perturbation using single-cell implementation of CAGE (C1-CAGE).
Specifically, the budget will be used to cover cell culture and high-throughput sequencing costs for both bulk and single-cell sequencing. Single-cell sequencing will offer us an opportunity to study the heterogeneity: how individual cells respond to the demethylating drug. Additional follow-up and validation experiments may also be performed in FY2019 depending on the result from the above-mentioned experiments as was initially planned.
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| 次年度使用額が生じた理由 |
Postponing the experiments till FY2019 allowed more time for the analysis of data from the first/pilot experiment and to optimize the protocol for single cell experiment.
In the FY2019, following activities are planned: 1) performing the perturbation experiment with a demethylating agent in a cancer cell line. 2) performing single-cell gene expression profiling after the perturbation using single-cell implementation of CAGE (C1-CAGE). Specifically, the budget will be used to cover cell culture and high-throughput sequencing costs for both bulk and single-cell sequencing.
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