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Nucleolar release of Hand1 acts as a molecular switch to determine cell fate

Nature Cell Biology volume 9, pages 1131–1141 (2007)Cite this article

Abstract

The bHLH transcription factor Hand1 is essential for placentation and cardiac morphogenesis in the developing embryo. Here we implicate Hand1 as a molecular switch that determines whether a trophoblast stem cell continues to proliferate or commits to differentiation. We identify a novel interaction of Hand1 with a protein that contains an I-mfa (inhibitor of myogenic factor) domain that anchors Hand1 in the nucleolus where it negatively regulates Hand1 activity. In the trophoblast stem-cell line Rcho-1, nucleolar sequestration of Hand1 accompanies sustained cell proliferation and renewal, whereas release of Hand1 into the nucleus leads to its activation, thus committing cells to a differentiated giant-cell fate. Site-specific phosphorylation is required for nucleolar release of Hand1, for its dimerization and biological function, and this is mediated by the non-canonical polo-like kinase Plk4 (Sak). Sak is co-expressed in Rcho-1 cells, localizes to the nucleolus during G2 and phosphorylates Hand1 as a requirement for trophoblast stem-cell commitment to a giant-cell fate. This study defines a novel cellular mechanism for regulating Hand1 that is a crucial step in the stem-cell differentiation pathway.

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Figure 1: HICp40 specifically interacts with Hand1 and negatively regulates its transcriptional activity through nucleolar sequestration.
Figure 2: Nucleolar release of Hand1 coincides with Rcho-1 stem-cell commitment to a trophoblast giant-cell fate.
Figure 3: Hand1–EGFP-induced differentiation of Rcho-1 cells produces bona fide trophoblast giant cells, and nucleolar release of Hand1 takes place along with commitment to a giant-cell fate.
Figure 4: Phosphorylation of threonine (T107) and serine (S109) residues in helix 1 underlies Hand1 nucleolar release during Rcho-1 giant-cell differentiation.
Figure 5: The B56δ phosphatase subunit undergoes nucleolar/nuclear redistribution to the cytoplasm during Rcho-1 differentiation and inhibits both Hand1–EGFP- and horse-serum-induced differentiation.
Figure 6: The polo-like kinase Sak (Plk4) localizes to Rcho-1 nucleoli at G2/M and phosphorylates Hand1.
Figure 7: Figure 7 Sak-null embryos reveal an expanded diploid trophoblast population that significantly reduced secondary giant-cell differentiation and nucleolus-restricted Hand1 localization.

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References

  1. Rossant, J. & Cross, J. C. Placental development: lessons from mouse mutants. Nature Rev. Genet. 2, 538–548 (2001).

    Article  CAS  Google Scholar 

  2. Cross, J. C. et al. Trophoblast functions, angiogenesis and remodeling of the maternal vasculature in the placenta. Mol. Cell Endocrinol. 187, 207–212 (2002).

    Article  CAS  Google Scholar 

  3. Hughes, M. et al. The Hand1, Stra13 and Gcm1 transcription factors override FGF signaling to promote terminal differentiation of trophoblast stem cells. Dev. Biol. 271, 26–37 (2004).

    Article  CAS  Google Scholar 

  4. Cross, J. C. et al. Hxt encodes a basic helix-loop-helix transcription factor that regulates trophoblast cell development. Development 121, 2513–2523 (1995).

    CAS  PubMed  Google Scholar 

  5. Riley, P., Anson-Cartwright, L. & Cross, J. C. The Hand1 bHLH transcription factor is essential for placentation and cardiac morphogenesis. Nat. Genet. 18, 271–275 (1998).

    Article  CAS  Google Scholar 

  6. Firulli, A. B., McFadden, D. G., Lin, Q., Srivastava, D. & Olson, E. N. Heart and extra-embryonic mesodermal defects in mouse embryos lacking the bHLH transcription factor Hand1. Nature Genet. 18, 266–270 (1998).

    Article  CAS  Google Scholar 

  7. Sahgal, N., Canham, L. N., Konno, T., Wolfe, M. W. & Soares, M. J. Modulation of trophoblast stem cell and giant cell phenotypes: analyses using the Rcho-1 cell model. Differentiation 73, 452–462 (2005).

