The ubiquitin ligase huwe1 regulates the maintenance and lymphoid commitment of hematopoietic stem cells

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 (_Mycn_M), 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. Access

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SIMILAR CONTENT BEING VIEWED BY OTHERS RNA BINDING PROTEIN SYNCRIP MAINTAINS PROTEOSTASIS AND SELF-RENEWAL OF HEMATOPOIETIC STEM AND PROGENITOR CELLS Article Open access 21 April 2023

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Google Scholar  Download references 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 Author notes * Bryan King and Francesco

Boccalatte: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * 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 * 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 * Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA Bryan King * Department of Medicine, Quinnipiac University Netter School of

Medicine, New Hampshire-MED, Hamden, Connecticut, USA Kelly Moran-Crusio * Comprehensive Cancer Center Mainfranken and Department of Biochemistry and Molecular Biology, Biocenter, University

of Würzburg, Würzburg, Germany Elmar Wolf * Center for Health Informatics and Bioinformatics, New York University School of Medicine, New York, New York, USA Charalampos Lazaris *

Department of Pathology and Cell Biology and Pediatrics, Institute for Cancer Genetics, Columbia University Medical Center, New York, New York, USA Anna Lasorella Authors * Bryan King View

author publications You can also search for this author inPubMed Google Scholar * Francesco Boccalatte View author publications You can also search for this author inPubMed Google Scholar *

Kelly Moran-Crusio View author publications You can also search for this author inPubMed Google Scholar * Elmar Wolf View author publications You can also search for this author inPubMed 

Google Scholar * Jingjing Wang View author publications You can also search for this author inPubMed Google Scholar * Clarisse Kayembe View author publications You can also search for this

author inPubMed Google Scholar * Charalampos Lazaris View author publications You can also search for this author inPubMed Google Scholar * Xiaofeng Yu View author publications You can also

search for this author inPubMed Google Scholar * Beatriz Aranda-Orgilles View author publications You can also search for this author inPubMed Google Scholar * Anna Lasorella View author

publications You can also search for this author inPubMed Google Scholar * Iannis Aifantis View author publications You can also search for this author inPubMed Google Scholar 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.

CORRESPONDING AUTHORS Correspondence to Bryan King or Iannis Aifantis. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. INTEGRATED SUPPLEMENTARY

INFORMATION SUPPLEMENTARY FIGURE 1 _HUWE1_ HAS HIGH EXPRESSION IN HSCS AND IS NECESSARY FOR QUIESCENCE. (A) Heat map visualizing expression of genes with a known function in

ubiquitin-mediated proteolysis (KEGG: Ubiquitin mediated proteolysis) in sorted hematopoietic populations (GSE60101), ranked by expression in HSC (LT- and ST-). (B) _Huwe1_ RNA-seq counts

per million in hematopoietic cell populations shown in (A). (C) Frequency of HSC in bone marrow of _Huwe1_+/Y Mx1-Cre+ (WT) or _Huwe1_F/Y Mx1-Cre+ (cKO) 4 weeks post-pI:pC treatment. (D)

HSPCs were sorted from bone marrow from WT or cKO mice 4 weeks post pI:pC treatment and serial colony formation in methylcellulose cultures was scored over two passages. Frequency of donor

cells within total bone marrow (E) or LSK population (F) of lethally-irradiated CD45.1+ recipient mice transplanted with 1x106 bone marrow cells from untreated CD45.2+ WT or cKO mice,

analyzed 28 weeks after pI:pC treatment. (G) WT or Mx1-cKO mice were given a single dose of 5-FU and cell cycle distribution was determined by intracellular Ki67/DAPI staining within gated

HSC. *_P_ < 0.05, **_P_ < 0.01, ***_P_ < 0.001 (two-tailed _t-_test). Data are representative of two experiments with five mice per group (C; mean and s.e.m.), two experiments with

three technical replicates each (D; mean and s.e.m.), one experiment with four recipient mice per group (E-F; mean and s.e.m.) or one experiment with five mice per group (G; mean and

s.e.m.). SUPPLEMENTARY FIGURE 2 _HUWE1_-DEFICIENT FETAL LIVER HSCS ARE NOT REDUCED IN NUMBER BUT ARE FUNCTIONALLY IMPAIRED. (A) Flow cytometry of fetal livers from _Huwe1_+/Y _Vav1_-Cre+

