Regulation of hematopoietic stem cell differentiation by a single ubiquitin ligase–substrate complex

Journal name:
Nature Immunology
Volume:
11,
Pages:
207–215
Year published:
DOI:
doi:10.1038/ni.1839
Received
Accepted
Published online

Abstract

Hematopoietic stem cell (HSC) differentiation is regulated by cell-intrinsic and cell-extrinsic cues. In addition to transcriptional regulation, post-translational regulation may also control HSC differentiation. To test this hypothesis, we visualized the ubiquitin-regulated protein stability of a single transcription factor, c-Myc. The stability of c-Myc protein was indicative of HSC quiescence, and c-Myc protein abundance was controlled by the ubiquitin ligase Fbw7. Fine changes in the stability of c-Myc protein regulated the HSC gene-expression signature. Using whole-genome genomic approaches, we identified specific regulators of HSC function directly controlled by c-Myc binding; however, adult HSCs and embryonic stem cells sensed and interpreted c-Myc-regulated gene expression in distinct ways. Our studies show that a ubiquitin ligase–substrate pair can orchestrate the molecular program of HSC differentiation.

At a glance

Figures

  1. Abundance of c-Myc protein in early hematopoiesis.
    Figure 1: Abundance of c-Myc protein in early hematopoiesis.

    (a) Expression of c-Myc–eGFP during early hematopoiesis in Lin+, Lin and myeloerythroid progenitor (MP; Linc-Kit+Sca-1) bone marrow subsets (n = 6 mice). max, maximum. (b) Immunofluorescence tracing of nuclear expression of c-Myc–eGFP in Lin bone marrow progenitors. DAPI, nuclear stain; Anti-eGFP, antibody to eGFP. Original magnification, ×40. (c) Immunoblot analysis of c-Myc in c-Kit+ and c-Kit subsets of adult bone marrow. (d) Expression of c-Myc–eGFP protein in LSK cells. (e) Expression of c-Myc–eGFP in Myc-eGFP−/− control, LT-HSC (LT) and MPP populations. Numbers in top right quadrants indicate percent c-Kit+eGFP+ cells. (f) Quantitative RT-PCR analysis of the expression of Myc and Fbw7 mRNA during HSC differentiation, presented (as average and s.d.) relative to the expression of Actb (encoding β-actin) and then normalized to the LT-HSC subset. (g) Abundance of c-Myc–eGFP protein and cell cycle status of sorted c-Myc–eGFPhi or c-Myc–eGFPlo LSK cells, analyzed by Ki67 and DAPI staining. Numbers in top right quadrants indicate percent Ki67+DAPI+ cells. Data are representative of at least three independent experiments.

  2. Correlation between the amount of c-Myc protein and loss of HSC self-renewal.
    Figure 2: Correlation between the amount of c-Myc protein and loss of HSC self-renewal.

    (a) Flow cytometry–dependent separation of c-Myc–eGFPhi and c-Myc–eGFPlo LSK populations. (b) In vitro culture of sorted c-Myc–eGFPhi and c-Myc–eGFPlo cells: black, first plating of LSK cells; gray, second plating. CFU, colony-forming unit. (c) Peripheral blood chimerism in competitive reconstitution assays at 25 weeks after transplantation (n = 3 mice). Numbers in plots indicate percent CD45.2+CD45.1 cells (top left) or CD45.2CD45.1+ cells (bottom right). (d) Chimerism of sorted c-Myc–eGFPhi and c-Myc–eGFPlo LSK cells (CD45.2+) in the bone marrow (LSK subset), thymus and spleen 17 weeks after transplantation (n = 3 mice). Data are representative of at least three experiments (a,c,d) or are from three independent experiments (b; error bars, s.d. of three mice).

  3. Fbw7 controls the stability of c-Myc protein in HSCs.
    Figure 3: Fbw7 controls the stability of c-Myc protein in HSCs.

