Nucleophosmin Regulates Cell Cycle Progression and Stress Response in Hematopoietic Stem/Progenitor Cells*

Nucleophosmin (NPM) is a multifunctional protein frequently overexpressed in actively proliferating cells. Strong evidence indicates that NPM is required for embryonic development and genomic stability. Here we report that NPM enhances the proliferative potential of hematopoietic stem cells (HSCs) and increases their survival upon stress challenge. Both short term liquid culture and clonogenic progenitor cell assays show a selective expansion of NPM-overexpressing HSCs. Interestingly, HSCs infected with NPM retrovirus show significantly reduced commitment to myeloid differentiation compared with vector-transduced cells, and majority of the NPM-overexpressing cells remains primitive during a 5-day culture. Bone marrow transplantation experiments demonstrate that NPM promotes the self-renewal of long term repopulating HSCs while attenuated their commitment to myeloid differentiation. NPM overexpression induces rapid entry of HSCs into the cell cycle and suppresses the expression of several negative cell cycle regulators that are associated with G1-to-S transition. NPM knockdown elevates expression of these negative regulators and exacerbates stress-induced cell cycle arrest. Finally, overexpression of NPM promotes the survival and recovery of HSCs and progenitors after exposure to DNA damage, oxidative stress, and hematopoietic injury both in vivo and in vitro. DNA repair kinetics study suggests that NPM has a role in reducing the susceptibility of chromosomal DNA to damage rather than promoting DNA damage repair. Together, these results indicate that NPM plays an important role in hematopoiesis via mechanisms involving modulation of HSC/progenitor cell cycle progression and stress response.

The MIEG3 and MIEG3-NPM plasmids (10 g each) were used to produce retroviral supernatant.
Retroviral Production and Infection-The MIEG3 and MIEG3-NPM retroviruses were prepared by Dr. van der Loo at the Viral Vector Core, Cincinnati Children's Research Foundation, Cincinnati Children's Hospital Medical Center, Cincinnati, OH. Retroviral supernatant was collected at 36,48, and 60 h, respectively, after transfection. Lin Ϫ or LSK cells were plated onto Retronectin-coated (Takara-Shuzo) non-tissue culture 24-well plates and prestimulated for 2 days in IMDM medium containing 20% fetal calf serum, 100 ng/ml stem cell factor (SCF), 20 ng/ml interleukin-6 (IL-6), and 50 ng/ml Flt-3 ligand (Flt-3L) (Peprotech). Cells were then exposed to the retroviral supernatant at multiplicity of infection (MOI) of 2-5 for 3 h at 37°C in the presence of 4 g/ml polybrene (Sigma). Cells were centrifuged at 600 ϫ g for 45 min. Infection was repeated two times and infection efficiency was assessed by the detection of green fluorescent protein (GFP)-positive cells by fluorescence-activated cell sorting (FACS).
Clonogenic Progenitor Assays-The transduced cells were cultured at 1000 cells/ml in a 35-mm tissue culture dish in 4 ml of semisolid medium containing 3 ml of MethoCult M 3134 (Stem Cell Technologies) and the following growth factors: 100 ng/ml SCF, 10 ng/ml rhIL-3, 100 ng/ml granulocyte colony-stimulating factor (G-CSF), and 4 units/ml erythropoietin (Peprotech). On day 10 after plating, the colony number was counted and photographed. Colony growth results were expressed as mean (of triplicate plates) Ϯ S.D. colonies per 1000 cells plated. Levels of significance were determined using Student's t distribution.
Single Cell Analysis-Single LSK GFPϩ cells were sorted and deposited into each well of 96-well plates and cultured in 15 l of IMDM medium containing cytokines. The presence of a single cell in individual wells was verified, and wells with no cell or more than one cell were excluded from analysis. Cell division was monitored at 24-h intervals for at least 4 days.
Apoptosis Assay and Cell Cycle Analysis-Cells were stained with annexin V and propidium iodide using BD ApoAlert Annexin V kit (BD PharMingen) in accordance with the manufacturer's instructions. Apoptosis was analyzed by quantification of annexin V positive cell population by flow cytometry. For cell cycle analysis, cells were fixed by 0.25% formaldehyde in phosphate-buffered saline, permeabilized with 0.3% Nonidet P-40, and then stained with propidium iodide containing 1 mg/ml RNase A, followed by FACS analysis of the G 0 /G 1 , S, and G 2 /M populations.
