Movatterモバイル変換

[0]ホーム
URL:

Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Advertisement

Nature
  • Letter
  • Published:

TheInk4/Arf locus is a barrier for iPS cell reprogramming

Naturevolume 460, pages1136–1139 (2009)Cite this article

Abstract

The mechanisms involved in the reprogramming of differentiated cells into induced pluripotent stem (iPS) cells by the three transcription factors Oct4 (also known as Pou5f1), Klf4 and Sox2 remain poorly understood1. TheInk4/Arf locus comprises theCdkn2a–Cdkn2b genes encoding three potent tumour suppressors, namely p16Ink4a, p19Arf and p15Ink4b, which are basally expressed in differentiated cells and upregulated by aberrant mitogenic signals2,3,4. Here we show that the locus is completely silenced in iPS cells, as well as in embryonic stem (ES) cells, acquiring the epigenetic marks of a bivalent chromatin domain, and retaining the ability to be reactivated after differentiation. Cell culture conditions during reprogramming enhance the expression of theInk4/Arf locus, further highlighting the importance of silencing the locus to allow proliferation and reprogramming. Indeed, the three factors together repress theInk4/Arf locus soon after their expression and concomitant with the appearance of the first molecular markers of ‘stemness’. This downregulation also occurs in cells carrying the oncoprotein large-T, which functionally inactivates the pathways regulated by theInk4/Arf locus, thus indicating that the silencing of the locus is intrinsic to reprogramming and not the result of a selective process. Genetic inhibition of theInk4/Arf locus has a profound positive effect on the efficiency of iPS cell generation, increasing both the kinetics of reprogramming and the number of emerging iPS cell colonies. In murine cells,Arf, rather thanInk4a, is the main barrier to reprogramming by activation of p53 (encoded byTrp53) and p21 (encoded byCdkn1a); whereas, in human fibroblasts,INK4a is more important thanARF. Furthermore, organismal ageing upregulates theInk4/Arf locus2,5 and, accordingly, reprogramming is less efficient in cells from old organisms, but this defect can be rescued by inhibiting the locus with a short hairpin RNA. All together, we conclude that the silencing ofInk4/Arf locus is rate-limiting for reprogramming, and its transient inhibition may significantly improve the generation of iPS cells.

This is a preview of subscription content,access via your institution

Access options

Access through your institution

Subscription info for Japanese customers

We have a dedicated website for our Japanese customers. Please go tonatureasia.com to subscribe to this journal.

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF

Prices may be subject to local taxes which are calculated during checkout

Figure 1:Functional reprogramming of theInk4/Arf locus.
Figure 2:Silencing of theInk4/Arf locus during reprogramming.
Figure 3:Effect ofInk4a/Arf on reprogramming efficiency.
Figure 4:Association between age of the parental cells, expression of theInk4/Arf locus and reprogramming efficiency.

Similar content being viewed by others

References

  1. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell126, 663–676 (2006)

    Article CAS  Google Scholar 

  2. Collado, M., Blasco, M. A. & Serrano, M. Cellular senescence in cancer and aging.Cell130, 223–233 (2007)

    Article CAS  Google Scholar 

  3. Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D. & Lowe, S. W. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a.Cell88, 593–602 (1997)

    Article CAS  Google Scholar 

  4. Sharpless, N. E.INK4a/ARF: a multifunctional tumor suppressor locus.Mutat. Res.576, 22–38 (2005)

    Article CAS  Google Scholar 

  5. Krishnamurthy, J. et al.Ink4a/Arf expression is a biomarker of aging.J. Clin. Invest.114, 1299–1307 (2004)

    Article CAS  Google Scholar 

  6. Bernstein, B. E. et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells.Cell125, 315–326 (2006)

    Article CAS  Google Scholar 

  7. Azuara, V. et al. Chromatin signatures of pluripotent cell lines.Nature Cell Biol.8, 532–538 (2006)

    Article CAS  Google Scholar 

  8. Mikkelsen, T. S. et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells.Nature448, 553–560 (2007)

    Article ADS CAS  Google Scholar 

  9. Ohm, J. E. et al. A stem cell-like chromatin pattern may predispose tumor suppressor genes to DNA hypermethylation and heritable silencing.Nature Genet.39, 237–242 (2007)

