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AML1/Runx1 Negatively Regulates Quiescent Hematopoietic Stem Cells in Adult Hematopoiesis

This information is current as Motoshi Ichikawa, Susumu Goyama, Takashi Asai, Masahito of September 28, 2021. Kawazu, Masahiro Nakagawa, Masataka Takeshita, Shigeru Chiba, Seishi Ogawa and Mineo Kurokawa J Immunol 2008; 180:4402-4408; ; doi: 10.4049/jimmunol.180.7.4402

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References This article cites 37 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/180/7/4402.full#ref-list-1 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

AML1/Runx1 Negatively Regulates Quiescent Hematopoietic Stem Cells in Adult Hematopoiesis1

Motoshi Ichikawa, Susumu Goyama, Takashi Asai, Masahito Kawazu, Masahiro Nakagawa, Masataka Takeshita, Shigeru Chiba, Seishi Ogawa, and Mineo Kurokawa2

Transcription factor AML1/Runx1, initially isolated from the t(8;21) chromosomal translocation in human , is essential for the development of multilineage hematopoiesis in mouse . AML1 negatively regulates the number of immature hematopoietic cells in adult hematopoiesis, whereas it is required for megakaryocytic maturation and lymphocytic development. However, it remains yet to be determined how AML1 contributes to homeostasis of hematopoietic stem cells (HSCs). To address this issue, we analyzed in detail HSC function in the absence of AML1. Notably, cells in the Hoechst 33342 side population fraction are increased in number in AML1-deficient marrow, which suggests enrichment of quiescent HSCs. We also found an increase in HSC number within the AML1-deficient using limiting dilution bone marrow transplantation assays. Downloaded from These results indicate that the number of quiescent HSCs is negatively regulated by AML1. The Journal of Immunology, 2008, 180: 4402–4408.

