Abstract

Haematopoietic stem cells (HSCs) self-renew for life, thereby making them one of the few blood cells that truly age1,2. Paradoxically, although HSCs numerically expand with age, their functional activity declines over time, resulting in degraded blood production and impaired engraftment following transplantation2. While many drivers of HSC ageing have been proposed2,3,4,5, the reason why HSC function degrades with age remains unknown. Here we show that cycling old HSCs in mice have heightened levels of replication stress associated with cell cycle defects and chromosome gaps or breaks, which are due to decreased expression of mini-chromosome maintenance (MCM) helicase components and altered dynamics of DNA replication forks. Nonetheless, old HSCs survive replication unless confronted with a strong replication challenge, such as transplantation. Moreover, once old HSCs re-establish quiescence, residual replication stress on ribosomal DNA (rDNA) genes leads to the formation of nucleolar-associated γH2AX signals, which persist owing to ineffective H2AX dephosphorylation by mislocalized PP4c phosphatase rather than ongoing DNA damage. Persistent nucleolar γH2AX also acts as a histone modification marking the transcriptional silencing of rDNA genes and decreased ribosome biogenesis in quiescent old HSCs. Our results identify replication stress as a potent driver of functional decline in old HSCs, and highlight the MCM DNA helicase as a potential molecular target for rejuvenation therapies.

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Gene Expression Omnibus

Referenced accessions

Gene Expression Omnibus

Data deposits

Data have been deposited in the Gene Expression Omnibus under accession number GSE48893.

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Acknowledgements

We thank A. Brunet and S. Villeda for providing some old C57BL/6 mice, B. McStay for advice on nucleolar analyses, C. Klijn for assistance with microarray analyses, S. Katzman for the SNP analyses, E. Davis for help with cytogenetic studies, I. Grummt for the rDNA plasmid, M. Kissner and M. Lee for management of our Flow Cytometry Core Facility, and all members of the Passegué laboratory for critical insights and suggestions. S.T.B. and M.M. were supported by a California Institute for Regenerative Medicine (CIRM) training grant and E.M.P. by National Institutes of Health (NIH) F32 HL106989. This work was supported by Science Foundation Ireland PI award 10/IN.1/B2972 to C.G.M. and a CIRM New Faculty Award RN2-00934 and NIH R01 HL092471 to E.P.

Author information

Affiliations

  1. The Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Department of Medicine, Hem/Onc Division, University of California San Francisco, San Francisco, California 94143, USA

    • Johanna Flach
    • , Sietske T. Bakker
    • , Mary Mohrin
    • , Eric M. Pietras
    • , Damien Reynaud
    •  & Emmanuelle Passegué
  2. Institute of Experimental Cancer Research, Comprehensive Cancer Center, 89081 Ulm, Germany

    • Johanna Flach
  3. Center for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland

    • Pauline C. Conroy
    •  & Ciaran G. Morrison
  4. Spanish National Cancer Research Centre (CNIO), E-28049 Madrid, Spain

    • Silvia Alvarez
    •  & Juan Méndez
  5. Department of Pathology, University of California San Francisco, San Francisco, California 94143, USA

    • Morgan E. Diolaiti
    •  & Bradley A. Stohr
  6. Institute for the Biology of Stem Cells, Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, California 95064, USA

    • Fernando Ugarte
    •  & E. Camilla Forsberg
  7. Section of Hematology/Oncology and the Comprehensive Cancer Center, University of Chicago, Chicago, Illinois 60637, USA

    • Michelle M. Le Beau

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Contributions

J.F. performed all of the experiments with help from S.T.B. for the comet assays and microarray data analyses, E.M.P. for the Ki67/DAPI staining and microarray analyses, and D.R. for the transplantation experiments. M.M. and P.C.C. initiated these studies. S.A. and J.M. performed the DNA replication track analyses and helped with the MCM experiments, M.E.D. and B.A.S. performed the telomere analyses, F.U. and E.C.F. performed the SNP analyses, and M.M.L.B. performed the cytogenetic breakage studies and spectral karyotyping analyses. J.F., C.G.M. and E.P. designed the experiments and interpreted the results. J.F. and E.P. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Emmanuelle Passegué.

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https://doi.org/10.1038/nature13619

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