PLZF mutation alters mouse hematopoietic stem cell function and cell cycle progression

Stem cells are maintained in a body for a long period and have developed specific programs against a variety of physiological stresses in order to maintain their lifelong integrity. As with most tissues that have a high cellular turnover, hematopoietic tissue is sensitive to aging, which results in multiple functional defects that accumulate in HSCs from older individuals.¹  Aging HSCs have a reduction in their regenerative capacity and support decreased lymphoid lineage output together with increased myeloid output.^2,3  Surprisingly, the number of phenotypically defined HSCs increases with age; although it has long been known that aging has a major impact on the frequency and cell cycle kinetics of hematopoietic cell compartments,⁴  mechanisms underlying this phenomenon remain poorly understood.

The transcription factor promyelocytic leukemia zinc finger (PLZF), also known as Zbtb16, was first described as a transcriptional repressor rearranged in cases of acute promyelocytic leukemias associated with the t(11;17) translocation.⁵  Since then, PLZF has been shown to be involved in major developmental and biological processes such as spermatogenesis, hind limb formation, and hematopoiesis regulation,⁶  with versatile biological function⁷  regulated by posttranslational modifications⁸  and/or differential epigenetic cofactor recruitment.^9-11  Within the hematopoietic system, PLZF has essential roles in the maintenance and development of multiple hematopoietic cells^12-14  and is emerging as an important regulator of innate immune cell function by restraining inflammatory signaling programs.¹⁵ 

Published data indirectly suggest that PLZF could be an important regulator of the HSC, although a direct demonstration is still lacking. To this end, we studied the role of the transcription factor PLZF in the regulation of HSC fate.

C57BL/6-CD45.1 mice were purchased from Charles River Laboratories. B6-CD45.1/CD45.2 mice were bred in the Centre de Recherche en Cancérologie de Marseille mouse facility. Zbtb16^lu/lu C57BL/6-CD45.2 mice were derived from frozen Zbtb16^wt/lu embryos purchased from The Jackson Laboratory. The strategy and the antibody combination are presented, respectively, in supplemental Figure 1 and supplemental Table 1, available on the Blood Web site. Flow cytometry analyses were performed using a BD-LSRII cytometer and analyzed using BD-DIVA Version 6.1.2 software (Special Order Research Products; BD Biosciences). Lineage-negative (Lin⁻) cells were enriched using the Lineage Cell Depletion Kit (Miltenyi Biotec), and cell sorting was performed on a FACS AriaII (Special Order Research Products; BD Biosciences). For serial transplantation assays, 2 × 10⁶ total bone marrow (BM) cells were isolated from Zbtb16^lu/lu or wild-type (WT) littermates (C57BL/6-CD45.2) and transplanted IV into 6- to 8-week-old recipient mice (C57BL/6-CD45.1), lethally irradiated (8 Gy using the RS 2000 X-ray irradiator; Rad Source Technologies). For competitive transplantation assays, 1 × 10⁶ total BM cells of 8-week-old Zbtb16^lu/lu or 1-year-old WT mice (C57BL/6-CD45.2) were mixed with 1 × 10⁶ total competitor CD45.1/2 BM cells and injected IV.

Zbtb16^lu/lu and WT Lin⁻ fractions were stained for HSCs (Lin⁻/Sca⁺/Kit⁺, Fms-related tyrosine kinase 3, CD150⁺) and subsequently incubated with Ki-67 and 4,6 diamidino-2-phenylindole. For 5-bromo-2′-deoxyuridine (BrdU) analysis, mice were injected intraperitoneally with 3 mg of BrdU 18 hours before isolating BM cells. BM Lin⁻ cells were stained for HSCs, and phases of cell cycle were analyzed by measuring the incorporation of BrdU and 7-aminoactinomycin D using the FITC BrdU Flow Kit (BD Pharmingen). For myelosuppression induction, Zbtb16^lu/lu and WT mice were injected intraperitoneally with a single dose of 5-fluorouracil at a concentration of 150 mg/kg 5 days before BrdU injection. Apoptosis was measured with Annexin V (BD Pharmingen).