ammalian hematopoietic development is believed to (AML) (4) and is recognized as one of the most important tran- derive from two distinct cellular origins. In mice, gen- scription factors which regulate mammalian hematopoietic ho- M eration of the primitive erythroid lineage consisting of meostasis. AML1 is specifically required for the development http://www.jimmunol.org/ large and nucleated erythrocytes, known as primitive hematopoi- of definitive hematopoiesis in the embryonic stage as shown by esis, occurs around day 7.5 postcoitus in the yolk sac (1). The gene-targeting experiments in mice (5, 6). In contrast, as we and second wave of hematopoiesis, which is called definitive hemato- others have previously shown, loss of AML1 in the adult stage poiesis and consists of enucleated erythrocytes, myeloid cells, and does not cause complete loss of hematopoiesis (7–9). AML1- lymphoid progenitors, emerges later in the fetal around day deleted adult mice show a decrease in due to a matu- 9.5 postcoitus and results in expansion of hematopoietic stem cells rational defect of the as well as block of lym- 3 (HSCs) and generation of cells (2, 3). At the center of stem phocyte development, while the fraction of immature cell self-renewal and lineage commitment decisions lie the pre- hematopoietic progenitors is expanded (7). These results sug- by guest on September 28, 2021 cisely regulated expression levels of lineage-specific transcription gest that AML1 is not required for the maintenance of HSCs, factors. The role of specific transcription factors in hematopoietic but for the differentiation and maturation of more committed fate decisions has derived largely from genetic strategies, cells. It is also suggested that embryonic development of hema- primarily gene-targeting and transgenic or retroviral overexpres- topoiesis and maintenance of adult hematopoiesis is, respectively, sion experiments. From the growing body of experimental results, regulated by distinct molecular mechanisms, which is also sup- several transcription factors have been found to play critical roles ported by analyses on conditional knockout mice which target he- in physiology. matopoietic genes, such as SCL and NOTCH1 (10, 11). Transcription factor AML1, also known as RUNX1, CBFA2, or Impaired function of AML1 in the hematopoietic system is as- PEBP2␣B, is initially isolated from the breakpoint of t(8;21) chro- sociated with a variety of human hematological malignancies in- mosomal translocations found in acute myelogenous leukemia cluding t(8;21)-positive AML, immature-type AML defined as M0 by French-American-British classification (12), myelodysplastic Department of Hematology and Oncology, Graduate School of Medicine, University syndromes (13), and familial disorder with propensity to of Tokyo, Tokyo, Japan AML (14). Mice with bone marrow cells that express leukemic Received for publication May 3, 2007. Accepted for publication January 21, 2008. chimeric protein AML1/ETO (AML1/MTG8) produced by t(8;21) The costs of publication of this article were defrayed in part by the payment of page chromosomal translocation do exhibit a myeloproliferative pheno- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. type, which was susceptible to the development of leukemia when 1 This work was supported in part by grants from the Ministry of Education, Culture, the mice were treated with alkylating agents to induce additional Sports, Science and Technology (KAKENHI 18013014 to M.K.), Japan Society for genetic aberrations (15–19). Because AML1/ETO acts dominant- the Promotion of Science (Grant-in-Aid for Fellows 17-10666 to M.I.), and the Min- negatively over normal AML1 function, the myeloproliferative istry of Health, Labour and Welfare (Health and Labour Sciences Research Grants to M.K.). phenotype has been explained by the expansion of the HSC frac- 2 Address correspondence and reprint requests to Dr. Mineo Kurokawa, Department tion in these mice caused by suppressed AML1 activities. As is of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, recently reported, however, introduction of a splicing variant form Bunkyo-ku, Tokyo, 113-8655, Japan. E-mail address: [email protected] of AML1/ETO in the bone marrow cells causes the development 3 Abbreviations used in this paper: HSC, hematopoietic stem cell; 7-AAD, 7-amino- of leukemia in mice independently of second-hit mutations by al- actinomycin D; SP, side population; KSL, lineage-negative, c-Kit-positive, and Sca- 1-positive; cKO, conditional knockout; CRU, competitive repopulating unit; Ct, cycle kylating agents (20). In addition, AML1-deficient bone marrow threshold; GMP, - progenitor; CMP, common myeloid cells fail to show infinite replating capacity in contrast to AML1/ progenitor. ETO-expressing bone marrow cells (7). Therefore, the expansion Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 of hematopoietic stem cells of AML1/ETO may not be caused only www.jimmunol.org The Journal of Immunology 4403 by suppression of AML1 function, but other molecular pathways Detector. For each sample and standard mixture, cycle threshold