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The ubiquitin ligase Huwe1 regulates the maintenance and lymphoid commitment of hematopoietic stem cells

Nature Immunology 17, 1312–1321 (2016) | Download Citation

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Abstract

Hematopoietic stem cells (HSCs) are dormant in the bone marrow and can be activated in response to diverse stresses to replenish all blood cell types. We identified the ubiquitin ligase Huwe1 as a crucial regulator of HSC function via its post-translational control of the oncoprotein N-myc (encoded by Mycn). We found Huwe1 to be essential for HSC self-renewal, quiescence and lymphoid-fate specification in mice. Through the use of a fluorescent fusion allele (MycnM), we observed that N-myc expression was restricted to the most immature, multipotent stem and progenitor populations. N-myc expression was upregulated in response to stress or following loss of Huwe1, which led to increased proliferation and stem-cell exhaustion. Mycn depletion reversed most of these phenotypes in vivo, which suggested that the attenuation of N-myc by Huwe1 is essential for reestablishing homeostasis following stress.

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Acknowledgements

We thank the members of the Aifantis laboratory for discussions; A. Heguy and members of the New York University (NYU) Genome Technology Center for assistance in RNA sequencing; the NYU Flow Cytometry facility for cell sorting; the NYU Histology Core; G. Inghirami for assistance with histopathological evaluations; S. Heimfeld (Fred Hutchinson Cancer Research Center) for human CD34+ cells (Core Center of Excellence NIDDK grant DK56465). Supported by the US National Institutes of Health (1R01CA169784, 1R01CA133379, 1R01CA105129, 1R01CA149655 and 5R01CA173636), the William Lawrence and Blanche Hughes Foundation, The Leukemia & Lymphoma Society (TRP#6340-11, LLS#6373-13), The Chemotherapy Foundation, The V Foundation for Cancer Research, the Alex's Lemonade Stand Foundation for Childhood Cancer, and the St. Baldrick's Cancer Research Foundation (all for the The Aifantis laboratory); the Damon Runyon Cancer Research Foundation (Berger Foundation Fellowship DRG-2234-15 to B.K.); Deutsche Forschungsgemeinschaft (Emmy Noether Research Group WO 2108/1-1 to E.W.); and the American-Italian Cancer Foundation (Alessandro and Catherine di Montezemolo endowment fund to F.B.).

Author information

    • Bryan King
    •  & Francesco Boccalatte

    These authors contributed equally to this work.

Affiliations

  1. Department of Pathology, New York University School of Medicine, New York, New York, USA.

    • Bryan King
    • , Francesco Boccalatte
    • , Kelly Moran-Crusio
    • , Jingjing Wang
    • , Clarisse Kayembe
    • , Charalampos Lazaris
    • , Xiaofeng Yu
    • , Beatriz Aranda-Orgilles
    •  & Iannis Aifantis

  2. Laura and Isaac Perlmutter Cancer Center, New York, New York, USA.

    • Bryan King
    • , Francesco Boccalatte
    • , Kelly Moran-Crusio
    • , Jingjing Wang
    • , Clarisse Kayembe
    • , Charalampos Lazaris
    • , Xiaofeng Yu
    • , Beatriz Aranda-Orgilles
    •  & Iannis Aifantis

  3. Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

    • Bryan King

  4. Department of Medicine, Quinnipiac University Netter School of Medicine, New Hampshire-MED, Hamden, Connecticut, USA.

    • Kelly Moran-Crusio

  5. Comprehensive Cancer Center Mainfranken and Department of Biochemistry and Molecular Biology, Biocenter, University of Würzburg, Würzburg, Germany.

    • Elmar Wolf

  6. Center for Health Informatics and Bioinformatics, New York University School of Medicine, New York, New York, USA.

    • Charalampos Lazaris

  7. Department of Pathology and Cell Biology and Pediatrics, Institute for Cancer Genetics, Columbia University Medical Center, New York, New York, USA.

    • Anna Lasorella

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Contributions

B.K. and I.A. designed the study and prepared the manuscript. B.K. performed most of the experiments. F.B. completed experiments and focused on N-myc genomic and transcriptomic studies. K.M.-C. initiated the Huwe1 cKO in vivo analysis. E.W. performed the N-myc ChIP-Seq. F.B. and B.A.-O. analyzed the MYCN ChIP-Seq data. J.W. and C.K. were responsible for animal husbandry. C.L. provided bioinformatics analysis and guidance. X.Y. designed the Mycn mCherry targeting vector. A.L. provided Huwe1-floxed mice and helped with data analysis.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Bryan King or Iannis Aifantis.

Integrated supplementary information

Supplementary figures

  1. 1.

    Huwe1 has high expression in HSCs and is necessary for quiescence.

  2. 2.

    Huwe1-deficient fetal liver HSCs are not reduced in number but are functionally impaired.

  3. 3.

    Huwe1 is required for lymphoid specification of HSPCs in vitro.

  4. 4.

    Aged Huwe1 Vav1-cKO mice exhibit myeloid expansion and anemia.

  5. 5.

    Unique gene-expression signatures in N-mychi HSCs versus N-myclo HSCs.

  6. 6.

    Identification of genome-wide transcriptional targets of N-myc in HSCs.

  7. 7.

    Restoration of HSC function in Huwe1- and N-myc-dKO mice.

Supplementary information

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    Supplementary Text and Figures

    Supplementary Figures 1–7 and Supplementary Tables 1 and 2

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DOI

https://doi.org/10.1038/ni.3559

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