Immunocytochemistry-Cells were cytospun onto slides and fixed in ice-cold methanol for 5 min in Ϫ20°C. After air drying, cells were blocked for 1 h with 5% normal serum. Then cells were incubated with primary antibodies (p53 or p21, both from Cell Signaling Technology) in phosphate-buffered saline with 2% normal serum at room temperature for 1 h. After extensive washes, cells were incubated with PE-conjugated secondary antibody (Jackson Laboratories, Bar Harbor, ME). DNA was then labeled with DAPI (4,6-diamidino-2phenylindole; Sigma). Slides were finally mounted in mounting medium (Vector). Cells were viewed and photographed using a Leica DM IRB microscope at ϫ10 magnification with an ORCA-ER C4742-95 camera (Hamamatsu). The captured images were pro-cessed using OpenLab 3.1 software (Improvision) and displayed with Adobe Photoshop V6.0.
RNA Isolation and Reverse Transcriptase (RT)-PCR-Total RNA was prepared with RNeasy kit (Qiagen) following the manufacturer's procedure. Reverse transcription was performed with random hexamers and Superscript II RT (Invitrogen) and was carried out at 42°C for 60 min and stopped at 95°C for 5 min. These reactions were followed by PCR using two primers specific for the respective NPM and 3ϫFLAG sequences: 5Ј-TCCGCTGCAGACAGACTGGCCAG-3Ј (sense; within NPM sequence) and 5Ј-CATCGCGCACATCCAGCCGAGC-3Ј (antisense; within 3ϫFLAG sequence), with thermal cycling parameters: 95°C for 5 min; 15, 20, or 30 cycles at 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min; and a final extension at 72°C for 10 min.
Short and Long Term Competitive Repopulating Analysis-Agematched female congenic B6.SJL-PtrcaPep3b/BoyJ (B6.BoyJ; CD45.1 ϩ ) mice (Jackson Laboratories) were used as transplant recipients. These mice were lethally irradiated (9.5 Gy, 110 cGy/min, 137 Cs ␥-rays) and injected intravenously with 1 ϫ 10 5 Lin-BM cells transduced with MIEG3 or MIEG3-NPM viruses, mixed with 5 ϫ 10 5 competitor cells. Donor-derived repopulation in recipients was assessed by the proportion of leukocytes in peripheral blood that expressed the GFP protein by flow cytometry. Short and long term engraftment and multilineage repopulation analysis of donor cells were performed at 1 month and 4 month post-transplantation. For secondary BM transplantation, BM cells from all individual recipients were pooled and then 5 ϫ 10 4 , 2 ϫ 10 5 , and 2 ϫ 10 6 bone marrow cells were injected into cohorts of eight female lethally irradiated recipients per cell dose. Recipients were analyzed at 4 and 16 weeks post-transplant. For transplants using LKS ϩ cells, recipient mice received 1,000 freshly isolated cells and were analyzed at 1, 4, and 6 month intervals.
Comet Assay-The generation of DNA strand breaks was assessed by the single cell gel electrophoresis (comet) assay (22), using a Fpg-FLARE (fragment length analysis using repair enzymes) comet assay kit in accordance with the manufacturer's instructions (Trevigen). For each experimental point at least three different cultures were analyzed, and 50 cells were evaluated from each culture. Comet tail length and tail moment were measured under a fluorescence microscope (Nikon model 027012) using an automated image analysis system based on a public domain NIH Image program (23).
Statistics-Data were analyzed statistically using a two-tail Student's t test. The level of statistical significance stated in the text was based on the p values. p Ͻ 0.05 was considered statistically significant.