    Article  CAS  Google Scholar 

  8. Scott, I. C., Anson-Cartwright, L., Riley, P., Reda, D. & Cross, J. C. The HAND1 basic helix-loop-helix transcription factor regulates trophoblast differentiation via multiple mechanisms. Mol. Cell Biol. 20, 530–541 (2000).

    Article  CAS  Google Scholar 

  9. Firulli, B. A., Hadzic, D. B., McDaid, J. R. & Firulli, A. B. The basic helix-loop-helix transcription factors dHAND and eHAND exhibit dimerization characteristics that suggest complex regulation of function. J. Biol. Chem. 275, 33567–33573 (2000).

    Article  CAS  Google Scholar 

  10. Hollenberg, S. M., Sternglanz, R., Cheng, P. F. & Weintraub, H. Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system. Mol. Cell Biol. 15, 3813–3822 (1995).

    Article  CAS  Google Scholar 

  11. Thebault, S., Gachon, F., Lemasson, I., Devaux, C. & Mesnard, J. M. Molecular cloning of a novel human I-mfa domain-containing protein that differently regulates human T-cell leukemia virus type I and HIV-1 expression. J. Biol. Chem. 275, 4848–4857 (2000).

    Article  CAS  Google Scholar 

  12. Chen, C. M., Kraut, N., Groudine, M. & Weintraub, H. I-mf, a novel myogenic repressor, interacts with members of the MyoD family. Cell 86, 731–741 (1996).

    Article  CAS  Google Scholar 

  13. Kraut, N., Snider, L., Chen, C. M., Tapscott, S. J. & Groudine, M. Requirement of the mouse I-mfa gene for placental development and skeletal patterning. EMBO J. 17, 6276–6288 (1998).

    Article  CAS  Google Scholar 

  14. Gautier, V. W., Sheehy, N., Duffy, M., Hashimoto, K. & Hall, W. W. Direct interaction of the human I-mfa domain-containing protein, HIC, with HIV-1 Tat results in cytoplasmic sequestration and control of Tat activity. Proc. Natl Acad. Sci. USA 102, 16362–16367 (2005).

    Article  CAS  Google Scholar 

  15. Snider, L. et al. Inhibition of Tcf3 binding by I-mfa domain proteins. Mol. Cell Biol. 21, 1866–1873 (2001).

    Article  CAS  Google Scholar 

  16. Thebault, S., Basbous, J., Gay, B., Devaux, C. & Mesnard, J. M. Sequence requirement for the nucleolar localization of human I-mfa domain-containing protein (HIC p40). Eur. J. Cell Biol. 79, 834–838 (2000).

    Article  CAS  Google Scholar 

  17. Thebault, S. & Mesnard, J. M. How the sequestration of a protein interferes with its mechanism of action: example of a new family of proteins characterized by a particular cysteine-rich carboxy-terminal domain involved in gene expression regulation. Curr. Prot. Pept. Sci. 2, 155–167 (2001).

    Article  CAS  Google Scholar 

  18. Stark, L. A. & Dunlop, M. G. Nucleolar sequestration of RelA (p65) regulates NF-kappaB-driven transcription and apoptosis. Mol. Cell Biol. 25, 5985–6004 (2005).

    Article  CAS  Google Scholar 

  19. Weber, J. D., Taylor, L. J., Roussel, M. F., Sherr, C. J. & Bar-Sagi, D. Nucleolar Arf sequesters Mdm2 and activates p53. Nature Cell Biol. 1, 20–26 (1999).

    Article  CAS  Google Scholar 

  20. Tao, W. & Levine, A. J. P19(ARF) stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2. Proc. Natl Acad. Sci. US A 96, 6937–6941 (1999).

    Article  CAS  Google Scholar 

  21. Weber, J. D. et al. Cooperative signals governing ARF-mdm2 interaction and nucleolar localization of the complex. Mol. Cell Biol. 20, 2517–2528 (2000).

    Article  CAS  Google Scholar 

  22. Lohrum, M. A., Ashcroft, M., Kubbutat, M. H. & Vousden, K. H. Identification of a cryptic nucleolar-localization signal in MDM2. Nature Cell Biol. 2, 179–181 (2000).