readings used by the ETO portion are necessary for the development of (Ct) were determined using the Sequence Detection Software (Applied ⌬ ϭ Ϫ leukemia. In contrast to our observation that AML1- deficient bone Biosystems) and the Ct ( (Ct for Zfy2) (Ct for GAPDH) amplified from each of the same sample) value was calculated. A standard curve was marrow contains expanded hematopoietic progenitors, it has been generated by plotting the known percentage of male DNA to the logarith- reported that bone marrow cells with the heterozygously inacti- mic y-axis and ⌬Ct values to the linear x-axis. The detection limit of male vated AML1 locus show a decrease in competitive repopulating cells in this was estimated to be ϳ0.3%. Excision of the Aml1 locus capacity (21) and lower chimerism in long-term competitive re- was confirmed for the samples with high male chimerism using nested PCR as described previously (7). population assays (9), Therefore, it is essential to know whether deletion of AML1 causes expansion of the most primitive hema- topoietic stem cells to understand the molecular mechanisms of hematological malignancies related to abnormal AML1 functions. Staining bone marrow cells for Hoechst 33342 side population (SP) cell fraction and G cell cycle fraction was done as described previously (24– To address this issue, we analyzed in detail HSC function in the 0 26). Flow cytometry was performed using BD LSR II apparatus (BD Bio- absence of AML1 using conditional AML1 knockout mice. sciences) for nonsorting analyses and FACSAria apparatus (BD Bio- sciences) for sorting experiments. The following mAbs were used for Materials and Methods analyses on hematopoietic stem cells: FITC-conjugated rat mAb to CD34 Animals ( RAM34; BD Biosciences), PE-conjugated Ab to Sca-1 (clone D7; BD Biosciences), biotin-conjugated Ab to CD3␧, CD4, CD8a, B220, Gr-1, AML1 conditional knockout mice (7) were crossed with Mx-cre transgenic Mac-1, and TER-119 (clones 145-2C11, RM4-5, 53-6.7, RA3-6B2, RB6- mice (22) and maintained at the C57BL/6J background at the Section of 8C5, M1/70, and TER-119; all from BD Biosciences, except for CD8a and Animal Research, Division of Research Resources and Support, Center for Gr-1 Abs from eBioscience) visualized by PerCP-conjugated streptavidin; Downloaded from Disease Biology and Integrative Medicine, Faculty of Medicine (Univer- BD Biosciences) and allophycocyanin-conjugated Ab to c-Kit (clone 2B8; sity of Tokyo, Tokyo, Japan). C57BL/6J mice were purchased from Clea BD Biosciences). For the analysis of the G0 cell cycle status of the cells in Japan. Mice congenic for the Ly5 locus (C57BL/6-Ly5.1), bred and main- the KSL fraction, cells were first stained with FITC-conjugated Ab to lin- tained at Sankyo Labo Service (Tsukuba, Japan), were provided by Dr. H. eage Ags listed above (all from BD Biosciences, except for TER-119 Ab Nakauchi (Institute of Medical Science, University of Tokyo, Tokyo, from Medical & Biological Laboratories) and negative fraction was col- Japan). lected using MACS anti-FITC Ab and an AutoMACS apparatus (Miltenyi The genotypes of the wild-type AML1 (AML1wt), loxP-flanked (AML1fl), Biotec) according to the manufacturer’s instructions. c-Kit-positive cells http://www.jimmunol.org/ and deleted (AML1Ϫ) loci were analyzed using genomic PCR primers as were then collected using a MACS anti-c-Kit Ab and AutoMACS (Milte- described previously (7). To induce excision of AML1 in the adult mice, nyi Biotec) and stained for allophycocyanin-conjugated Ab to Sca-1 (clone AML1fl/fl Mx-cre-transgenic mice were i.p. injected with poly(I:C) (Sigma- D7; eBioscience) and pyronin-Y. Cell cycle status was assayed for cells in Aldrich) along with littermate control mice at the age of 5–10 wk, and bone the FITC-negative, allophycocyanin-positive gates. For other sorting marrow cells were collected 4–9 wk after poly(I:C) injection. The age of analyses, cells were stained using FITC-conjugated Ab to CD34, PE- mice was between 12 and 19 wk at the time of bone marrow collection. In conjugated Ab to CD16/32 (Fc␥RIII/II; clone 2.4G2; eBioscience), limiting dilution bone marrow transplantation assays, 500 ␮g of poly(I:C) biotin-conjugated lineage Ag listed above followed by visualization was injected seven times on alternate days to maximize the gene deletion. with SA-PerCP, PE-Cy7-conjugated Ab to Sca-1 (clone E13-161.7; Because a high dose of poly(I:C) caused exhaustion of some mice, 250 ␮g eBioscience), and allophycocyanin-conjugated Ab to c-Kit. For analyz- of poly(I:C) was administered three times (days 1, 3, and 5) in other ex- ing lineage-negative (LinϪ) cells, dead cells were excluded from anal- periments, since this administering schedule caused effective deletion of yses along with lineage-positive cells by staining the cells with 7-ami- by guest on September 28, 2021 the AML1 locus (7). noactinomycin D (7-AAD, Via-Probe; BD Biosciences). Acquired data All animal studies were approved by the institutional review board, were analyzed using FACSDiva or CellQuest software (BD following institutional guidelines for animal researches. Biosciences).