NPM Promotes HSC and Progenitor
Cell Expansion-To gain insight into the role of NPM in hematopoiesis, we overexpressed FLAG-tagged NPM in murine BM LSK cells by retroviral gene transfer (Fig. 1A). We purified GFP-positive cells by FACS and confirmed the expression of the NPM transgene by RT-PCR using primers complementary to the NPM and FLAG-coding sequences (Fig. 1B). We generally achieved transduction efficiency of greater than 50% (Fig.  1C). We cultured vector-and NPM-transduced LSK cells for 10 days and analyzed cell proliferative potential. As shown in Fig. 2A, NPMtransduced cells expanded significantly faster than vector-trans-  duced cells during 4 days of culture (4.2-and 6.4-fold at days 3 and 4, respectively, compared with 1.7-and 2.3-fold with vector-transduced cells). Notably, we observed that the growth rate of NPMtransduced cells peaked at days 5 and 6. Further analysis indicated a modest increase in cell death (Fig. 2B) and erythroid lineage (Ter119 ϩ ) differentiation in these cells (Table 1). Thus, a modest decline in proliferation of the NPM-transduced cells might be caused by an increase in terminal differentiation, which could lead to an increase in cell death. Indeed. We found that NPM expression increased erythroid reconstitution in our bone marrow transplantation experiments (see Fig. 4, below). We next determined whether NPM confers growth advantage to HSC and progenitor cells. During the 10-day period, the content of GFP ϩ cells in vector group (cells transduced with vector alone) remained steady at ϳ50% (Fig. 2C), which was close to initial transduction efficiency of 52%. Significantly, NPM-transduced cells (54% transduction efficiency) exhibited a significant growth advantage to total cells, with more than 80% of the cells expressed eGFP/NPM at day 10 ( Fig. 2C).
We next asked if NPM expression increases clonogenic progenitor activity. We found that total number of colonies formed by NPM-transduced cells was more than 2-fold higher than that of vector-transduced cells (Fig. 2D). Significantly increased series-plating efficiency was also observed with NPM-transduced LSK cells compared with vector-transduced cells (Fig. 2E). In addition, NPM-expressing LSK cells generally formed larger colonies than vector-transduced cells did (data not shown). Taken together, these results indicate a selective expansion of NPM-overexpressing HSC and progenitor cells.
NPM Overexpression Decreases Differentiation of Myeloid-committed Progenitors-Given that NPM overexpression promotes HSC expansion, we wondered if NPM affected differentiation of these primitive cells. Interestingly, NPM expression decreased the differentiation of LSK cells toward myeloid lineage, with a 3-fold decrease in cell population expressing myeloid marker Gr-1 and CD11b (Fig.   3, A and B). Moreover, more than 50% of NPM-expressing cells retained expression of primitive marker ScaI and c-Kit compared with 20% of vector-transduced cells (Fig. 3C). Consistent with this, NPM-expressing cells showed loss of granulocyte maturation and accumulation of myeloblasts and early progenitors (mostly promyelocytes) at day 10 of culture (Fig. 3D). In contrast, myeloid differentiation in vector-transduced cells seemed to be morphologically normal, as evidenced by mature neutrophil granulocytes and macrophages (Fig. 3D). These results indicate a selective expansion of NPM-expressing HSC and progenitor cells. NPM Expression Increases HSC Repopulating Capacity-Next, we performed bone marrow transplantation to evaluate the in vivo function of NPM-expressing hematopoietic progenitor cells. Significantly, NPM-transduced cells constituted more than 40% of the peripheral blood cells at 16 weeks after transplantation, compared with less than 10% reconstitution by vector-transduced cells (Fig. 4B). Examination of lineage distribution of GFP ϩ and GFP Ϫ cells in the peripheral blood cells from the 16-week post-transplanted mice shows that 10% of the cells were Gr-1/CD11bϩ, 30% were either B220ϩ or CD3eϩ, and 16% were Ter119ϩ, indicating that HSCs expressing NPM has the ability of multilineage reconstitution. However, the percentage of myeloid (Gr-1/CD11b ϩ ) cells was significantly reduced in NPM/GFP ϩ fraction compared with that reconstituted by non-transduced cells (Fig.  4, C and D). This is in agreement with the in vitro data (Fig. 3) showing that NPM inhibits myeloid differentiation. Interestingly, NPM expression increased proportion of erythroid (Ter119 ϩ ) cells (16% compared with 6% in vector/GFPϩ fraction; p Ͻ 0.05; Fig. 4, C and D), suggesting that NPM may be a positive regulator of erythropoiesis. Similar results were obtained with secondary transplantation, which shows that NPM promoted the self-renewal of long term repopulating HSCs while attenuated their commitment to myeloid differentiation ( Table 2).
