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Repression of p15INK4b expression by Myc through association with Miz-1

Abstract

Deregulated expression of c-myc can induce cell proliferation in established cell lines and in primary mouse embryonic fibroblasts (MEFs), through a combination of both transcriptional activation and repression by Myc. Here we show that a Myc-associated transcription factor, Miz-1, arrests cells in G1 phase and inhibits cyclin D-associated kinase activity. Miz-1 upregulates expression of the cyclin-dependent kinases (CDK) inhibitor p15INK4b by binding to the initiator element of the p15INK4b promoter. Myc and Max form a complex with Miz-1 at the p15 initiator and inhibit transcriptional activation by Miz-1. Expression of Myc in primary cells inhibits the accumulation of p15INK4b that is associated with cellular senescence; conversely, deletion of c-myc in an established cell line activates p15INK4b expression. Alleles of c-myc that are unable to bind to Miz-1 fail to inhibit accumulation of p15INK4b messenger RNA in primary cells and are, as a consequence, deficient in immortalization.

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Figure 1: Induction of G1 arrest and acidic β-galactosidase activity by Miz-1 in RAT1 cells.
Figure 2: Miz-1 activates expression of p15INK4b.
Figure 3: Regulation of the p15INK4b promoter by Myc and Miz-1.
Figure 4: A ternary Max–Myc–Miz-1 complex binds to the p15 initiator element in vivo.
Figure 5: Myc blocks transactivation by disrupting a Miz-1–p300 complex.
Figure 6: Chimeras between Myc and Mad associate with Max, but not with Miz-1.
Figure 7: Repression of endogenous p15INK4b expression by Myc through association with Miz-1.
Figure 8: Enhanced p15 expression and p15 promoter activity in c-myc−/− cells.

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References

  1. Grandori, C., Cowley, S. M., James, L. P. & Eisenman, R. N. The Myc/Max/Mad network and the transcriptional control of cell behavior. Annu. Rev. Cell Dev. Biol. 16, 653–699 (2000).

    Article  CAS  Google Scholar 

  2. O'Hagan, R. C. et al. Gene-target recognition among members of the Myc superfamily and implications for oncogenesis. Nature Genet. 24, 113–119 (2000).

    Article  CAS  Google Scholar 

  3. Coller, H. A. et al. Expression analysis with oligonucleotide microarrays reveals that Myc regulates genes involved in growth, cell cycle, signaling, and adhesion. Proc. Natl Acad. Sci. USA 97, 3260–3265 (2000).

    Article  CAS  Google Scholar 

  4. Freytag, S. O. & Geddes, T. J. Reciprocal regulation of adipogenesis by Myc and C/EBPα. Science 256, 379–382 (1992).

    Article  CAS  Google Scholar 

  5. Wu, K. J., Polack, A. & Dalla-Favera, R. Coordinated regulation of iron-controlling genes, H-ferritin and IRP2, by c-Myc. Science 283, 676–679 (1999).

    Article  CAS  Google Scholar 

  6. Warner, B. J., Blain, S. W., Seoane, J. & Massagué, J. Myc downregulation by transforming growth factor required for activation of the p15Ink4b G1 arrest pathway. Mol. Cell. Biol. 19, 5913–5922 (1999).

    Article  CAS  Google Scholar 

  7. Claassen, G. F. & Hann, S. R. A role for transcriptional repression of p21Cip1 by c-Myc in overcoming transforming growth factor β-induced cell-cycle arrest. Proc. Natl Acad. Sci. USA 97, 9498–9503 (2000).

    Article  CAS  Google Scholar 

  8. Li, L., Nerlov, C., Prendergast, G., MacGregor, D. & Ziff, E. B. c-Myc represses transcription in vivo by a novel mechanism dependent on the initiator element and Myc box II. EMBO J. 13, 4070–4079 (1994).

    Article  CAS  Google Scholar 

  9. Peukert, K. et al. An alternative pathway for gene regulation by Myc. EMBO J. 16, 5672–5686 (1997).

    Article  CAS  Google Scholar 

  10. Salghetti, S. E., Kim, S. Y. & Tansey, W. P. Destruction of Myc by ubiquitin-mediated proteolysis: cancer-associated and transforming mutations stabilize Myc. EMBO J. 18, 717–726 (1999).

    Article  CAS  Google Scholar 

  11. Dimri, G. P. et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl Acad. Sci. USA 92, 9363–9367 (1995).

    Article  CAS  Google Scholar 

  12. Lam, E. W-F. et al. HPV16 E7 oncoprotein deregulates B-myb expression: correlation with targeting of p107/E2F complexes. EMBO J. 13, 871–878 (1994).