DNA microarrays were used to define and compare the transcriptional profiles of LSK cells isolated from Zbtb16^lu/lu mice and WT mice (n = 4). For details, see also supplemental Experimental Procedures. Raw transcriptomic data were deposited in ArrayExpress database (http://www.ebi.ac.uk/arrayexpress/; accession number: E-MTAB-3682).

Statistical analyses were performed with Mann-Whitney nonparametric tests.

To elucidate PLZF function in hematopoietic development, we analyzed the hematopoietic compartment of the Zbtb16^lu/lu mouse model.¹⁶  Zbtb16^lu/lu mice showed a twofold BM cellularity reduction (supplemental Figure 2A) due to their reduced size,¹⁶  but no difference was observed in the relative numbers of mature cells, phenotypically defined progenitors, or HSCs in comparison with WT BM, with the exception of the relative number of granulocyte-macrophage progenitors (GMPs), which was slightly increased in Zbtb16^lu/lu mice (supplemental Figure 2B-H). Because we revealed a reduction in Zbtb16^lu/lu HSC colony formation (supplemental Figure 3), which was in accordance with the loss of transforming potential observed in plzf-mutant HSCs,¹⁷  we assessed the functional competence of Zbtb16^lu/lu HSCs by serial transplantation assay (Figure 1A). This assay revealed a defect of Zbtb16^lu/lu HSCs to reconstitute hematopoietic organs after 3 successive transplants (Figure 1B). To better understand this functional defect, we analyzed Zbtb16^lu/lu HSC populations after 14 and 52 weeks of reconstitution. When analyzing the Zbtb16^lu/lu HSC pool at 14 weeks, we observed an increased frequency of immature LT-HSCs at the expense of the MPP1 fraction (Figure 1C-D; supplemental Figure 4A) in secondary and tertiary recipients. When frequency was converted to absolute number, a significant accumulation of LT-HSCs was observed in the BM of secondary Zbtb16^lu/lu recipients (supplemental Figure 4B). In addition, we observed a compromised reconstitution of common lymphoid progenitors, whereas GMPs were increased in tertiary recipients of Zbtb16^lu/lu BM (Figure 1E), resulting in a loss of B cells in favor of myeloid cells in peripheral blood, BM, and spleen of the Zbtb16^lu/lu tertiary reconstituted mice (supplemental Figure 4C). These results suggest that plzf mutation alters HSC functions through increasing the LT-HSC pool and by biasing the HSC differentiation potential toward the myeloid compartment. Because these features are characteristics of aged HSCs,^1,3,18  we further investigated Zbtb16^lu/lu HSCs upon aging. First, we analyzed the HSC compartment of 1-year-old Zbtb16^lu/lu mice and revealed that among the HSC population, LT-HSCs were significantly more expanded in aged Zbtb16^lu/lu mice compared with WT mice (Figure 1F). In addition, 1-year-old Zbtb16^lu/lu transplanted mice harbored an accelerated age–like phenotype, because the 2 aging characteristics were observed 1 transplant earlier when mice were analyzed at 52 rather than at 14 weeks after transplant (Figure 1G-H). We further compared young Zbtb16^lu/lu HSCs with 1-year-old HSCs for their competitive repopulation ability. Although old HSCs have a decreased regenerative potential, they keep a myeloid competitive advantage after the first transplant.¹  We showed that for 1-year-old HSCs, the Zbtb16^lu/lu HSCs have a myeloid competitive advantage (supplemental Figure 5) and contribute more to the LT-HSC compartment relative to the ST-HSC and MPP1 fractions (Figure 1I). Together, these results suggest that Zbtb16^lu/lu promotes a premature aging–like syndrome in mice after regenerative stress and imply a role for plzf in the control of age-related mechanisms in HSCs.