    (a) Flow cytometry of c-Myc–eGFP in LSK, myeloerythroid progenitor and thymic DN (CD4CD8) subsets of progenitor populations. BM, bone marrow; WT, wild-type. (b) Flow cytometry analysis of c-Myc–eGFP protein in an LT-HSC subset (LSK, CD150+, CD48). (ce) Flow cytometry of bone marrow LSK cells (c), cell-cycle status of LSK cells (d) and methylcellulose assay of CD150+ LSK cells (e). (c) Numbers above outlined areas indicate percent c-Kit+Sca-1+ cells. (d) Numbers above bracketed lines indicate percent dividing cells. (e) Black, first plating; gray, second plating (n = 5 mice). Control (ce), wild-type. Data are representative of at least three independent experiments (error bars (e), s.d.).

  4. The abundance of c-Myc protein directly controls the molecular program of stem cell differentiation, cell-cycle entry and self-renewal.
    Figure 4: The abundance of c-Myc protein directly controls the molecular program of stem cell differentiation, cell-cycle entry and self-renewal.

    (a) Heat map of the gene-expression signatures of c-Myc–eGFPhi and c-Myc–eGFPlo subsets and Fbw7−/− LSK cells. (b) GSEA profiles of the correlation of the c-Myc–eGFPlo signature to stem cell gene sets (HSC; top) and to cell cycle and DNA replication gene sets (bottom). (c) GSEA profiles of the correlation between the transforming growth factor-β (TGF-β) and Wnt signaling pathways with the c-Myc–eGFPlo gene-expression data set. Data are representative of two replicates of three mice.

  5. Genes overexpressed in c-Myc-EGFPhi cells are directly bound by the c-Myc transcription factor.
    Figure 5: Genes overexpressed in c-Myc-EGFPhi cells are directly bound by the c-Myc transcription factor.

    (a) GSEA profile of the correlation of c-Myc–eGFPhi gene expression profiles to a direct c-Myc target ChIP–and–microarray analysis data set. (b) ChIP assay (left) and heat map (right) of selected genes overexpressed in c-Myc–eGFPhi T-ALL cells. Red, upregulation; blue, downregulation. Data are representative of two individual experiments (error bars, s.d.). (c) ChIP assay of genomic DNA from purified Linc-Kit+c-MyceGFP+ bone marrow progenitor cells. IgG, immunoglobulin G. Right, heat map of the overexpression of selected genes in the c-Myc–eGFPhi LSK subset. Data are from three independent experiments (error bars, s.d.).

  6. The role of the c-Myc-Fbw7 interaction in fetal liver stem cells and progenitor cells.
    Figure 6: The role of the c-Myc–Fbw7 interaction in fetal liver stem cells and progenitor cells.

    (a) Flow cytometry of LSK cells in fetal liver (E14.5) and adult bone marrow (6 weeks old). Numbers above outlined areas indicate percent c-Kit+Sca-1+ cells. (b) Cell-cycle status of fetal and adult LSK cells. Numbers above bracketed lines indicate percent dividing cells. (c) Expression of c-Myc–eGFP protein in fetal and adult LSK cells. Far right, induction of c-Myc protein expression in fetal LSK cells. (d) Expression of c-Myc–eGFP protein in CD150+ LSK cells. Numbers adjacent to outlined areas (c,d) indicate percent c-Kit+eGFP+ cells. (e) Methylcellulose culture of cell populations purified from fetal liver (Fetal) or bone marrow (Adult). Black, first plating; gray, second plating. (f) Peripheral blood chimerism of c-Myc–eGFPlo and c-Myc–eGFPhi fetal liver LSK subsets in competitive reconstitution assays 20 weeks after transplantation (n = 6 mice). CD45.2+ cells are donor-derived cells. Numbers in plots indicate percent CD45.2+CD45.1 cells (top left) or CD45.2CD45.1+ cells (bottom right). Data are representative of at least three independent experiments (error bars (e), s.d. of six mice).

  7. Expression patterns of Fbw7 and c-Myc in mouse ESCs.
    Figure 7: Expression patterns of Fbw7 and c-Myc in mouse ESCs.