    Article  CAS  Google Scholar 

  13. Beijersbergen, R. L., Carlée, L., Kerkhoven, R. M. & Bernards, R. Regulation of the retinoblastoma protein-related p107 by G1 cyclin complexes. Genes Dev. 9, 1340–1352 (1995).

    Article  CAS  Google Scholar 

  14. McConnell, B. B., Starborg, M., Brookes, S. & Peters, G. Inhibitors of cyclin-dependent kinases induce features of replicative senescence in early passage human diploid fibroblasts. Curr. Biol. 8, 351–354 (1998).

    Article  CAS  Google Scholar 

  15. Littlewood, T. D., Hancock, D. C., Danielian, P. S., Parker, M. G. & Evan, G. I. A modified oestrogen receptor ligand binding domain as an improved switch for the regulation of heterologous proteins. Nucleic Acids Res. 23, 1686–1690 (1995).

    Article  CAS  Google Scholar 

  16. Li, J. M., Nichols, M. A., Chandrasekharan, S., Xiong, Y. & Wang, X. F. Transforming growth factor β activates the promoter of cyclin-dependent kinase inhibitor p15INK4B through an Sp1 consensus site. J. Biol. Chem. 270, 26750–26753 (1995).

    Article  CAS  Google Scholar 

  17. Boyd, K. E., Wells, J., Gutman, J., Bartley, S. M. & Farnham, P. J. c-Myc target gene specificity is determined by a post-DNA binding mechanism. Proc. Natl Acad. Sci. USA 95, 13887–13892 (1998).

    Article  CAS  Google Scholar 

  18. Datto, M. B., Hu, P. P., Kowalik, T. F., Yingling, J. & Wang, X. F. The viral oncoprotein E1A blocks transforming growth factor β-mediated induction of p21/WAF1/Cip1 and p15/INK4B. Mol. Cell. Biol. 17, 2030–2037 (1997).

    Article  CAS  Google Scholar 

  19. Ayer, D. E., Kretzner, L. & Eisenman, R. N. Mad: a heterodimeric partner for Max that antagonizes Myc transcriptional activity. Cell 72, 211–222 (1993).

    Article  CAS  Google Scholar 

  20. Desbarats, L., Gaubatz, S. & Eilers, M. Discrimination between different E-box binding proteins at an endogenous target gene of Myc. Genes Dev. 10, 447–460 (1996).

    Article  CAS  Google Scholar 

  21. Malumbres, M. et al. Cellular response to oncogenic ras involves induction of the Cdk4 and Cdk6 inhibitor p15(INK4b). Mol. Cell. Biol. 20, 2915–2925 (2000).

    Article  CAS  Google Scholar 

  22. Erickson, S. et al. Involvement of the Ink4 proteins p16 and p15 in T-lymphocyte senescence. Oncogene 17, 595–602 (1998).

    Article  CAS  Google Scholar 

  23. Quelle, D. E. et al. Cloning and characterization of murine p16INK4a and p15INK4b genes. Oncogene 11, 635–645 (1995).

    CAS  Google Scholar 

  24. Harvey, M. et al. In vitro growth characteristics of embryo fibroblasts isolated from p53-deficient mice. Oncogene 8, 2457–2467 (1993).

    CAS  Google Scholar 

  25. Lukas, J. et al. Cyclin E-induced S phase without activation of the pRb/E2F pathway. Genes Dev. 11, 1479–1492 (1997).

    Article  CAS  Google Scholar 

  26. Land, H., Parada, L. F. & Weinberg, R. A. Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature 304, 596–602 (1983).

    Article  CAS  Google Scholar 

  27. Wang, J., Xie, L. Y., Allan, S., Beach, D. & Hannon, G. J. Myc activates telomerase. Genes Dev. 12, 1769–1774 (1998).

    Article  CAS  Google Scholar 

  28. Wu, K. J. et al. Direct activation of TERT transcription by c-MYC. Nature Genet. 21, 220–224 (1999).

    Article  CAS  Google Scholar 

  29. Greenberg, R. A. et al. Telomerase reverse transcriptase gene is a direct target of c-Myc but is not functionally equivalent in cellular transformation. Oncogene 18, 1219–1226 (1999).

    Article  CAS  Google Scholar 

  30. Kamijo, T. et al. Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell 91, 649–659 (1997).

    Article  CAS  Google Scholar 

  31. Carnero, A., Hudson, J. D., Price, C. M. & Beach, D. H. p16INK4A and p19ARF act in overlapping pathways in cellular immortalization. Nature Cell Biol. 2, 148–155 (2000).