Bone marrow transplantation Colony assays Bone marrow cells were collected by flushing the and tibiae of the Determined numbers of cells were collected in IMDM supplemented with mice with 22-gauge needles into 5 ml of RPMI 1640 medium supple- 2% FBS and cultured in MethoCult GF M3434 methylcellulose-based me- mented with 2% FBS and nucleated cells were counted. For transplanta- dium (Stem Cell Technologies) per the manufacturer’s instructions. Cell tion, nucleated cells were counted as indicated and i.v. injected into lethally clusters consisting of 50 cells or more were counted as colonies and col- irradiated (9 Gy) female, 8- to 10-wk-old C57BL/6J mice. The x-ray irra- onies were differentially scored according to their morphology at day 7 of diation was performed using MBR-1505R2 apparatus (Hitachi Medical). culture. For transplantation of retrovirus-transduced hematopoietic cells, C57BL/ 6-Ly5.1 mice were i.p. injected with 150 mg/kg 5-fluorouracil (Sigma- Aldrich). Five days after injection, mononuclear cells from two to four Retrovirus infection mice were precultured in a -containing medium for retrovirus in- Mouse AML1 cDNA was subcloned into pGCDNsam-IRES-GFP (a gift fection overnight and transduced with retrovirus medium for 3 days. Trans- from Dr. H. Nakauchi, Institute of Medical Science, University of Tokyo, duced cells were purified with FACSAria flow cytometer using “yield” Tokyo, Japan), and viral supernatants from the empty vector (mock) and mode at a purity of 70–90%, and 3–4 ϫ 105 purified cells were coinjected AML1 vector were produced using the MP34 packaging cell line as pre- with 1 ϫ 105 normal C57BL/6J bone marrow cells to irradiated (9 Gy) viously described (27). Transduction was performed using Retronectin C57BL/6J recipient mice. (Takara Bio) according to the manufacturer’s instructions in RPMI 1640 Real-time PCR for chimerism assays supplemented with 10% FBS, 10 ng/ml mouse , 6 ng/ml mouse IL-3, and 10 ng/ml mouse IL-6 ( provided by Kirin Brew- Bone marrow cells from one and two tibiae were collected into 1 ml ery) at a cell density of ϳ10,000–40,000 cells/well on 24-well nontreated of RPMI 1640 medium supplemented with 2% FBS, and the genomic DNA culture plates (Nunc) for purified hematopoietic progenitor cells or 4–8 ϫ was purified from 100 ␮l of cell suspension using an MFX-9600 apparatus 105 cells/well on 6-well nontreated culture plates (Nunc) for nonsorted and a NPK-391 magnetic bead-based genomic DNA extraction mononuclear cells. Three days after culture in the retrovirus-containing (Toyobo) according to the manufacturer’s instructions. Chimerism assays medium, cells were sorted using FACSAria for GFP positivity. using real-time PCR were done as previously described (23). Briefly, male bone marrow cells were serially diluted with female bone marrow cells (to Statistical analyses contain 0, 0.1, 0.3, 1, 3, 10, 30, and 100% of male cells) for establishing standard curves, and the genomic DNA was subjected for real-time PCR Competitive repopulating units (CRUs) were calculated using the StatMod amplification for Zfy2 (primers 5Ј-TGG AGA GCC ACA AGC TAA package for the GNU R statistical analysis software. Flow cytometry data CCA-3Ј and 5Ј-TCC CAG CAT GAG AAA GAT TCT TC-3Ј) and from mouse littermate pairs were first examined using the F test for equal- GAPDH (primers 5Ј-GGA GAT TGT TGC CAT CAA CG-3Ј and 5Ј-GTC ity of variance and then compared using the paired or unpaired t test if TCG CTC CTG GAA GAT GG-3Ј) using the SYBR Green PCR reagent applicable. When equality of variance was rejected, paired data were tested kit (Invitrogen Life Technologies) and Applied Biosystems 7000 Sequence by Wilcoxon’s signed rank test. 4404 AML1 NEGATIVELY REGULATES HSCs

