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Small molecules that delay S phase suppress a zebrafish bmyb mutant

Nature Chemical Biology volume 1, pages 366–370 (2005)Cite this article

Abstract

Bmyb is a ubiquitously expressed transcription factor involved in cellular proliferation and cancer1,2,3,4. Loss of bmyb function in the zebrafish mutant crash&burn (crb) results in decreased cyclin B1 expression, mitotic arrest and genome instability5. These phenotypic observations in crb mutants could be attributed to the decreased expression of cyclin B1, a cell-cycle regulatory protein that is responsible for driving cell progression from G2 through mitosis. To identify small molecules that interact with the bmyb pathway, we developed an embryo-based suppressor screening strategy. In 16 weeks we screened a diverse ∼16,000 compound library, and discovered one previously unknown compound, persynthamide (psy, 1), that suppressed bmyb-dependent mitotic defects. Psy- treated embryos showed an S-phase delay, and knockdown of the cell-cycle checkpoint regulator ataxia telangiectasia—and Rad-related kinase (ATR) abrogated the suppression of crb. The DNA synthesis inhibitors aphidicolin (2) and hydroxyurea (3) also suppressed crb. S-phase inhibition upregulated cyclin B1 mRNA, promoting the progression of cells through mitosis. Our study demonstrates that chemical suppressor screening in zebrafish can identify compounds with cell-cycle activity and can be used to identify pathways that interact with specific cell-cycle phenotypes.

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Figure 1: Persynthamide (psy) suppresses mitotic accumulation in crb mutants.
Figure 2: Psy suppresses many aspects of the crb phenotype.
Figure 3: Psy suppresses crb by delaying cells in S phase.
Figure 4: crb is suppressed by activation of the intra–S-phase checkpoint.

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Acknowledgements

We would like to thank D. Hayes, J. Follen and C. Shamu for help with small-molecule screening, T. Mitchison for helpful advice, and C. Burns, J. DeCaprio, N. Dyson, I. Hariharan and W. Kaelin for critical comments on the manuscript. We also thank R. Finley for help with in situ hybridization, R. Wingert for providing reagents and C. Belair and B. Barut for expert laboratory management. This work was supported by NIH grants 5K08 DK061849 (H.M.S.), 1R01 DK55381 (L.I.Z.), 1R01 HD044930 (L.I.Z.) and 5K08 HL04082 (J.F.A.). L.I.Z. is an Investigator of the Howard Hughes Medical Institute.

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Author notes

  1. Howard M Stern and Ryan D Murphey: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pathology, Brigham and Women's Hospital, 75 Francis Street, Boston, 02115, Massachusetts, USA

    Howard M Stern

  2. Stem Cell Program and Division of Hematology and Oncology, Children's Hospital, Dana-Farber Cancer Institute, Howard Hughes Medical Institute and Harvard Medical School, 1 Blackfan Circle, Boston, 02115, Massachusetts, USA

    Howard M Stern, Ryan D Murphey, Jennifer L Shepard, James F Amatruda, Christian T Straub, Kathleen L Pfaff, Gerhard Weber & Leonard I Zon

  3. Institute of Chemistry and Cell Biology, Harvard Medical School, 200 Longwood Avenue, Boston, 02115, Massachusetts, USA

    John A Tallarico & Randall W King

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Correspondence to Leonard I Zon.

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Supplementary information

Supplementary Fig. 1

Schematic representation of the screen. (PDF 946 kb)

Supplementary Fig. 2

Confirmation of chemical composition of persynthamide. (PDF 289 kb)

Supplementary Fig. 3

Persynthamide is required during a specific developmental time window to suppress crb. (PDF 129 kb)

Supplementary Fig. 4

Counts of spindles and centrosomes in tails of psy-treated and untreated embryos. (PDF 737 kb)

Supplementary Fig. 5

Persynthamide-, aphidocolin- and hydroxyurea- treated embryos have similar gene expression profiles. (PDF 914 kb)

Supplementary Fig. 6

Cyclin B1 levels are upregulated by S-phase inhibitors. (PDF 592 kb)

Supplementary Fig. 7

Model of how S-phase delay via the intra-S-phase checkpoint may suppress the bmyb loss of function mutant crb by increasing cyclin B1 message. (PDF 1028 kb)

Supplementary Methods (DOC 37 kb)

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Stern, H., Murphey, R., Shepard, J. et al. Small molecules that delay S phase suppress a zebrafish bmyb mutant. Nat Chem Biol 1, 366–370 (2005). https://doi.org/10.1038/nchembio749

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