NPM Is a Positive Regulator of HSC Cell Cycle Progression-To address how NPM expression enhances HSC and progenitor cell expansion, we analyzed cell cycle profile of NPM-transduced LSK cells. As shown in Fig. 5A, NPM overexpression prominently reduced cells in the G 0 /G 1 phase (55.6% compared with 65.4% with vector-transduced cells). This coincided with a significant increase in cells in the S phase (34% compared with 26.1% with vector-transduced cells). BrdU labeling experiments show that the percentage of cells entering the cell cycle was significantly higher in NPM-transduced cells than in vector-transduced   JUNE 16, 2006 • VOLUME 281 • NUMBER 24 cells (45% compared with 30%; Fig. 5B). To further determine the cellular mechanism by which NPM promotes HSC and progenitor cell expansion, we employed a single-cell assay to see if overexpression of NPM regulates the recruitment of HSCs into the cell cycle in response to cytokine stimulation. At 24 h, about 20% of NPM-transduced LSK cells underwent at least one division compared with ϳ12% of vectortransduced cells ( p Ͻ 0.05; Fig. 5C). Less dramatic but still significant ( p Ͻ 0.05) result was obtained with the pair of cell lines at 48 h when nearly 70% of NPM-transduced cells have undergone division compared with 56% of vector-transduced cells (Fig. 5D).

NPM Suppresses the Expression of Negative Cell Cycle
Regulators-To investigate the molecular mechanism whereby NPM promotes HSC and progenitor cell cycle progression, we examined the expression of cell cycle regulatory molecules in vector-and NPM-transduced cells. We focused on p53, p21 WAF1 , p27 KIP1 , p16 INK4A , and p19 Arf , which have been known to negatively regulate cell cycle progression in HSC and progenitor cells (24,25). We found that more than 70% of the control vector-transduced LSK cells were stained positive for p53, significantly higher than NPM-transduced cells (20%; p Ͻ 0.05; Fig. 6, A and B). Higher accumulation of p21 WAF1 was also observed in vector-transduced cells than in NPM-transduced cells. Gene expression profiling by RT-PCR shows that the expression of p16 Ink4a was significantly decreased by NPM expression (Fig. 6C). We also examined the expression of the positive regulators of G 1 -to-S transition including cyclins A, B, D, and E, and cyclin-dependent kinases cdc2 (Cdk1), Cdk2, -4, and -6. Among them, we found only cyclin A that is up-regulated by NPM overexpression (Fig. 6D). It should be noted that the levels of phosphorylated Cdk1 and Cdk2 and the kinase activities of Cdk1, -2, and -4 were significantly higher in NPM-transduced cells than in vector cells (data not shown).
NPM Enhances HSC and Progenitor Survival in Response to DNA Damage and Hematopoietic Stress-Because we previously showed that forced expression of NPM reduces cell death induced by tumor necrosis factor ␣ in lymphocytes derived from a Fanconi anemia group C patient (18), and because cells from FA patients exhibit hypersensitivity to DNA cross-linking agents such as mitomycin C (MMC) and oxidative stress (26 -30), we investigated the effect of NPM expression in HSC and progenitor cells isolated from mice deficient for the Fanconi group C gene (Fancc Ϫ/Ϫ ). Transduced WT and Fancc Ϫ/Ϫ LSK cells were challenged with DNA damage (MMC) or oxidative stress (H 2 O 2 ). Fancc Ϫ/Ϫ HSC and progenitor cells were extremely sensitive to MMC with virtually no viable cells left after 4 days (Fig. 7A). NPM expression dramatically increased survival of Fancc Ϫ/Ϫ cells exposed to MMC or H 2 O 2 (Fig.  7, A and B). A milder but still significant increase in survival was also observed in WT NPM-transduced cells. This was apparently because of a significant reduction in stress-induced apoptosis in the NPM-transduced cells, as determined by annexin V binding (Fig. 7C).
To further investigate the in vivo role of NPM in the response of HSC and progenitor cells to stress, we determined the effect of NPM overexpression on hematopoietic recovery in mice treated with 5-FU. Analysis of peripheral blood from 5-FU-treated recipients showed that NPMtransduced cells were able to recover from hemoablation rapidly and by only 7 days post 5-FU treatment, the number of GFP-positive cells has reached at least the pretreatment level (data not shown). Furthermore, mice transplanted with NPM-transduced HSC and progenitors showed accelerated multilineage recovery within 7 days after 5-FU treatment ( Table 3). In contrast, vector-transduced cells recovered slowly and reached ϳ70% of the level before 5-FU treatment (data not shown).