    (a) Expression of Fbw7, Myc and Nanog mRNA transcripts in self-renewing (day 0) and differentiating (days 1–6) mouse ESCs on days 0–6 of differentiation, presented relative to Actb expression. (b) Fbw7 gene-trap cassette. SA, splice acceptor; βGEO, β-galactosidase–neomycin; LTR, long terminal repeat. (c) Analysis of Fbw7 expression by lacZ staining of self-renewing (+LIF) and differentiating (+LIF–RA) mouse ESCs containing the Fbw7 gene-trap cassette. RA, retinoic acid. Original magnification, ×10. (d) Immunoblot analysis of total c-Myc and c-Myc phosphorylated at Tyr58 (p-c-Myc) in self-renewing (ESC) and differentiating (Diff) mouse ESCs left untreated (−) or treated (+) with 20 μM MG132 (proteasome inhibitor). (e) ChIP assay (with the specific regulators in Fig. 6) after c-Myc immunoprecipitation in mouse ESCs. Data representative of at least three independent experiments (error bars (a), s.d.).

  8. Fbw7 is dispensable for the self-renewal of mouse ESCs.
    Figure 8: Fbw7 is dispensable for the self-renewal of mouse ESCs.

    (a) Accumulation of c-Myc protein in W4 and MyceGFP/− mouse ESCs left untreated or treated with dimethyl sulfoxide (DMSO) or MG132. Numbers in plots indicate percent eGFP+ cells. SSC, side scatter. (b) Visualization of c-Myc–eGFP in W4 and MyceGFP/− mouse ESCs with and without treatment with MG132. Green, GFP; blue, nuclear staining (DAPI). Original magnification, ×20. (c) Accumulation of c-Myc protein in MyceGFP/− ESCs expressing nonsilencing siRNA or siRNA specific for GFP (siGFP), c-Myc (siMyc) or Fbw7 (siFbw7). Numbers in plots indicate percent c-Myc–eGFP+ cells. (d) Quantitative RT-PCR analysis of Fbw7 mRNA expression in ESCs expressing nonsilencing shRNA (Nonsil; control) or shRNA specific for Fbw7 (shFbw7) or Nanog (shNanog). (e) Immunoblot analysis of the accumulation of total c-Myc protein and c-Myc phosphorylated at Tyr58 in W4 ESCs expressing nonsilencing shRNA or shRNA specific for Fbw7. (f) Alkaline phosphatase staining of mouse ESCs expressing nonsilencing shRNA or shRNA specific for Fbw7 or Nanog. Original magnification, ×10. (g) Nanog-eGFP expression in a Nanog-eGFP reporter ESC line expressing no siRNA, nonsilencing siRNA (Nontargeting) or siRNA specific for GFP (siGFP), the transcription factor Oct4 (siOct4), c-Myc (siMyc) or Fbw7 (siFbw7). Numbers in plots indicate percent cells expressing Nanog. H-SSC, side-scatter height. Data are representative of at least three independent experiments (error bars (d), s.d.).

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Author information

Affiliations

  1. Howard Hughes Medical Institute and Department of Pathology, New York, New York, USA.

    • Linsey Reavie,
    • Kelly Crusio,
    • Beatriz Aranda-Orgilles,
    • Shannon M Buckley,
    • Benjamin Thompson,
    • Eugine Lee,
    • Jie Gao &
    • Iannis Aifantis
  2. New York University (NYU) Cancer Institute and Helen & Martin S. Kimmel Stem Cell Center, NYU School of Medicine, New York, New York, USA.

    • Linsey Reavie,
    • Kelly Crusio,
    • Beatriz Aranda-Orgilles,
    • Shannon M Buckley,
    • Benjamin Thompson,
    • Eugine Lee,
    • Jie Gao,
    • Jiri Zavadil &
    • Iannis Aifantis
  3. Institute for Cancer Genetics, Columbia University, New York, New York, USA.

    • Giusy Della Gatta,
    • Teresa Palomero &
    • Adolfo Ferrando
  4. Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.

    • Andrea L Bredemeyer,
    • Beth A Helmink &
    • Barry P Sleckman
  5. Center for Health Informatics and Bioinformatics, NYU School of Medicine, New York, New York, USA.

    • Jiri Zavadil

Contributions

L.R. did most of the experiments and participated in preparing the manuscript; G.D.G., T.P., A.F. and B.A.-O. designed and did the ChIP-plus-microarray and ChIP experiments; K.C., E.L. and B.T. did the ESC experiments; B.P.S., B.T., A.L.B. and B.A.H. generated and did initial studies with the c-Myc–eGFP mice; J.Z. analyzed microarray data; and I.A. designed the study and prepared the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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