2017 Fiscal Year Research-status Report
Detecting combinatorial histone modification dynamics at single nucleosome resolution for key genes in Epithelial-Mesenchymal-Transition
| Project/Area Number |
17K18358
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
チャン レンチエン 国立研究開発法人理化学研究所, ライフサイエンス技術基盤研究センター, 研究員 (10792137)
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| Project Period (FY) |
2017-04-01 – 2019-03-31
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| Keywords | single-molecule imaging / epigenetics / chromatin / histone modification / ChIP-seq / CRISPR/Cas |
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| Outline of Annual Research Achievements |
Epigenome, including histone modifications and DNA methylation, represent the unique signature of a cell type. Epigenomic states have to be tightly regulated for the cell to function properly; therefore the combination of multiple modifications can form a code to reflect cell status and be informative in revealing abnormal cell states during diseases. However traditional bulk biochemical assays usually measure only the average states, making delineating heterogeneous epigenomes during a cell process like differentiation, development, or reprogramming difficult.
To solve the above problem, I have developed a TIRF microscipe and established an assay to simultaneously probe multiple histone modifications on cell extracted single nucleosome based on single-molecule imaging with fluorescent antibody. In this kakenhi project, I am working on extending this method to target specific genomic locus by combining sample preparation with engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) and applying the method to understand intermediate epigenetic states for key genes during Epithelial-Mesenchymal Transition (EMT) which is relevant in cancer metastasis.
In this fiscal year, progress has been made in several aspects toward the goal: (1) The imaging platform and analysis pipeline have been improved. (2) The protocols for single nucleosome preparation with enChIP are under active development. (3) Bulk ChIP-seq for EMT has been performed and bioinformatics analysis has been carried out to determine key genes in EMT suitable for study.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
All components essential for this project has been progressed smoothly:
(1) Previously I have set up a custom TIRF microscope in the lab and demonstrated its ability for single-molecule imaging. In this fiscal year, a Bioptech chamber suitable for microfluidics control has been integrated and tested. This part is important for multiple cycles of fluorescent antibodies exchange to achieve imaging more than three modifications.
(2) The protocols for preparing single nucleosome from target gene loci for imaging is under development. Custom Tn5 transposome was produced for one-step attachment of imaging oligo adapter to the nucleosome during enChIP step. Also, instead of the original enChIP using Active Motif enChIP kit, recently a simpler version of enChIP protocol is published (in vitro enChIP. T. Fujita and H. Fujii, BIO-PROTOCOL, 2017), which does not require establishing a stable cell line expressing dCAS9 and guide RNA. More importantly, it makes scaling up to work on multiple loci more feasible. We then contacted the lab (Fujii lab, Hirosaki University) and obtained assistance in adopting this protocol.
(3) Bulk ChIP-seq has been performed on MCF10A cell for reference and comparison. Six histone marks (H3K4me1, H3K4me3, H3K9me3, H3K27me3, H3K27ac, H3K9ac) were profiled for 0h and 24h after EMT induction. Bioinformatics analysis has been carried out to identify gene loci with chromatin states transition. Guide RNAs targeting selected loci were designed to investigate intermediate states at single molecule level using the imaging assay developing in the project.
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| Strategy for Future Research Activity |
In FY2018, protocol development for sample preparation will be continued and achieved. CDH1 promoter is targeted for proof-of-principle experiment. First, in vitro enChIP followed by DNA sequencing will be performed to verify the protocol on our hand. Enhancer elements in contact with CDH1 promoter are expected to be pulled down. Once successful, MNase treatment followed by imaging adapter ligation will be combined to label and isolate (during the IP step) imaging-compatible mononucleosome from specific gene loci. In parallel, custom Tn5 transposase will also be attempted for improved nucleosome recovery. Single-molecule imaging of the labelled nucleosome from CDH1 promoter for H3K4me3 and H3K27me3 for before (0h) and after (24h) EMT will demonstrate the validity and sensitivity of the technique.
After establishing the method, application to EMT key genes will be carried out to measure the intermediate chromatin states which cannot be directly investigated by other methods. Initial screening using bulk ChIP-seq data identified 10 E-genes and 25 M-genes with significant chromatin states transition during EMT, which are candidates for applying single-molecule imaging assay. If possible, two EMT system: fast transition by Zeb-1 overexpression (as our ChIP-seq data) and slow but more physiological transition by TGF beta will be compared. Four major histone marks (H3K4me1, H3K4me3, H3K27me3, H3K27ac) will be imaged. We expect to discover different types of transition states for different genes which may suggest different regulators and mechanisms.
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| Causes of Carryover |
As stated above, a newer protocol (in vitro enChIP) for nucleosome preparation was published in FY2017. Adapting this protocol can be beneficial (simpler and more scalable) to the project. We are developing this protocol so a large portion of FY2017 budget proposed to purchase original enChIP kit from Active Motif will be carried over to FY2018 for the application in EMT after the new method is established. In combination, the budget implementation plan for FY2018 are as follows.
* Consumables (1,800,000 yen): including (1) For sample preparation: MCF10A culture medium, Doxycycline and other culture reagent (PBS, trypsin, dishes, etc.), gRNA synthesis, FLAG antibogy, and reagent for mononucleosome prep, and (2) For imaging: PEG slides, Antibody (x5), Antibody labeling kit (x5), Biotin-dye-DNA oligo, bioptech gasket set.
* For Travel expenses (700,000 yen): including (1) One domestic conference and one international conference, and (2) Publication fee.
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Research Products
(3 results)
