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MOZ is essential for maintenance of hematopoietic stem cells.

Monocytic leukemia zinc-finger protein (MOZ), a MYST family histone acetyltransferase, is involved in the chromosome translocations associated with acute myeloid leukemia. MOZ acts as a transcriptional coactivator for AML1, which is essential for establishment of definitive hematopoiesis. To investigate the roles of MOZ in normal hematopoiesis, we generated MOZ-null mice. MOZ-/- mice died around embryonic day 15 (E15). In MOZ-/- E14.5 embryos, hematopoietic stem cells, lineage-committed progenitors, and B lineage cells were severely reduced. On the other hand, arrest of erythroid maturation and elevated myeloid lineage populations were observed. MOZ-deficient fetal liver cells could not reconstitute hematopoiesis of recipients after transplantation. Analysis using microarray and flow cytometry revealed that expression of thrombopoietin receptor (c-Mpl), HoxA9, and c-Kit was down-regulated. These results show that MOZ is required for maintenance of hematopoietic stem cells, and that it plays a role in differentiation of erythroid and myeloid cells. Some aspects of the MOZ-/- phenotype are similar to that observed in PU.1-deficient mice. MOZ was able to interact with PU.1 and activate PU.1-dependent transcription, thus suggesting a physical and functional link between PU.1 and MOZ.

Monocytic leukemia zinc-finger protein (MOZ) is a transcriptional coactivator with histone acetyltransferase activity (Champagne et al. 2001;Kitabayashi et al. 2001a). MOZ was first isolated as a gene involved in chromosome translocaton t(8;16) (p11;p13), which is associated with the FAB M4/M5 subtype of acute myeloid leukemia with monocytic arrest (Borrow et al. 1996). This translocation results in the fusion of MOZ to transcription coactivator CBP. MOZ is also fused to CBP-like coactivator p300 and nuclear receptor coactivator TIF2 in leukemiaassociated chromosome rearrangements t(8;22) (Chaffanet et al. 2000;Kitabayashi et al. 2001b), and inv(8) (Carapeti et al. 1998;Liang et al. 1998), respectively. In addition, MOZ is involved in the t(2;8) chromosome translocation found in myelodysplastic syndrome (Imamura et al. 2003). In all of the leukemia-associated fusions, MOZ lacks the C-terminal region but retains some of its functional domains, including the histone acetyltransferase domain, PHD-type zinc-finger motif, and histone H1-like domain. MOZ-TIF2 fusion is able to confer properties of leukemic stem cells to committed hematopoietic progenitors, thus inducing acute myeloid leukemia in irradiated recipient mice after transplantation (Deguchi et al. 2003;Huntly et al. 2004). MOZ-CBP inhibits differentiation of M1 myeloid precursor cells into macrophages (Kitabayashi et al. 2001a).
It has been reported that MOZ interacts with AML1, and acts as a transcriptional coactivator (Kitabayashi et al. 2001a). AML1 (Runx1) is the most frequent target of chromosome rearrangements associated with acute leukemia. AML1 is essential for generation of hematopoietic stem cells (Okuda et al. 1996;Wang et al. 1996a), and is important for differentiation of megakaryocytes and lymphocytes (Ichikawa et al. 2004;Growney et al. 2005). AML1 forms a stable complex with CBF␤, which is also essential for definitive hematopoiesis (Sasaki et al. 1996;Wang et al. 1996b;Niki et al. 1997). Although MOZ and p300/CBP act as coactivators for AML1, the leukemiaassociated MOZ-CBP inhibits AML1-mediated transcription (Kitabayashi et al. 1998(Kitabayashi et al. , 2001aBristow and Shore 2003). The ETS family transcription factor PU.1 (Spi-1) is essential for maintenance of hematopoietic stem cells and development of myeloid and lymphoid lineages (Scott et al. 1994;McKercher et al. 1996;Kim et al. 2004;Iwasaki et al. 2005). Recent studies of mice carrying hypomorphic PU.1 alleles have indicated that reduction in the expression of PU.1 is capable of predisposing mice to AML (Rosenbauer et al. 2004).
Zebrafish having a mutation in the MOZ gene exhibit defects in Hox expression and pharyngeal segmental identity (Miller et al. 2004). Mice carrying a mutation in a gene encoding the MOZ-like protein MORF/Querkopf exhibited defects in bone and brain development (Thomas et al. 2000). However, the role of zebrafish MOZ and mouse MORF in hematopoiesis has not been described.
In order to clarify the physiological role of MOZ, we generated mutant mice that completely lack expression of MOZ. Involvement in leukemia-associated chromosome rearrangements and interaction with AML1 suggest critical roles of MOZ in hematopoiesis. Thus, we focused on analysis of the hematopoietic system in MOZ-null mice.