    Article  CAS  Google Scholar 

  32. Alevizopoulos, K., Vlach, J., Hennecke, S. & Amati, B. Cyclin E and c-Myc promote cell proliferation in the presence of p16INK4a and of hypophosphorylated retinoblastoma family proteins. EMBO J. 16, 5322–5333 (1997).

    Article  CAS  Google Scholar 

  33. Lasorella, A., Noseda, M., Beyna, M. & Iavarone, A. Id2 is a retinoblastoma protein target and mediates signalling by Myc oncoproteins. Nature 407, 592–598 (2000).

    Article  CAS  Google Scholar 

  34. Herman, J. G. et al. Distinct patterns of inactivation of p15INK4b and p16INK4a characterize the major types of hematological malignancies. Cancer Res. 57, 837–841 (1997).

    CAS  Google Scholar 

  35. Hannon, G. J. & Beach, D. p15INK4b is a potential effector of cell cycle arrest mediated by TGF-β. Nature 371, 257–261 (1994).

    Article  CAS  Google Scholar 

  36. Feng, X. H., Lin, X. & Derynck, R. Smad2, smad3 and smad4 cooperate with Sp1 to induce p15 (Ink4B) transcription in response to TGF-β. EMBO J. 19, 5178–5193 (2000).

    Article  CAS  Google Scholar 

  37. Seoane, J. et al. TGFβ influences Myc, Miz-1 and Smad to control the CDK inhibitor INK4b. Nature Cell Biol. 4, 400–408.

  38. Coffey, R. J. J. et al. Selective inhibition of growth-related gene expression in murine keratinocytes by transforming growth factor β. Mol. Cell. Biol. 8, 3088 (1988).

    Article  CAS  Google Scholar 

  39. Morgenstern, J. P. & Land, H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18, 3587–3596 (1990).

    Article  CAS  Google Scholar 

  40. Bouchard, C. et al. Direct induction of cyclin D2 by Myc contributes to cell cycle induction and sequestration of p27. EMBO J. 18, 5321–5333 (1999).

    Article  CAS  Google Scholar 

  41. Grignani, F. et al. High-efficiency gene transfer and selection of human hematopoietic progenitor cells with a hybrid EBV/retroviral vector expressing the green fluorescence protein. Cancer Res. 58, 14–19 (1998).

    CAS  Google Scholar 

  42. Steiner, P. et al. Identification of a Myc-dependent step during the formation of active G1 cyclin/CDK complexes. EMBO J. 14, 4814–4826 (1995).

    Article  CAS  Google Scholar 

  43. Lukas, J., Pagano, M., Staskova, Z., Draetta, G. & Bartek, J. Cyclin D1 protein oscillates and is essential for cell cycle progression in human tumor cell lines. Oncogene 9, 707–718 (1994).

    CAS  Google Scholar 

  44. Gaubatz, S. et al. Transcriptional activation by Myc is under negative control by the transcription factor AP-2. EMBO J. 14, 1508–1529 (1995).

    Article  CAS  Google Scholar 

  45. Evan, G. I. & Hancock, D. C. Studies on the interaction of the human c-Myc protein with cell nuclei: p62 c-Myc as a member of a discrete subset of nuclear proteins. Cell 43, 253–261 (1985).

    Article  CAS  Google Scholar 

  46. Hata, A. et al. OAZ uses distinct DNA- and protein-binding zinc fingers in separate BMP-Smad and Olf signaling pathways. Cell 100, 229–240 (2000).

    Article  CAS  Google Scholar 

  47. Mateyak, M. K., Obaya, A. J. & Sedivy, J. M. c-Myc regulates cyclin D-Cdk4 and -Cdk6 activity but affects cell cycle progression at multiple independent points. Mol. Cell. Biol. 19, 4672–4683 (1999).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank P. Krimpenfort and A. Berns for p15+/+ and p15−/− fibroblasts, B. Eisenman for Mad-1 cDNA, several colleagues for anti-Max antibodies, K-H. Klempnauer for GST–p300 chimaeric proteins, R. Eckner for p300 clones and antibodies, D. Parry and Y. Xiong for anti-p15 antibodies, J. Sedivy for TGR and c-myc−/− cells, and K. Vousden for E7 plasmids. We also thank C. Pouponnot for help with bandshift experiments, and A. Maier, A. Grzeschiczek and A. A. Kjerulff for technical support. M.E. was supported by the Deutsche Forschungsgemeinschaft, the Biomed 2 programme of the European Community, and the Human Frontiers of Science Organisation.

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Correspondence to Martin Eilers.

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Staller, P., Peukert, K., Kiermaier, A. et al. Repression of p15INK4b expression by Myc through association with Miz-1. Nat Cell Biol 3, 392–399 (2001). https://doi.org/10.1038/35070076

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