    Article CAS  Google Scholar 

  10. Savatier, P., Lapillonne, H., van Grunsven, L. A., Rudkin, B. B. & Samarut, J. Withdrawal of differentiation inhibitory activity/leukemia inhibitory factor up-regulates D-type cyclins and cyclin-dependent kinase inhibitors in mouse embryonic stem cells.Oncogene12, 309–322 (1996)

    CAS PubMed  Google Scholar 

  11. Sharpless, N. E.Ink4a/Arf links senescence and aging.Exp. Gerontol.39, 1751–1759 (2004)

    Article CAS  Google Scholar 

  12. Hara, E. et al. Regulation of p16CDKN2 expression and its implications for cell immortalization and senescence.Mol. Cell. Biol.16, 859–867 (1996)

    Article CAS  Google Scholar 

  13. Sherr, C. J. Divorcing ARF and p53: an unsettled case.Nature Rev. Cancer6, 663–673 (2006)

    Article CAS  Google Scholar 

  14. Zhao, Y. et al. Two supporting factors greatly improve the efficiency of human iPSC generation.Cell Stem Cell3, 475–479 (2008)

    Article CAS  Google Scholar 

  15. Sherr, C. J. & Roberts, J. M. CDK inhibitors: positive and negative regulators of G1-phase progression.Genes Dev.13, 1501–1512 (1999)

    Article CAS  Google Scholar 

  16. Cherry, S. R., Biniszkiewicz, D., van Parijs, L., Baltimore, D. & Jaenisch, R. Retroviral expression in embryonic stem cells and hematopoietic stem cells.Mol. Cell. Biol.20, 7419–7426 (2000)

    Article CAS  Google Scholar 

  17. Brambrink, T. et al. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells.Cell Stem Cell2, 151–159 (2008)

    Article CAS  Google Scholar 

  18. Stadtfeld, M., Maherali, N., Breault, D. T. & Hochedlinger, K. Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse.Cell Stem Cell2, 230–240 (2008)

    Article CAS  Google Scholar 

  19. Wei, W., Hemmer, R. M. & Sedivy, J. M. Role of p14ARF in replicative and induced senescence of human fibroblasts.Mol. Cell. Biol.21, 6748–6757 (2001)

    Article CAS  Google Scholar 

  20. Evan, G. I. & d’Adda di Fagagna, F. Cellular senescence: hot or what?Curr. Opin. Genet. Dev.19, 25–31 (2009)

    Article CAS  Google Scholar 

  21. Wong, D. J. et al. Module map of stem cell genes guides creation of epithelial cancer stem cells.Cell Stem Cell2, 333–344 (2008)

    Article CAS  Google Scholar 

  22. Ben-Porath, I. et al. An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors.Nature Genet.40, 499–507 (2008)

    Article CAS  Google Scholar 

  23. Blelloch, R., Venere, M., Yen, J. & Ramalho-Santos, M. Generation of induced pluripotent stem cells in the absence of drug selection.Cell Stem Cell1, 245–247 (2007)

    Article CAS  Google Scholar 

  24. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell131, 861–872 (2007)

    Article CAS  Google Scholar 

  25. Park, I. H. et al. Reprogramming of human somatic cells to pluripotency with defined factors.Nature451, 141–146 (2008)

    Article ADS CAS  Google Scholar 

  26. Palmero, I. & Serrano, M. Induction of senescence by oncogenic Ras.Methods Enzymol.333, 247–256 (2001)

    Article CAS  Google Scholar 

  27. Munoz, P., Blanco, R., Flores, J. M. & Blasco, M. A. XPF nuclease-dependent telomere loss and increased DNA damage in mice overexpressing TRF2 result in premature aging and cancer.Nature Genet.37, 1063–1071 (2005)

    Article CAS  Google Scholar 

  28. Li, H., Vogel, H., Holcomb, V. B., Gu, Y. & Hasty, P. Deletion of Ku70, Ku80, or both causes early aging without substantially increased cancer.Mol. Cell. Biol.27, 8205–8214 (2007)