MOZ deficiency is embryonic lethal
In order to clarify the roles of MOZ in hematopoiesis, we generated MOZ-deficient mice by homologous recombination in embryonic stem (ES) cells using a gene-targeting vector, in which the MOZ exon 2 containing the first ATG was replaced with the neo gene cassette (Fig. 1A). Western blot analysis showed no detectable MOZ in homozygous embryos and decreased expression of MOZ in heterozygous embryos (Fig. 1B). Western blot analysis using antibodies to detect N-terminal and C-terminal MOZ, and RT-PCR using different sets of primers on the 5Ј, central, and 3Ј regions indicated that no truncated MOZ protein or mRNA was present in MOZ −/− embryos ( Supplementary Fig. S1B). MOZ +/− mice were born and were fertile, exhibiting no morphological abnormalities (Fig. 1C). On the other hand, MOZ −/− pups were not seen. To identity the stage of embryonic development at which the MOZ mutation is lethal, embryonic day 12.5-16.5 (E12.5-E16.5) embryos were analyzed for genotype. MOZ −/− embryos remained alive until E14.5; however, after E15.5, viable MOZ-deficient fetuses were not detected (Fig. 1C). MOZ −/− embryos at E14.5 were pale, and the fetal liver, which is the major hematopoietic organ at this stage, was smaller than that of wild-type or heterozygous littermates (Fig. 1D). Some MOZ −/− embryos exhibited hemorrhaging, subcutaneous edema, and bent tail. Gross histological analysis of wild-type and null mutant littermates revealed that MOZ −/− embryos have no other notable defects or abnormalities (Fig. 1E). Two independent targeted lines were produced, and exhibited essentially the same phenotype.

Decreased colony-forming cells in MOZ-deficient fetal liver
The morphological features of MOZ −/− embryos suggest defects in fetal liver hematopoiesis. The total number of fetal liver cells was decreased by 40% in E14.5 MOZ −/− embryos ( Fig. 2A). To examine the presence of hematopoietic progenitors, colony-forming cells (CFCs) were analyzed. Fetal liver cells were cultured in methylcellulose medium, and myeloid, erythroid, and mixed-lineage colonies were scored on the basis of standard morphological criteria after 12 d of culture. The total number of CFCs in MOZ −/− fetal liver cells was decreased by fivefold to 10-fold. However, the ratio of lineage-committed CFCs was not affected (Fig. 2B). These results suggest that the hematopoietic progenitor population was reduced in MOZ-deficient embryos. No significant differ- Structure of the mouse MOZ locus, the targeting construct, and the targeted locus. The targeting vector contains the HpaI-EcoRI MOZ genomic fragment, in which exon 2 containing the first ATG is replaced with a 1.1-kb pMC1-neor-poly(A) cassette, and a 0.8-kb DT-A fragment at the 3Ј terminus. The open and filled boxes represent the noncoding and coding regions of MOZ exon 2, respectively. (B) Expression of MOZ protein in MOZ +/+ , MOZ +/− and MOZ −/− embryos. Lysates were prepared from E12.5 embryos derived from MOZ +/− mice intercrosses, and were analyzed by Western blotting using anti-MOZ antibody. (C) Genotyping of offspring from MOZ +/− intercrosses was performed by PCR analysis using genomic DNAs from tails of adult mice and heads of embryos at the stages indicated. (D) Phenotypic comparison of MOZ +/− and MOZ −/− embryos at E14.5. The MOZ −/− fetus appears pale and the liver is small. (E) Sagital sections of E14.5 embryos of wild-type and null-mutant mice stained with hematoxylin and eosin. ence in total number of cells in the yolk sac was observed ( Supplementary Fig. S1C), thus suggesting no defects in primitive hematopoiesis in MOZ −/− embryos.