(WT) and _Huwe1__F/Y__ Vav1_-Cre+ (cKO) at E18.5 showing average frequency of Lin-c-kit+Sca1+ HSPCs (upper panels), sub-fractionated further with CD48 and CD150 (lower panels). (B) Total

cells recovered from E18.5 WT or cKO fetal livers. (C) 2x104 cells from either WT or cKO E18.5 fetal livers were plated in methycellulose, scored for colony formation and harvested 7d later.

5x103 cells were replated and scored again the following week. _*P <_ 0.05, **_P <_ 0.01 (one-way ANOVA). Data are representative of two experiments with a minimum of three embryos

per genotype (A-B; mean and s.e.m.) or three technical replicates from two embryos per genotype (C). SUPPLEMENTARY FIGURE 3 _HUWE1_ IS REQUIRED FOR LYMPHOID SPECIFICATION OF HSPCS _IN

VITRO_. (A) Thymii isolated from 8-week-old _Huwe1_+/Y _Vav1_-Cre+ (WT) or _Huwe1__F/Y__ Vav1_-Cre+ (cKO) mice. Sorted Lin-Sca1+c-kit+ cells from the bone marrow of WT or cKO mice were

co-cultured with OP9 stromal cell lines expressing either empty vector (OP9-MIG) (B) or a cDNA to ectopically express the Notch ligand _Dll1_ (delta-like 1) (OP9-DL1) (C). Under these

conditions, bone marrow progenitors will differentiate into B cells and T cells, respectively, in the presence of Flt3-L (5 ng/ml) and IL-7 (1 ng/ml). Cells derived from either genotype were

harvested at the time points shown, stained for markers of myeloid (Gr1, CD11b), B cell (CD19) and T cell (CD4, CD8, CD25, CD44) differentiation and analyzed by flow cytometry. *_P_ <

0.05, **_P_ < 0.01, ***_P_ < 0.001 (two-tailed _t-_test). Data are representative of two experiments with three technical replicates per genotype (B-C; mean and s.e.m.). SUPPLEMENTARY

FIGURE 4 AGED _HUWE1_ VAV1-CKO MICE EXHIBIT MYELOID EXPANSION AND ANEMIA. Complete blood counts (CBC) were measured from 4 month old _Huwe1_+/Y _Vav1_-Cre+ (WT) or _Huwe1__F/Y__ Vav1_-Cre+

(cKO) littermates. (A) Hemoglobin (Hb) content, (B) red blood cell (RBC) counts and (C) white blood cell (WBC) counts from peripheral blood of aged WT and cKO mice are shown. (D) Light

micrographs of stained blood smears (left, 20x) and histological sections of bone marrow (middle, 10x) and spleen (right, 5x) comparing tissues from WT (upper panels) and cKO (lower panels)

mice. Insets are of light micrographs taken at 63x magnification. (E) Peripheral blood mononuclear cells (PBMCs) and spleen suspensions (F) from aged WT or cKO mice were analyzed for

expression of mature cell markers by FACS. Average frequency of B cells (B220+), T cells (CD4+ helper or CD8+ cytotoxic) and granulocytes/monocytes (CD11b+ Gr1lo/hi) in each organ by cohort

is shown. *_P_ < 0.05, **_P <_ 0.01 (two-tailed _t-_test). Data shown represents analyses of nine WT and six cKO mice (A-C, E-F; mean and s.e.m.). SUPPLEMENTARY FIGURE 5 UNIQUE

GENE-EXPRESSION SIGNATURES IN N-MYCHI HSCS VERSUS N-MYCLO HSCS. (A) Schematic representing targeting strategy for _Mycn_M allele. The 3 exons of _Mycn_, mCherry cDNA and loxP-flanked

Neomycin resistance cassette are depicted. Recombination between the endogenous _Mycn_ locus and the long (5.6kb) and short (2kb) homologous arms off the targeting construct yields