    Article  CAS  Google Scholar 

  23. Datta, A. et al. Myc-ARF (alternate reading frame) interaction inhibits the functions of Myc. J. Biol. Chem. 279, 36698–36707 (2004).

    Article  CAS  Google Scholar 

  24. Sahgal, N., Canham, L. N., Canham, B. & Soares, M. J. Rcho-1 trophoblast stem cells: a model system for studying trophoblast cell differentiation. Methods Mol. Med. 121, 159–178 (2006).

    PubMed  Google Scholar 

  25. Hamlin, G. P., Lu, X. J., Roby, K. F. & Soares, M. J. Recapitulation of the pathway for trophoblast giant cell differentiation in vitro: stage-specific expression of members of the prolactin gene family. Endocrinology 134, 2390–2396 (1994).

    Article  CAS  Google Scholar 

  26. Faria, T. N. & Soares, M. J. Trophoblast cell differentiation: establishment, characterization, and modulation of a rat trophoblast cell line expressing members of the placental prolactin family. Endocrinology 129, 2895–2906 (1991).

    Article  CAS  Google Scholar 

  27. Parast, M. M., Aeder, S. & Sutherland, A. E. Trophoblast giant-cell differentiation involves changes in cytoskeleton and cell motility. Dev. Biol. 230, 43–60 (2001).

    Article  CAS  Google Scholar 

  28. Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A. & Rossant, J. Promotion of trophoblast stem cell proliferation by FGF4. Science 282, 2072–2075 (1998).

    Article  CAS  Google Scholar 

  29. Visintin, R., Stegmeier, F. & Amon, A. The role of the polo kinase Cdc5 in controlling Cdc14 localization. Mol. Biol. Cell 14, 4486–4498 (2003).

    Article  CAS  Google Scholar 

  30. Firulli, B. A. et al. PKA, PKC, and the protein phosphatase 2A influence HAND factor function: a mechanism for tissue-specific transcriptional regulation. Mol. Cell 12, 1225–1237 (2003).

    Article  CAS  Google Scholar 

  31. Huang, Z., Traugh, J. A. & Bishop, J. M. Negative control of the Myc protein by the stress-responsive kinase Pak2. Mol. Cell Biol. 24, 1582–1594 (2004).

    Article  CAS  Google Scholar 

  32. Centonze, V. E., Firulli, B. A. & Firulli, A. B. Fluorescence resonance energy transfer (FRET) as a method to calculate the dimerization strength of basic helix-loop-helix (bHLH) proteins. Biol. Proced. Online. 6, 78–82 (2004).

    Article  CAS  Google Scholar 

  33. Jaken, S. Protein kinase C isozymes and substrates. Curr. Opin. Cell Biol. 8, 168–173 (1996).

    Article  CAS  Google Scholar 

  34. Griffioen, G. & Thevelein, J. M. Molecular mechanisms controlling the localisation of protein kinase A. Curr. Genet. 41, 199–207 (2002).

    Article  CAS  Google Scholar 

  35. Andersen, J. S. et al. Nucleolar proteome dynamics. Nature 433, 77–83 (2005).

    Article  CAS  Google Scholar 

  36. Newton, A. C. Regulation of protein kinase C. Curr. Opin. Cell Biol. 9, 161–167 (1997).

    Article  CAS  Google Scholar 

  37. MacAuley, A., Cross, J. C. & Werb, Z. Reprogramming the cell cycle for endoreduplication in rodent trophoblast cells. Mol. Biol. Cell 9, 795–807 (1998).

    Article  CAS  Google Scholar 

  38. Fode, C., Motro, B., Yousefi, S., Heffernan, M. & Dennis, J. W. Sak, a murine protein-serine/threonine kinase that is related to the Drosophila polo kinase and involved in cell proliferation. Proc. Natl Acad. Sci. USA 91, 6388–6392 (1994).