ϩ Downloaded from FIGURE 2. G0 cell cycle analyses on the c-Kit bone marrow cells from cKO mice. A, Bone marrow cells from cKO and control mice that are ϩ c-Kit are assayed for G0 cell fractions. G0 and G1 cells were gated ac- cording to Hoechst 33342 fluorescence. G0 cells were defined as cells the p-Y fluorescence of which is below those of G2-M cells (Hoechst-high cells). The value of p was calculated using Wilcoxon’s signed rank test. B, Ϫ ϩ

Similar assays were performed on bone marrow cells sorted by Lin c-Kit http://www.jimmunol.org/ cells and stained with allophycocyanin-conjugated Ab to Sca-1. Represen- tative data within Sca-1ϩ gates from two independent experiments.

AML1 excision at the adult stage by injecting poly(I:C). Although deletion of the loxP-flanked AML1 loci in the bone marrow cells was almost complete after three injections of poly(I:C) at a dose of FIGURE 1. Hoechst 33342 SP analysis of the bone marrow cells from 250 ␮g, we used a higher dose (seven injections of 500 ␮gof conditional AML1 knockout (cKO) mice. A, Schematic representation of poly(I:C)) in transplantation experiments to maximize the gene by guest on September 28, 2021 the cKO alleles. B, SP cell profiles of LinϪ and CD34Ϫ KSL populations deletion, because a trace number of HSCs may expand and affect from cKO and control mice. C and D, Statistical analyses of the SP fraction wt/wt fl/fl from cKO and control bone marrow cells. Percentage of cells in the SP the data. AML1 Mx-cre mice or AML1 mice without the fraction (shown in B) from each paired (cKO and control littermates) ex- Mx-cre transgene were treated with poly(I:C) and used as controls. periments are displayed as paired data. C, Numbers of the LinϪ SP cells HSCs that can reconstitute adult hematopoiesis for a long term are (frequencies within mononuclear gates and absolute numbers per mouse). concentrated in the Hoechst 33342 SP fraction, which represents D, SP fraction cells within CD34Ϫ KSL cells as frequencies within mono- quiescent stem cells with the ability to efflux the dye at a greater nuclear gates and absolute numbers per mouse. Mean SP percentages are rate than the other major part of the cells (main population) (24). shown as bars in these graphs. Because equality of the variance of the data Although the total number of cells in the AML1-deficient bone were rejected by the F test, p values were calculated using Wilcoxon’s marrow was not significantly different from that of conditional signed rank test. Tg, Transgenic. knockout (cKO: 4.73 Ϯ 2.11 ϫ 107 vs control: 5.74 Ϯ 2.86 ϫ 107, p ϭ 0.50), the SP cell fraction within the bone marrow cells and the absolute number of SP cells per mouse was significantly in- Results creased in AML1-deficient mice compared with control mice, sug- Number of quiescent hematopoietic stem cells in the bone gesting that quiescent HSCs are increased in the absence of AML1 marrow is negatively regulated by AML1 (Fig. 1, B and C). Among SP cells, quiescent HSCs are known to We previously reported the increased cell number of the CD34Ϫ, be further concentrated in the fraction that has the CD34Ϫ KSL LinϪ, c-Kitϩ, and Sca-1ϩ (CD34Ϫ KSL) fraction, in which long- surface marker phenotype (CD34Ϫ KSL SP cells) (26). CD34Ϫ term hematopoietic stem cells are concentrated in the bone marrow KSL SP cells were also increased in the AML1-deficient mice of the conditional knockout mice that delete AML1 after birth (7). (Fig. 1, B and D), again indicating the increase in the quiescent AML1-deficient cells contribute to the granulocytic lineage of the HSC number in the AML1-deficient bone marrow. Given the el- recipient mice more efficiently than control cells at 1 mo after evated number of SP cells in the absence of AML1, we next an- transplantation, when both of them are transplanted into lethally alyzed the cell cycle status of immature hematopoietic cells to irradiated mice in the competitive repopulation experiments. How- determine the number of cells within the G0 state. We stained the ever, the contribution of the AML1-deficient cells to bone marrow cells with a stem cell-related surface marker c-Kit, in the peripheral blood tends to decrease over time (7). Therefore, DNA dye Hoechst 33342 and RNA dye pyronin-Y. As shown in we sought to determine precisely whether HSCs are increased in Fig. 2A, the G0 fraction defined as cells that show 2n DNA content the bone marrow of AML1-deficient mice. We crossed conditional by Hoechst 33342 staining and low RNA by pyronin-Y staining is AML1 knockout mice with loxP-flanked exon 5 in the AML1 locus also increased in the c-Kitϩ bone marrow cells of AML1-deficient (AML1fl, Fig. 1A) (7) with IFN-inducible Mx-cre-transgenic mice mice. We then sorted KSL cells, in which HSCs are concentrated, fl/fl (22) to generate AML1 Mx-cre mice, in which we can induce and found an increase of the G0 KSL cells in the AML1-deficient The Journal of Immunology 4405

Table I. Quantification of CRUs of the bone marrowa

Injected Cell No. AML1fl/fl Mx-cre AML1fl/wt Mx-cre AML1wt/wt Mx-cre

30,000 8/8 3/6 1/6 10,000 7/8 1/6 2/8 3,000 2/8 0/6 2/8 1,000 2/8 3/8 0/6 HSC frequency 1 in 5,606 1 in 29,531 1 in 52,043 (95% confidence interval) (3,085–10,186) (13,326–65,441) (20,688–130,925)