To further investigate the mechanism by which NPM protects HSC and progenitor cells from DNA-damaging stresses, we determine oxidative stress-induced DNA damage in HSC/progenitor cells by the comet assay (31,32), which measures specifically oxidative DNA damage including single-and double-strand DNA breaks (32). To augment the stress effect, we also used Fancc Ϫ/Ϫ HSC and progenitor cells which are hypersensitive to H 2 O 2 treatment (Fig. 7). NPM expression significantly reduced oxidative DNA damage in both untreated and H 2 O 2treated Fancc Ϫ/Ϫ HSC and progenitor cells, as well as in H 2 O 2 -treated WT cells (data not shown). The reduced levels of oxidative DNA dam- age observed in NPM-overexpressing HSC/progenitor cells suggest that NPM could protect DNA from oxidative attack, thereby decreasing DNA strand breaks, or alternatively, NPM could enhance the damage response/repair process. To distinguish between these possibilities, we treated BM HSC and progenitor cells transduced with vector alone or NPM with H 2 O 2 and conducted a time course study to assess DNA repair kinetics by examining the levels of DNA strand breaks. NPMoverexpressing HSC/progenitor cells consistently showed lower levels of DNA strand breaks than vector-transduced cells (data not shown). However, there was no significant difference between the two cell lines  in terms of the kinetics of DNA damage repair, as measured by the remaining amounts of DNA strand breaks over a period of 60-min posttreatment. This suggests that cells expressing NPM accumulated less oxidative DNA damage probably because of its role in reducing the susceptibility of chromosomal DNA to damage rather than promoting DNA damage repair.

DISCUSSION
Hematopoiesis is an ordered process of proliferation and differentiation, which leads to the generation of mature blood cells from a rare population of pluripotent HSCs (33). The hematopoietic system utilizes a variety of homeostatic mechanisms for regulation of HSCs in order to sustain blood cell production throughout life. A fine balance between self-renewal and differentiation allows for the size of the HSC pool to be maintained and for the body to be continually supplied with the lymphoid, myeloid, and erythroid lineages. The fate of HSCs for self-renewal or for differentiation is decided by both extrinsic and intrinsic factors. Studies with mutant gene knock-in in mice have provided insights into some of positive and negative regulators for hematopoiesis (34). For example, certain cytokines such as stem cell factor, thrombopoietin, flt-3 ligand, and their receptors have been shown to play important roles in the regulation of HSC self-renewal and differentiation (35)(36)(37). Recently, other receptor/ligand signaling pathways such as the Notch and Wnt signaling pathways have been found to regulate HSC selfrenewal (38,39). And a variety of transcription factors such as Ikaros and Bmi-1 and cell cycle regulators like the G 1 -specific inhibitor p21 WAF1 have emerged as key regulatory components involved in proliferation and differentiation of HSCs (24,25,40). Here we report that NPM is another regulatory molecule that plays a role in HSC proliferation, differentiation, and stress response.
Our results show for the first time that NPM enhances the proliferative potential of HSC and progenitor cells, as demonstrated by using both ex vivo and BM transplantation models. This finding is consistent with a recent report by Grisendi et al. (16) showing that NPM is essential for primitive hematopoiesis. Mice deficient for NPM die at early embryonic stage because of severe anemia (16). We attribute the enhanced proliferative effect of NPM to its ability to induce rapid entry of hematopoietic progenitors into the cell cycle, probably via promoting G 1 -to-S transition. Our study of the mechanism underlying this phenomenon reveals that the positive role of NPM in HSC and progenitor cell cycle progression is associated with suppression of the negative cell cycle regulators p53, p21 WAF1 and p16 Ink4a . Recent findings in mice deficient for Bmi-1, ATM, and JunB have highlighted the impact of G 1 -S regulation by p53, p21 WAF1 , and p16 Ink4a in primitive hematopoiesis (24,25,33). In contrast to previous reports that NPM binds the tumor suppressors p53 and p19 Arf and may play a role in the stability of these proteins (17,41), overexpression of NPM in HSC and progenitor cells reduced p53 protein levels, which was correlated with decreased expression of p53 target protein p21 WAF1 , whereas down-regulation of NPM by siRNA elevated p53 levels accompanied by increased p21 WAF1 in response to stress. We failed to detect the effect of NPM on p19 Arf in either NPM-overexpressing or NPM-knockdown HSC and progenitor cells. Among the positive regulators of G 1 -to-S transition, we found only cyclin A that is up-regulated by NPM overexpression in BM progenitors. Notably, overexpression of cyclin A in cultured cells affects cell cycle progression and leads to accelerated entry into S phase (42). However, down-regulation of endogenous NPM in HSC and progenitor cells did not affect cyclin A expression in the steady state or in response to stress. This suggests that cyclin A may not be the target of NPM overexpression. It has been reported that NPM is a substrate of cyclin-de-pendent kinases Cdk1 (cdc2) and Cdk2 (43,44), which could complex with cyclin A in the process of cell cycle progression. We are in the process of investigating whether NPM plays a role in regulation of the formation or/and activity of cyclin A/Cdk complexes.