Reduction of hematopoietic stem cells and progenitors in MOZ-deficient fetal liver
In order to analyze hematopoietic progenitors and stem cells directly, we performed flow cytometry. Analysis of c-Kit, a common marker for hematopoietic progenitors, revealed that the population of cells expressing high levels of c-Kit (Lin − c-Kit hi ) was severely decreased in MOZ −/− fetal liver, while the population of cells expressing low levels of c-Kit was not strongly affected (Fig. 3A). The population of Lin − Sca-1 + c-Kit + cells, which includes hematopoietic stem cells (HSCs), was severely reduced in MOZ −/− embryos when compared with wildtype controls (Fig. 3B). Reductions were also observed in lineage-committed progenitors, such as common lymphoid progenitors (CLPs, Lin − IL-7R␣ + Sca-1 lo c-Kit lo ) (Fig. 3B), common myeloid progenitors (CMPs, Lin − Sca-1 − c-Kit + CD34 + Fc␥R III/II lo ), granulocyte/macrophage progenitors (GMPs, Lin − Sca-1 − c-Kit + CD34 + Fc␥R III/ II + ), and megakaryocyte/erythroid progenitors (MEPs, Lin − Sca-1 − c-Kit + CD34 − Fc␥R III/II lo ) (Fig. 4A). The number of HSCs and progenitors was also reduced in E12.5 MOZ −/− fetal liver as compared with those in wild-type littermates, but the reductions were milder than those observed in E14.5 embryos ( Supplementary Fig. S2). These data suggest that generation or maintenance of HSCs and progenitors was disrupted in MOZ −/− embryos, and the number of HSCs and progenitors was slightly reduced in MOZ +/− embryos.

Reduction of B lymphocytes, but not granulocytes and monocytes, in MOZ-deficient fetal liver
In order to assess differentiated hematopoietic cells in fetal liver, we investigated cells expressing a B lineage marker, CD19, and myeloid lineage markers, Gr-1 and Mac-1 by flow cytometry. CD19 + cells were reduced in MOZ −/− embryos when compared with wild-type embryos (Fig. 3C). In contrast, cell numbers of Mac-1 + Gr-1 hi granulocytes and Mac-1 + Gr-1 lo monocytes were  During analysis of myeloid progenitors, we found that the population of the uncharacterized cell fraction KLSF (Lin − Sca-1 − c-Kit + CD34 − Fc␥R III/II + ) was also elevated (Fig. 4A). To characterize the KLSF fraction, we sorted KLSFs as well as GMPs from both wild-type and MOZ −/− embryos by flow cytometry. Morphological assessment by May-Giemsa staining demonstrated that the KLSF fractions from both wild-type and MOZ −/− embryos contained more mature myeloid lineage cells than observed in the GMP fraction ( Supplementary Fig. S4). These cells were also cultured in liquid and methylcellulose medium. After culture in liquid medium with cytokines for 4 d, the majority of KLSFs and GMPs differentiated into either granulocytes or macrophages, although the differentiation of KLSFs was somewhat perturbed compared with that of GMPs. No significant differences in differentiation between wild-type and MOZ −/− cells were observed in the KLSF and GMP fractions before and after cultured in liquid medium. However, MOZ −/− GMPs and KLSFs exhibited poor proliferation and colony formation activity when compared with those from wild-type fetal liver ( Supplementary Fig. S5). These results suggest that KLSFs contain not-yet characterized myeloid lineage cells that are relatively resistant to differentiation.

Defects in erythropoiesis in MOZ-deficient fetal liver
In order to investigate erythroid lineage differentiation, E14.5 fetal liver cells were simultaneously stained for transferrin receptor (CD71) and erythroid antigen Ter119, and were analyzed by flow cytometry (Socolovsky et al. 2001). While differentiated red cells (Ter119 + CD71 − ) were severely reduced, late-stage erythroblasts (Ter119 + CD71 + ) were elevated in MOZ −/− fetal liver when compared with wild-type or MOZ +/− controls (Fig. 4C). Morphological analysis of fetal liver cells indicated that erythroblasts were markedly accumulated in MOZ −/− embryos when compared with wild-type embryos ( Supplementary Fig. S3). These data suggest that MOZ is indispensable for maturation of erythroid cells.