Comparative microarray-based gene expression data from Zbtb16^lu/lu and control HSCs identified 1428 genes differentially expressed (supplemental Table 2). Gene ontology enriched terms associated with the genes upregulated in the Zbtb16^lu/lu group included metabolic- and cell cycle–related processes, whereas gene ontology terms associated with downregulated genes were mainly involved in lymphoid functions (supplemental Table 3). Consistently, gene set enrichment analysis demonstrated a negative correlation between stemness and lymphoid signatures and a positive correlation between cell cycle signatures (in particular, genes involved in G1-S transition) and the Zbtb16^lu/lu genotype (Figure 2A), suggesting changes in HSC cell cycles induced by plzf mutation. Quantitative polymerase chain reaction analysis confirmed that Zbtb16^lu/lu LT-HSCs presented a deregulated cell cycle gene expression profile (Figure 2B), important for HSC integrity.¹⁹ 

Next, we focused on the effect of the Zbtb16^lu/lu mutation in HSC cell cycle regulation. Staining BM with Ki-67/4,6 diamidino-2-phenylindole showed a decreased proportion of the Zbtb16^lu/lu CD150⁺ hematopoietic stem and progenitor cells (HSPCs) in G1 phase, with an increased proportion of the cells in S-G2/M phase (Figure 2C). Measurement of BrdU incorporation 18 hours after injection coupled with 7-aminoactinomycin D staining demonstrated an accumulation of the Zbtb16^lu/lu CD150⁺ HSPCs in S phase, a phenotype that was in accordance with previous observations of PLZF controlling G₁/S and G₂/M checkpoints of the cell cycle^20,21  (Figure 2D; supplemental Figure 6). This accumulation was even stronger after hematopoietic recovery induced by myelosuppression (Figure 2E). Considering that increased apoptosis upon plzf deletion was previously observed in the germ line in association with increased proliferation and tubule degeneration,^16,22  we measured the level of apoptosis in Zbtb16^lu/lu CD150⁺ HSPCs. We demonstrated a significant increase in apoptosis in Zbtb16^lu/lu HSCs in comparison with WT cells (Figure 2Fi) that could account for the limited accumulation of Zbtb16^lu/lu HSCs in young plzf-mutant mice. Interestingly, apoptosis was not as increased in transplanted Zbtb16^lu/lu HSCs, a condition in which HSCs accumulate (Figure 2Fii). Although, we have no direct evidence that the Zbtb16^lu/lu-induced cell cycle deregulation is linked to aging, especially considering recent studies reporting inconsistent cell cycle changes upon aging,^23,24  our results support the links among plzf function, cell cycle regulation, and HSC integrity. Because epigenetic processes are known to regulate age-dependent functional changes,^25,26  it will be important to study the epigenetic cofactors of plzf that have the ability to regulate cell cycle gene expression during aging.

The authors thank M. L. Thibult and F. Mallet for assistance with the use of the cytometry and cell sorting facility; F. Bardin, R. Castellano, and L. Pouyet for help with irradiated mice injections; J. C. Orsoni and P. Gibier for assistance in the animal facility; and A. Saurin and C. Chabannon for critically reading the manuscript.

This work was supported by the Fondation de France (201200029166), the Fondation pour la Recherche Médicale grant INE201000518534, the Fondation ARC pour la recherche sur le cancer, the Cancéropôle Provence-Alpes-Côte d’Azur (C.V.-F.), the Ligue Nationale Contre le Cancer (A.V.), and Institut Thématique Multi-Organismes Cancer (M.P.).

Contribution: C.V.-F. designed the study, performed the experiments, analyzed the data, and cowrote the paper; N.P., A.V., M.P., M.K., and A.-M.I. performed the experiments and analyzed the data; P.F., G.T., and F.B. performed the transcriptome analysis; E.D. initiated and directed the project and cowrote the paper. All authors read and approved the final manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Estelle Duprez, INSERM U1068, Centre de Recherche en Cancérologie de Marseille, BP 30059, 27, Blvd Lei Roure, 13273 Marseille Cedex 09, France; e-mail: estelle.duprez@inserm.fr.