_Mycn_MNeo. Expression of Cre recombinase leads to looping out of the Neo cassette and results in a functional _Mycn_M allele. mCherryhi and mCherrylo CD150+ HSPCs were sorted from pooled

bone marrow from _Mycn_M/M mice. Whole RNA was isolated from either population and amplified cDNA was hybridized to Affymetrix 430 2.0 microarrays. (B) Heat map of genes that were

differentially expressed (fold change > 2, _P <_ 0.05) between the N-mychi and N-myclo cells. Gene sets were tested for enrichment in expression among either population. Enrichment

plots for two gene sets that were highly enriched in the N-mychi HSPCs are shown: (C) Genes upregulated in small cell lung carcinoma where _MYCN_ is amplified and (D) Genes highly expressed

in stem cells from adult tissues. SUPPLEMENTARY FIGURE 6 IDENTIFICATION OF GENOME-WIDE TRANSCRIPTIONAL TARGETS OF N-MYC IN HSCS. (A) Smear plot illustrating global gene expression changes in

_Huwe1_-deficient HSCs. Differentially expressed transcripts are highlighted in red. (B) Chart showing distribution of N-myc peaks across genomic regions. (C) Heat map of ChIP-sequencing

read densities for N-Myc, H3K27ac, H3K4me3 and H3K27me3. All heatmaps are centered on N-myc peaks +/- 5kb and scaled to reads per million. SUPPLEMENTARY FIGURE 7 RESTORATION OF HSC FUNCTION

IN HUWE1- AND N-MYC-DKO MICE. (A) HSPCs sorted from bone marrow of _Huwe1_+/Y_Mycn_+/+_Mx1-_Cre+ (WT), _Huwe1_F/Y_Mycn_+/+_Mx1-_Cre+ (Huwe1 cKO), _Huwe1_+/Y_Mycn_F/F_Mx1-_Cre+ (N-myc cKO) or

_Huwe1_F/Y_Mycn_F/F_Mx1-_Cre+ (dKO) mice two weeks after pI:pC treatment were plated in complete methylcellulose medium (M3434) and colonies were enumerated, harvested and replated every 7d

for 3 passages. (B) Absolute number of phenotypic HSC as determined by FACS in the bone marrow from mice with indicated genotypes. (C) Representative FACS histograms showing GFP

fluorescence in HSC and myeloid progenitors from _Mycn_+/+_Myc_G/+_Mx1-_Cre+ or _Mycn_F/F_Myc_G/+_Mx1-_Cre+ mice 2 weeks after pI:pC administratrion. (D) Relative levels of _Mycn_ and _Myc_

mRNA were measured by qRT-PCR in Lin-Kit+Sca1+ cells from bone marrow of WT or N-myc cKO mice, using _Gapdh_ as an internal control. (E) HSPCs from Huwe1F/Y Cre- mice were transduced

simultaneously with Cre (or empty) retrovirus with a bicistronic Thy.1.1 reporter and a retroviral shRNA GFP construct targeting a previously identified Huwe1 substrate or _Renilla_

luciferase. Thy1.1+GFP+ cells were sorted 48h later, plated in methylcellulose medium and scored for colony formation as in (A). *_P_ < 0.05, **_P < 0.01_ (A, E; one-way ANOVA, B,D;

two-tailed _t-test_). Data are representative of two experiments with three technical replicates (A, E; mean and s.e.m.), analyses of four mice per genotype (B; mean and s.e.m.), or one

experiment with three biological replicates (C-D; mean and s.e.m. in D). SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES Supplementary Figures 1–7 and Supplementary Tables 1 and 2

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the maintenance and lymphoid commitment of hematopoietic stem cells. _Nat Immunol_ 17, 1312–1321 (2016). https://doi.org/10.1038/ni.3559 Download citation * Received: 30 September 2015 *

Accepted: 17 August 2016 * Published: 26 September 2016 * Issue Date: November 2016 * DOI: https://doi.org/10.1038/ni.3559 SHARE THIS ARTICLE Anyone you share the following link with will be

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