Research
Team Leader Charles Boone, Ph.D.
Small molecular ligands with unique activities must have the specific target molecules that exist in their cells or organisms. Identification of the target molecules is critical for elucidating the mode of action of the molecular ligands and for drug development. However, drug target identification has been difficult in general, because the mode of interactions between the molecular ligands and their targets is not uniform. Our team aims at developing innovative techniques based on global analysis of yeast genetic interaction and physical interaction using mass spectrometry, which leads to quick and accurate detection of the ligand-target interactions. Furthermore, novel assay systems for mode-of-action studies based on protein post-translational modifications and epigenetics will be established.
Research Projects
1. Global analysis of genetic and physical interaction between molecular ligands and their target molecules
2. Mode of action of bioactive compounds
3. Novel assay systems for mode-of-action studies based on protein modifications and epigenetics
Specific Research Areas
1. Construction of a molecular barcoded yeast ORF (MoBY-ORF) library that enables mode-of-action analysis of bioactive compounds
We construct a yeast chemical-genomics system designed to identify genes that when mutated confer drug resistance, thereby providing insight about the modes of action of compounds. We developed a molecular barcoded yeast open reading frame (MoBY-ORF) library in which each gene, controlled by its native promoter and terminator, is cloned into a centromere-based vector along with two unique oligonucleotide barcodes. The MoBY-ORF resource has numerous genetic and chemical-genetic applications, but we focus on cloning wild-type versions of mutant drug-resistance genes using a complementation strategy and on simultaneously assaying the fitness of all transformants with barcode microarrays (Fig. 1). The complementation cloning was validated by mutation detection using whole-genome yeast tiling microarrays, which identified unique polymorphisms associated with a drug-resistant mutant. We used the MoBY-ORF library to identify the genetic basis of several drug-resistant mutants and in this analysis discovered a new class of sterol-binding compounds.
Fig. 1. Construction (A) and application of the MoBY-ORF library (B): (i) Isolate a drug-resistant mutant. (ii) Transform mutant with MoBY-ORF library. (iii) Grow the pool of transformants in the absence and presence of a drug. The wild-type B allele complements the drug-resistant allele (iv) PCR amplify and fluorescently label the barcode sequences. (v) Hybridize to microarray. (vi) Identify the transformant most depleted in the drug treated pool.
2. Identification of molecular targets for theonellamide F by chemical genomic approach
Taking advantage of the fission yeast ORFeome strain collections and the omics datasets, we generated chemical genomic profiles for many bioactive compounds. To identify the mode-of-action of theonellamide F (TNM-F), marine natural product, in fission yeast, we constructed a chemical genomic profile of this compound and compared with reference profiles and existing dataset, suggesting a link between TNM-F and β-glucan synthesis. Indeed, TNM-F induced overproduction of 1,3-β-D-glucan in a Rho1-dependent manner. Furthermore, binding assay using a fluorescent derivative of TNM-F revealed that TNM-F represents a sterol-binding molecule that induces Rho1-mediated overproduction of 1,3-β-D-glucan.
3. Identification of transcription factors involved in epigenetic regulation
Methylation of histones and its effect on transcription is maintained for long period over cell division. For screening compounds that affect this epigenetic regulation, it is important to identify the transcription factors involved. We have found that transcription factor ATF-7 is involved in epigenetic regulation. ATF-7 binds to the transcriptional regulatory region of serotonin receptor 5b (Htr5b) gene, and silences this gene via recruiting histone H3K9 trimethylase ESET. In response to social isolation stress, ATF-7 is phosphorylated by p38, and is released from Htr5b, leading to upregulation and abnormal behaviors of mice. This study indicates that ATF-7 is a good molecular target for the compounds that modulate epigenetic regulation. We have established the screening systems for the compounds that bind to ATF-7.