Intriguingly, we observed the attenuation of myeloid differentiation in HSC/progenitor cells overexpressing NPM. Significantly lower levels of mature myeloid markers (Gr-1/CD11b) were expressed in NPMoverexpressing LSK progeny or mice reconstituted with NPM-transduced progenitors. In addition, majority of the NPM-overexpressing cells in day-5 liquid culture and in the increased series-plating colonies showed loss of granulocyte maturation and accumulation of myeloblasts and early progenitors (mostly promyelocytes) and expressed primitive progenitor markers ScaIϩc-kitϩ, thus arguing for a direct effect of NPM overexpression on differentiation of myeloid progenitor cells. The detection of significantly lower myeloid (Gr-1/CD11b ϩ ) cells in recipients reconstituted with NPM-transduced BM progenitors compared with that reconstituted by non-transduced cells further supports the notion that the observed block in myeloid differentiation is intrinsic to the NPM-transduced BM progenitor cells. The cause for this phenomenon may involve preferential proliferation over differentiation of NPM-overexpressing myeloid precursors. It has been long known that NPM expression is elevated in actively proliferating cells but decreased when cells are induced to differentiate (1,2,6). However, the mechanism by which NPM selectively inhibits myeloid differentiation remains to be determined.
Overexpression of NPM protects HSCs and progenitors exposed to MMC or hydrogen peroxide, suggesting that NPM can protect cells from DNA damage and oxidative stress. We recently demonstrated that NPM protects cells from apoptotic cell death induced by diverse stresses through a mechanism involving inhibition of the p53 tumor suppressor protein (19,20). Others have reported that UV radiation induces expression and nuclear translocation of NPM, which in turn enhances DNA damage repair and prevents apoptosis (17,45). More recently, work with NPM-deficient mice strongly indicates that NPM is essential for the maintenance of genomic stability and cell survival (16,17). Still, it seems to be contradictory that NPM protects cells from DNA damage and at the same time suppresses p53 in response to stress, the latter of which may allows cells to proceed with cycling without repairing the DNA damage. However, p53 is also a major factor that influences life-or-death decision of the cell. By regulating p53 in response to DNA-damaging stress, NPM may provide a survival mechanism which allows the cell to ultimately repair the damage. This may be particularly important for such types of cells as HSC and progenitor cells, which are needed for the production of billions of blood cells each day.
How does NPM protect cells from DNA damage induced by genotoxic stress? NPM-overexpressing HSC and progenitor cells accumulated less stress-induced DNA damage. By examining the kinetics of DNA repair in these cells, we show that NPM could protect DNA from oxidative damage, thereby reducing the levels of DNA strand breaks. NPM binds both DNA and RNA (46), and functions as a histone chaperone during the assembly of new nucleosomes and after DNA lesions are repaired (47). NPM also plays roles in chromatin remodeling and assembly (48). NPM has been shown to enhance UV-induced DNA repair (49), is a component B cell-specific multiprotein complex that possesses DNA recombination activity (50). In vitro experiments with recombinant NPM showed that the protein promoted DNA singlestrand reannealing and mediated D-loop formation (50). However, our results with repair kinetics do not support the idea that NPM enhances or facilitates the repair of DNA strand breaks induced by oxidative stress. Instead, we argue a role for NPM in reducing the susceptibility of chromosomal DNA to damage. Taken together, our study indicates that NPM plays an important role in hematopoiesis via mechanisms involving modulation of HSC and progenitor cell differentiation, cell cycle progression, and stress response.