    Article  CAS  Google Scholar 

  39. Hudson, J. W. et al. Late mitotic failure in mice lacking Sak, a polo-like kinase. Curr. Biol. 11, 441–446 (2001).

    Article  CAS  Google Scholar 

  40. Leung, G. C., Ho, C. S., Blasutig, I. M., Murphy, J. M. & Sicheri, F. Determination of the Plk4/Sak consensus phosphorylation motif using peptide spots arrays. FEBS Lett. 581, 77–83 (2007).

    Article  CAS  Google Scholar 

  41. Ma, G. T. & Linzer, D. I. GATA-2 restricts prolactin-like protein A expression to secondary trophoblast giant cells in the mouse. Biol. Reprod. 63, 570–574 (2000).

    Article  CAS  Google Scholar 

  42. Queralt, E., Lehane, C., Novak, B. & Uhlmann, F. Downregulation of PP2A(Cdc55) phosphatase by separase initiates mitotic exit in budding yeast. Cell 125, 719–732 (2006).

    Article  CAS  Google Scholar 

  43. Pederson, T. The plurifunctional nucleolus. Nucleic Acids Res. 26, 3871–3876 (1998).

    Article  CAS  Google Scholar 

  44. Hill, A. A. & Riley, P. R. Differential regulation of Hand1 homodimer and Hand1-E12 heterodimer activity by the cofactor FHL2. Mol. Cell Biol. 24, 9835–9847 (2004).

    Article  CAS  Google Scholar 

  45. Kumar, R., Conklin, D. S. & Mittal, V. High-throughput selection of effective RNAi probes for gene silencing. Genome Res. 13, 2333–2340 (2003).

    Article  CAS  Google Scholar 

  46. Kunath, T. et al. Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype. Nature Biotechnol. 21, 559–561 (2003).

    Article  CAS  Google Scholar 

  47. Kurki, S. et al. Nucleolar protein NPM interacts with HDM2 and protects tumor suppressor protein p53 from HDM2-mediated degradation. Cancer Cell 5, 465–475 (2004).

    Article  CAS  Google Scholar 

  48. Swallow, C. J., Ko, M. A., Siddiqui, N. U., Hudson, J. W. & Dennis, J. W. Sak/Plk4 and mitotic fidelity. Oncogene 24, 306–312 (2005).

    Article  CAS  Google Scholar 

  49. Moorman, A. F., Houweling, A. C., de Boer, P. A. & Christoffels, V. M. Sensitive nonradioactive detection of mRNA in tissue sections: novel application of the whole-mount in situ hybridization protocol. J. Histochem. Cytochem. 49, 1–8 (2001).

    Article  CAS  Google Scholar 

  50. Riley, P. R., Gertsenstein, M., Dawson, K. & Cross, J. C. Early exclusion of hand1-deficient cells from distinct regions of the left ventricular myocardium in chimeric mouse embryos. Dev. Biol. 227, 156–168 (2000).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Anthony Firulli, Vivek Mittal and Sabine Thebault, for generously providing plasmids, Jean-Michel Mesnard for the α-HIC antibody and Michael Soares and Satoshi Tanaka for generously providing the Rcho-1 and TS cell lines respectively. This research was supported by the British Heart Foundation.

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Authors and Affiliations

  1. Molecular Medicine Unit, UCL Institute of Child Health, London, WC1N 1EH, UK

    David M. J. Martindill, Catherine A. Risebro, Nicola Smart, Maria Del Mar Franco-Viseras & Paul R. Riley

  2. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, M5G 1X5, Ontario, Canada

    Carla O. Rosario, Carol J. Swallow & James W. Dennis

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Contributions

D.M.J.M carried out the functional cell-based characterization studies. C.A.R. and N.S. contributed to northern and western data and histological sections from Sak-null embryos. M.D.M.F.-V. peformed the yeast two-hybrid screen. COR collected and prepared Sak-null embryos. C.J.S. and J.W.D. provided Sak-null embryos and critical appraisal of Sak-null embryo data. P.R.R. carried out initial characterization studies and Sak-null embryo analyses, devised the functional studies and wrote the manuscript.

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Correspondence to Paul R. Riley.

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Martindill, D., Risebro, C., Smart, N. et al. Nucleolar release of Hand1 acts as a molecular switch to determine cell fate. Nat Cell Biol 9, 1131–1141 (2007). https://doi.org/10.1038/ncb1633

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