a For each indicated number of transplanted cells from AML1fl/fl Mx-cre, AML1fl/wt Mx-cre, and AML1wt/wt Mx-cre male mice, the proportion of mice that are positive for test male cells is given as (number of positive mice)/(number of analyzed mice). Frequencies of HSCs were calculated using Poisson statistics. Representative data from two independent experiments are shown. mice (Fig. 2B), indicating that loss of AML1 causes an increase of early stages (7, 9), we used bone marrow cells rather than periph- immature hematopoietic cells in the quiescent state. eral blood cells for the analysis for repopulation to avoid under- Because the fractions in which HSCs are concentrated have in- estimation. At 4 mo after transplantation, genomic DNA was pu- creased in the AML1-deficient mice, we assessed CRUs contained rified from the bone marrow cells. Donor cell chimerism was in the AML1-deficient mice using a bone marrow transplantation assayed using quantitative real-time genomic PCR targeted for the assay with limiting dilution to determine the frequency of long- Zfy locus, which is specific for the Y chromosome (23). Mice with Downloaded from term HSCs (28). Bone marrow cells from AML1fl/fl Mx-cre, Ն1% test cell chimerism were treated as positive for donor cell AML1fl/wt Mx-cre,orAML1wt/wt Mx-cre male mice treated with engraftment, and CRUs were evaluated using proportions of en- poly(I:C) were serially diluted, cotransplanted with 2 ϫ 105 female grafted mice and numbers of transplanted cells (28). As shown in competitor bone marrow cells into the lethally irradiated wild-type Table I and Fig. 3A, there was ϳ10-fold increase in CRUs in female C57BL/6J mice, and then donor cell chimerism was eval- AML1-deficient bone marrow in comparison to control mice. PCR uated in the reconstituted bone marrow. Because nucleated cells in genotyping confirmed almost complete deletion of the AML1 locus http://www.jimmunol.org/ the peripheral blood are predominantly in mice and of the AML1fl/fl Mx-cre bone marrow cells (Fig. 3B). CRUs in the AML1-deficient progenitors show maturation block at bone marrow of mice with heterozygously deleted AML1 locus (AML1fl/wt Mx-cre mice treated with poly(I:C)) showed a slight but not statistically significant increase in number compared with con- trol mice. These results indicate that loss of AML1 causes expan- sion of long-term HSCs in the bone marrow. Because the number of hematopoietic stem cells appeared to be inversely correlated to the attenuated doses of AML1, we next by guest on September 28, 2021 examined the effect of forced expression of AML1 on HSC activ- ity. Bone marrow cells from Ly5.1ϩ C57BL/6 congenic mice were transduced with retroviruses expressing GFP alone or along with AML1 (27). After the infection, GFPϩ cells were sorted and i.v. injected into lethally irradiated Ly5.2ϩ wild-type C57BL/6J mice along with supporting cells. Four weeks after transplantation, donor cell chimerism in the bone marrow was assayed by flow

FIGURE 3. Limiting dilution analysis based on competitive repopula- tion. A, Varying numbers of bone marrow cells from male AML1fl/fl, Mx- cre (ࡗ), AML1fl/wt, Mx-cre (f), or AML1wt/wt, Mx-cre (Œ) mice were mixed with 2 ϫ 105 bone marrow cells from female C57BL/6J mice and injected into lethally irradiated C57BL/6J female mice. Reconstitution in the bone marrow was evaluated at 4 mo after transplantation. Mice were considered negative when the percent chimerism was Ͻ1.0. Estimated fre- quencies of the repopulating cells are indicated as vertical dashed lines (1 repopulating cell per indicated numbers of bone marrow cells) for each FIGURE 4. Hematopoietic reconstitution by AML1-overexpressing genotype. Representative data from two independent experiments are bone marrow cells. Bone marrow cells from C57BL/6-Ly5.1 mice were shown. B, Genomic PCR analysis of the bone marrow cells reconstituted infected with mock or AML1-expressing retrovirus containing GFP, col- by AML1fl/fl, Mx-cre mice in the same experiment (two lanes at the left). lected for expression of GFP and transplanted into C57BL/6J (Ly5.2) mice. Control PCR fragments using tail tip DNA from AML1fl/wt and AML1wt/Ϫ Reconstituted bone marrow cells at 4 wk after transplantation were stained mice (two lanes at the right) are also displayed to indicate the position of with 7-AAD viability dye and PE-conjugated Ab to Ly-5.1 (CD45.1). Rep- the PCR products generated from each type of AML1 allele. wt, Wild type. resentative dot plots within 7-AAD-negative cell gates are shown. 4406 AML1 NEGATIVELY REGULATES HSCs Downloaded from FIGURE 6. Colony-forming capacity of myeloid progenitors overex- pressing AML1. Each myeloid progenitor fraction from wild-type bone marrow cells was sorted and transduced with mock or AML1-expressing retrovirus. GFPϩ cells were sorted and cultured for 7 days in methylcel- lulose-containing medium supplemented with cytokines. 3, mixed colo- nies; Ⅺ, myeloid colonies; and f, erythroid (BFU-E and CFU-E) colonies.