    Article CAS  Google Scholar 

  29. Dickins, R. A. et al. Probing tumor phenotypes using stable and regulated synthetic microRNA precursors.Nature Genet.37, 1289–1295 (2005)

    Article CAS  Google Scholar 

  30. Voorhoeve, P. M. & Agami, R. The tumor-suppressive functions of the humanINK4A locus.Cancer Cell4, 311–319 (2003)

    Article CAS  Google Scholar 

  31. Yuan, J. S., Reed, A., Chen, F. & Stewart, C. N. Statistical analysis of real-time PCR data.BMC Bioinformatics7, 85 (2006)

    Article  Google Scholar 

Download references

Acknowledgements

We thank S. Lowe and R. Agami for reagents. We are grateful to M. Muñoz, O. Dominguez, D. Megias and H. Schonthaler. H.L. is the recipient of a ‘Juan de la Cierva’ contract from the Spanish Ministry of Science (MICINN). M.Co. is the recipient of a ‘Ramon y Cajal’ contract (MICINN). Work in the laboratory of M.S. is funded by the CNIO and by grants from the MICINN (SAF and CONSOLIDER), the Regional Government of Madrid, the European Union, the European Research Council (ERC), and the ‘Marcelino Botin’ Foundation.

Author Contributions H.L. performed most of the experimental work. M.Co. and A.V. made critical experimental contributions. K.S., S.O. and M.Ca. contributed experimentally. H.L., M.Co., M.A.B. and M.S. designed the experimental plan, analysed and interpreted the data. M.S. directed the project and wrote the paper.

Author information

Authors and Affiliations

  1. Tumor Suppression Group,,

    Han Li, Manuel Collado, Aranzazu Villasante & Manuel Serrano

  2. Telomeres and Telomerase Group,,

    Katerina Strati & Maria A. Blasco

  3. Transgenic Mice Unit,,

    Sagrario Ortega

  4. Comparative Pathology Unit, Spanish National Cancer Research Centre (CNIO), 3 Melchor Fernandez Almagro Street, Madrid E-28029, Spain ,

    Marta Cañamero

Authors
  1. Han Li

    You can also search for this author inPubMed Google Scholar

  2. Manuel Collado

    You can also search for this author inPubMed Google Scholar

  3. Aranzazu Villasante

    You can also search for this author inPubMed Google Scholar

  4. Katerina Strati

    You can also search for this author inPubMed Google Scholar

  5. Sagrario Ortega

    You can also search for this author inPubMed Google Scholar

  6. Marta Cañamero

    You can also search for this author inPubMed Google Scholar

  7. Maria A. Blasco

    You can also search for this author inPubMed Google Scholar

  8. Manuel Serrano

    You can also search for this author inPubMed Google Scholar

Corresponding author

Correspondence toManuel Serrano.

Supplementary information

Supplementary Information

This file contains Supplementary Table S1 and Supplementary Figures S1-S15 with Legends. (PDF 1879 kb)

Rights and permissions

About this article

Cite this article

Li, H., Collado, M., Villasante, A.et al. TheInk4/Arf locus is a barrier for iPS cell reprogramming.Nature460, 1136–1139 (2009). https://doi.org/10.1038/nature08290

Download citation

Access through your institution
Buy or subscribe

Editorial Summary

On iPS cells and p53: the Ink4/Arf barrier

TheInk4/Arf tumour suppressor locus encodes three potent tumour suppressors, namely p16Ink4a, p15Ink4b and p19Arf. Here Liet al. show that the locus is rate limiting for reprogramming, and its transient inhibition significantly improves the generation of iPS cells. The also show ageing upregulates theInk4/Arf locus and, accordingly, reprogramming is less efficient in cells from old organisms.

Associated content

The promises and perils of p53

  • Valery Krizhanovsky
  • Scott W. Lowe
NatureNews & Views

Advertisement

Search

Advanced search

Quick links

Nature Briefing

Sign up for theNature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox.Sign up for Nature Briefing
[8]ページ先頭
©2009-2025 Movatter.jp