Members
Charles Boone, Ph.D. (Team Leader)
Shunsuke Ishii, Ph.D. (Deputy Team Leader, concurrently Chief Scientist of Molecular Genetics Laboratory)
Jeffrey Piotrowski, Ph.D. (Foreign Postdoctoral Researcher)
Akihiro Ito, Ph.D. (concurrently Senior Research Scientist of Chemical Genetics Laboratory)
Yoko Yashiroda, Ph.D. (concurrently Senior Research Scientist of Chemical Genetics Laboratory)
Teruaki Nomura, Ph.D. (concurrently Senior Research Scientist of Molecular Genetics Laboratory)
Publications
Arita, Y., Nishimura, S., Matsuyama, A., Yashiroda, Y., Usui, T., Boone, C., and Yoshida, M.: "Microarray-based target identification using drug hypersensitive fission yeast expressing ORFeome." Mol. BioSyst., 7: 1463-1472, 2011. PMID: 21340088
Ito, T., Umehara, T., Sasaki, K., Nakamura, Y., Nishino, N., Terada, T., Shirouzu, M., Padmanabhan, B., Yokoyama, S., Ito, A., and Yoshida, M.: "Real-time imaging of histone H4K12-specific acetylation determines the modes of action of histone deacetylase and bromodomain inhibitors." Chem. Biol., 18: 495-507, 2011. PMID: 21513886
Ho, CH., Piotrowski, J., Dixon, SJ., Baryshnikova, A., Costanzo, M., and Boone, C.: "Combining functional genomics and chemical biology to identify targets of bioactive compounds." Curr. Opin. Chem. Biol., 15: 66-78, 2011. PMID: 21093351
Nishimura, S., Arita, Y., Honda, M., Iwamoto, K., Matsuyama, A., Shirai, A., Kawasaki, H., Kakeya, H., Kobayashi, T., Matsunaga, S., and Yoshida, M.: "Marine antifungal theonellamides target 3β-hydroxysterol to activate Rho1 signaling." Nat. Chem. Biol., 6: 519-526, 2010. PMID: 20543850
Maekawa, T., Jin, W., and Ishii, S.: "The role of ATF-2 family transcription factors in adipocyte differentiation: antiobesity effects of p38 inhibitors." Mol. Cell. Biol., 30: 613-625, 2010. PMID: 19948881
Maekawa, T., Kim, S., Nakai, D., Makino, C, Takagi, T., Ogura, H., Yamada, K., Chatton, B., and Ishii, S:. "Social isolation stress induces ATF-7 phosphorylation and impairs silencing of the 5-HT 5B receptor gene." EMBO J., 29: 196-208, 2010. PMID: 19893493
Sasaki, K., Ito, T., Nishino, N., Khochbin, S., and Yoshida, M.: "Real-time imaging of histone H4 hyperacetylation in living cells." Proc. Natl. Acad. Sci. USA, 106: 16257-16262, 2009. PMID: 19805290
Ho, CH., Magtanong, L., Barker, SL., Gresham, D., Nishimura, S., Natarajan, P., Koh, JL., Porter, J., Gray, CA., Andersen, RJ., Giaever, G., Nislow, C., Andrews, B., Botstein, D., Graham, TR., Yoshida, M., and Boone, C.: "A molecular barcoded yeast ORF library enables mode-of-action analysis of bioactive compounds." Nat. Biotechnol., 27: 369-377, 2009. PMID: 19349972
Shirai, A., Matsuyama, A., Yashiroda, Y., Hashimoto, A., Kawamura, Y., Arai, R., Komatsu, Y., Horinouchi, S., and Yoshida, M.: "Global analysis of gel mobility of proteins and its use in target identification." J. Biol. Chem., 283: 10745-10752, 2008. PMID: 18292091
Yashiroda, Y., Matsuyama, A., and Yoshida, M.: "New insights into chemical biology from ORFeome libraries." Curr. Opin. Chem. Biol., 12: 55-59, 2008. PMID: 18282487
Synthesis Team