Typical colony numbers from three independent duplicate experiments are http://www.jimmunol.org/ shown. Error bars, SDs.

counts in the peripheral blood remain unchanged in AML1-deficient mice (7). These observations allowed us to ex- FIGURE 5. Colony-forming capacity of myeloid progenitors from cKO amine whether altered dosages of AML1 confer any qualitative mice. A, Sorting gates of KSL, CMP, and GMP cells from control and cKO change on the defined populations of myeloid progenitors as seen mice. B, Frequencies of the progenitors (KSL, CMP, GMP, and MEP for in HSCs. For this purpose, we purified each population that con- by guest on September 28, 2021 /erythrocyte progenitors). C, Numbers of the progenitors tains myeloid progenitors from the bone marrow and evaluated the (KSL, CMP, GMP, and MEP) per mouse calculated as products of the colony-forming capacity of those cells with methylcellulose-based frequencies of the progenitors and the numbers of the total bone marrow cultures. As is consistent with the previous report, the proportion Ϯ ϭ ϭ cells per mouse. Data are also shown as mean SD (n 7 for KSL, n of GMPs was elevated in the absence of AML1 (9). In addition, we 3 for others) Asterisks indicate p Ͻ 0.05 by paired t test. D, Colony- found that the proportion of common myeloid progenitors (CMPs; forming capacity of myeloid progenitor cells. Cells sorted using the gates Ϫ Ϫ ϩ ϩ ␥ low shown in A were cultured in a methylcellulose-containing medium and Lin Sca-1 c-Kit CD34 Fc RIII/II ) was also slightly but sig- scored according to their morphology on day 7 of culture. 3, mixed col- nificantly elevated from poly(I:C) injection by analyzing AML1- onies; Ⅺ, myeloid colonies, and f, erythroid (BFU-E and CFU-E) colo- deficient mice after 4–9 wk (Fig. 5, A and B). The total number of nies. Typical colony numbers from three independent duplicate experi- these progenitor cells from AML1-deficient mice was also in- ments are shown. Error bars, SDs. WT, Wild type. creased, although increase in the GMP cells was not significant. These results indicate that the numbers of CMPs were negatively regulated by AML1. However, when the same number of KSL cytometry using isotypes of CD45 (Ly5) and positivity of GFP. cells, CMPs, and GMPs were seeded onto methylcellulose-con- ϩ Although all six mice showed a distinct contribution of GFP taining medium, colony-forming capacities were not significantly ϩ ϩ Ly5.1 cells in the mock-infected group, GFP cells were de- different even in the absence of AML1, indicating that loss of tected in none of six mice transplanted with AML1-overexpressing AML1 does not affect the quality of these progenitor populations cells (Fig. 4). These results indicated that the elevated dose of (Fig. 5C). Taken together with the above findings, these results AML1 prevents hematopoietic cells from reconstituting the bone suggest that the elevated frequencies of committed progenitors in marrow of the recipient mice, again suggesting that AML1 nega- AML1-deficient bone marrow are mainly the consequences of the tively regulates hematopoietic stem cells. increased number of HSCs rather than cell-autonomous or stage- specific changes in committed progenitors. Loss of AML1 causes expansion of myeloid progenitor cells Next, we examined the effect of overdosage of AML1 on my- without affecting their colony-forming capacity eloid progenitors. Unsorted cells, KSL cells, and CMPs obtained We have previously shown that myeloid CFUs are increased when from bone marrow cells of wild-type C57BL/6J mice were trans- evaluated using the whole bone marrow cells of AML1-deficient duced with mock or AML1-expressing retrovirus. Infected cells mice, indicating that loss of AML1 results in an increased number were sorted for GFP positivity and seeded onto methylcellulose- of myeloid progenitors (7). These findings were consistent with based medium. As shown in Fig. 6, KSL and CMP cells that were another report showing that the granulocyte-macrophage progen- transduced with AML1-overexpressing retrovirus formed fewer itors (GMPs; LinϪSca-1Ϫc-KitϩCD34ϩFc␥RIII/IIhigh) are in- colonies than control cells, indicating that overexpression of creased upon deletion of AML1 in adult mice (9). Nonetheless, AML1 attenuates the proliferating capacity of these cells. The Journal of Immunology 4407