MOZ is required for reconstituting the hematopoietic system
We performed competitive reconstitution assay in order to determine whether MOZ-deficient embryos possess functional HSCs. Fetal liver cells (2 × 10 5 cells) from E13.5-E14.5 MOZ +/+ , MOZ +/− , and MOZ −/− (Ly5.2 + ) embryos were injected into lethally irradiated normal recipient mice (Ly5.1 + /5.2 + ) together with an equal number of competitor fetal liver cells from wild-type E13.5-E14.5 (Ly5.1 + ) embryos. Engraftment of recipient mice was monitored for 2, 4, 8, or 12 wk after transplantation by flow cytometry analysis of Ly5 allotype expression in peripheral blood cells. As shown in Figure 5A, MOZ +/+ and MOZ +/− Ly5.2 + cells were detected in the recipient mice. However, MOZ −/− cells were not detected at any of the time points examined (Fig. 5B). The population of MOZ +/− cells was lower than that of wild-type cells. We also analyzed cells in the thymus, spleen, and bone marrow at 12 wk after transplantation and found that Ly5.2 + cells derived from MOZ −/− embryos were not present in any organs tested (Fig. 5C). In order to determine whether loss of reconstitution activity was due to reductions in the number of HSCs or loss of HSC self-renewal activity, competitive reconstitution assay was performed using increasing numbers of donor cells (threefold and ninefold) (Fig. 5D). MOZ −/− cells were not detected in the recipient mice under any conditions, while increased population of MOZ +/− cells were detected when increased numbers of cells were in-jected. MOZ −/− fetal liver cells from an earlier embryonic time point (E13.5) also failed to exhibit reconstitution activity (Fig. 5E). These results suggest that loss of HSC activity was not due to reductions in HSCs or loss of HSC viability. Thus, MOZ is required for HSC activity to reconstitute hematopoiesis in recipients.

Reduced expression of HoxA9 and c-Mpl in MOZ −/− fetal liver cells
In order to identify genes with altered expression levels in MOZ −/− fetal liver cells, total RNAs were extracted from E12.5 MOZ +/+ , MOZ +/− , and MOZ −/− fetal livers. Analysis by oligonucleotide microarray identified 48 and 163 genes exhibiting changes in expression of more than twofold (Fig. 6A) and 1.5-fold ( Supplementary Fig. S6), respectively. Among the former 48 genes, down-regulation of the homeobox transcription factor HoxA9 and the thrombopoietin receptor c-Mpl are likely to be associated with defects in hematopoiesis. Semiquantitative RT-PCR also showed that expression levels of HoxA9 and c-Mpl were apparently reduced in MOZ −/− fetal liver cells (Fig. 6B). Among the latter 163 genes, reduced expression of c-Kit was also observed in MOZ −/− fetal liver cells ( Supplementary Fig. S6).

Interaction of MOZ with PU.1
The defects in hematopoiesis in MOZ-null mice are similar to those observed in PU.1 −/− mice. In PU.1 −/− fetal liver, HSCs and progenitors, in particular, the population of c-Kit + Lin − cells is severely reduced (Kim et al. 2004;Iwasaki et al. 2005). These similarities suggest a functional link between MOZ and PU.1. To test whether MOZ interacts with PU.1, we performed coimmunoprecipitation analysis. HA-tagged MOZ was transfected with Flag-tagged PU.1, AML1, C/EBP␣, and C/EBP. Cell lysates were prepared, and were immunoprecipitated with anti-Flag antibodies. Western blot analysis of the immunoprecipitates indicated that MOZ was coprecipitated with PU.1 and AML1, but not with C/EBP␣ and C/EBP (Fig. 7A). Reciprocal immunoprecipitation analysis indicated that PU.1 was coprecipitated with MOZ ( Fig. 7B). To test the interaction between endogenous MOZ and PU.1, we performed coimmunoprecipitation analysis using lysates of E14.5 fetal liver cells. Endogenous MOZ was detected in immunoprecipitates of endogenous PU.1 (Fig. 7C). These results suggest that MOZ is able to physically interact with PU.1. To test the effects of MOZ on PU.1-regulated transcription activity, reporter analysis was performed. MOZ strongly stimulated PU.1-mediated transcriptional activation (Fig. 7D). These results suggest that MOZ functions as a transcriptional coactivator for PU.1. Cold Spring Harbor Laboratory Press on July 19, 2018 -Published by genesdev.cshlp.org Downloaded from

Discussion
In this study, we generated MOZ-deficient mice and demonstrated that MOZ is essential for definitive hematopoiesis. HSCs and progenitors were decreased, and maturation of erythroid cells was inhibited in MOZ-deficient fetal liver. In competitive repopulation assay, MOZ −/− fetal liver cells did not exhibit any activity to reconstitute hematopoietic system. These results indicate that MOZ is essential for maintenance of HSCs. Intermediate phenotypes in the number of HSCs and progenitors as well as in reconstitution activity were observed in MOZ +/− fetal liver cells, thus suggesting a gene dosage requirement for MOZ. Gene expression profiles showed that expression of HoxA9, c-Mpl, and c-Kit was diminished. Immunoprecipitation analysis and reporter analysis suggest that MOZ functions as a coactivator for PU.1 as well as AML1.