Discussion Given that loss of AML1 causes expansion of HSCs in adult In this article, we have found that in vivo long-term HSC activity bone marrow, genetic aberrations involving AML1 found in hu- is inversely correlated with the dosage of AML1. HSCs identified man may also promote expansion of leukemic stem phenotypically in terms of dye efflux or cell cycle status are also cells by inhibiting AML1 function, although it remains yet to be increased upon deletion of AML1, which indicates that AML1 determined whether normal and leukemic stem cells are main- negatively regulates the number of quiescent HSCs. Increase in the tained through similar molecular mechanisms. Elucidation of such quiescent fraction of HSCs in the AML1-deficient bone marrow mechanisms would be a clue to a more precise understanding of suggests that the expansion of HSCs is not due to compensation of normal hematopoiesis and hematological malignancies. Analyses on mutant mice have shown that various genes, including those for the differentiation block in the myeloid lineage, but the conse- transcription factors, cell surface ligands, their cognate receptors, quence of altered intrinsic transcriptional mechanisms that are cell cycle proteins, and molecules involved in signal transduction, physiologically regulated by AML1. Additionally, our current control the HSC pool size (reviewed in Ref. 32). AML1 is docu- study shows that overexpression of AML1 in the bone marrow mented to regulate the expression of target genes that act mainly in cells impairs hematopoietic reconstitution as well as colony-form- committed hematopoietic cells, such as IL-3, myeloperoxidase, ing capacities, again suggesting that the dosage of AML1 directly neutrophil elastase, M-CSF receptor, GM-CSF, TCRs (reviewed in regulates HSC activity. Ref. 33), Ig ␣-chain (34–36), and ␭5 (37). In contrast, little is The current observations appear to be in contrast to a previous known about candidates for transcriptional targets of AML1 in report showing that mice that congenitally lack AML1 in one allele adult HSCs. The regulatory mechanism of transcription through contain fewer CRUs than control mice (21). This difference may which AML1 controls the size of the HSC pool remains an unan- Downloaded from depend on the time when deletion of AML1 occurs. We analyzed swered question that should be elucidated in future studies. HSC activity immediately upon loss of AML1 by using mice that In this study, we have explicitly shown that AML1 negatively lack AML1 in an inducible fashion after birth. Mice with hemi- regulates the number of HSCs in the quiescent state in adult he- zygous levels of AML1 show aberrant distribution of definitive matopoiesis. Expansion of the immature hematopoietic cells HSCs during embryogenesis (29), and factors like this may modify caused by disruption of AML1 function may be involved in the

the property of HSCs in the adult stage in heterozygously AML1 pathogenesis of hematological malignancies associated with al- http://www.jimmunol.org/ knockout mice. It is known that mice that lack CBF␤, a functional tered AML1 function. partner of AML1, phenotypically recapitulate AML1 knockout mice, showing total loss of definitive hematopoiesis. A recent re- Acknowledgments port using CBF␤ hypomorphic mice revealed that 15% of the wild- We thank H. Nakauchi for the retrovirus vector and C57BL/6-Ly-5.1-con- type level of CBF␤ is sufficient for emergence of definitive hema- genic mice, R. Ku¨hn and J. Takeda for the Mx-cre-transgenic mice, topoiesis (30). In this report, attenuated doses of CBF␤ between 15 N. Watanabe and T. Kitamura for the protocol for retroviral transduction, and 30% of wild type cause expansion of hematopoietic stem/ and Y. Sawamoto and Y. Shimamura for technical assistance. progenitor cells in neonates, which return to normal when CBF␤ is restored up to the hemizygous level. These facts are compatible Disclosures by guest on September 28, 2021 with our findings that a reduced level of AML1 results in the The authors have no financial conflict of interest. expansion of HSCs after birth, although the amount of CBF␤ may not linearly correlate with AML1 function. Our observation is also References 1. Dzierzak, E., and A. Medvinsky. 1995. Mouse embryonic hematopoiesis. Trends consistent with the earlier in vivo studies in which immature he- Genet. 11: 359–366. matopoietic cells were shown to expand when they are introduced 2. Medvinsky, A., and E. Dzierzak. 1996. Definitive hematopoiesis is autonomously with AML1/ETO chimeric protein that represses the function of initiated by the AGM region. Cell 86: 897–906. 3. Muller, A. M., A. Medvinsky, J. Strouboulis, F. Grosveld, and E. Dzierzak. 1994. normal AML1 (15–19). 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