Targets of MOZ
Expression levels of HoxA9, c-Mpl, and c-Kit were significantly decreased in MOZ −/− fetal liver cells and HSCenriched Lin − /Sca-1 + cells. HoxA9 has key roles in hematopoiesis (Lawrence et al. 1997;So et al. 2004) and leukemia pathogenesis (Kroon et al. 1998;Calvo et al. 2000). It has been reported that loss of HoxA9 reduces the proliferation and repopulating potential of hematopoietic stem cells, although HoxA9 is not essential for the maintenance of HSCs (Lawrence et al. 2005). The TPO receptor c-Mpl is important for megakaryocyte development (Carver-Moore et al. 1996) and for HSC activity in reconstituting hematopoietic organs in irradiated recipients (Kimura et al. 1998). c-Kit-mutated Vickid and Kit Wv/Wv mice also exhibited reduced HSC repopulating activity (Waskow et al. 2002;Antonchuk et al. 2004). Moreover, reduced progenitors and repopulating activities, as well as defects in erythropoiesis, were exacerbated in c-Mpl-and c-Kit double-deficient mice (Antonchuk et al. 2004). In addition, the c-Mpl ligand, TPO, induces HOXA9 nuclear localization (Kirito et al. 2004). Therefore, it is possible that defects in HSC repopulating activity and erythropoiesis in MOZ −/− mice are due to the synergistic effects of decreased expression of these factors.
In E14.5 MOZ −/− fetal liver, populations of HSCs and all lineages of hematopoietic progenitors were reduced, particularly progenitors expressing high levels of c-Kit. These phenotypes were, at least in part, similar to those observed in PU.1 −/− mice. In PU.1 −/− fetal liver, HSCs and progenitors, except for MEPs, were severely reduced (Kim et al. 2004;Iwasaki et al. 2005). The population of Lin − c-Kit + cells was particularly low, thus suggesting that expression of c-Kit is positively regulated by PU.1. We showed here that MOZ is able to interact with PU.1 and activates PU.1-dependent transcription, which suggests that MOZ is a coactivator of PU.1 and that it regu- lates transcription of PU.1-target genes. PU.1 −/− mice show more severe reductions in HSCs and hematopoietic progenitors than MOZ −/− mice. MOZ is a transcription coactivator, and thus the difference may be because PU.1 transcription activity is not completely abolished in MOZ −/− mice. Therefore, MORF, which is highly homologous to MOZ, may compensate for some MOZ functions. In contrast to MOZ −/− embryos, PU.1-null embryos have no myeloid cells (Kim et al. 2004;Iwasaki et al. 2005). However, increases in myeloid cells were observed in mice carrying hypomorphic PU.1 alleles, in which PU.1 expression was decreased to 20% of wildtype levels (Rosenbauer et al. 2004) as observed in MOZ −/− embryos. Thus, the MOZ −/− mouse phenotype, except for defects in erythropoiesis and reductions in MEPs, can be explained by reduced PU.1 activity.
We previously showed that MOZ forms a complex with AML1 and acts as a potent coactivator of AML1 (Kitabayashi et al. 2001a). HoxA9 expression is reduced in AML patients with chromosome translocations t(8:21) and inv(16) (Debernardi et al. 2003). In such leukemia cells, AML1 is inhibited by AML1-ETO and CBF␤-MYH11, respectively. Expression of c-Mpl is also reduced in patients having familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML), which has a mutation in AML1 (Heller et al. 2005). These studies indicate that expression of HoxA9 and c-Mpl can be regulated by AML1. Thus, it is likely that reduced expression of HoxA9 and c-Mpl in MOZ −/− fetal liver arises from the inactivity of AML1 in the absence of its coactivator, MOZ.

Roles of MOZ and MOZ fusion in myelogenesis and leukemia pathogenesis
We observed increases in the populations of Mac-1 + Gr-1 + myeloid cells and in the uncharacterized myeloid cell fraction KLSF (Lin − Sca-1 − c-Kit + CD34 − Fc␥R III/II + ) in MOZ-deficient fetal liver (Fig. 4A,B), although immature myeloid progenitors (CMP, GMP) were reduced. This may be because myeloid differentiation is inhibited around the KLSF stage in MOZ −/− mice, as MOZ has important roles in the regulation of this stage. Alternatively, accumulation of myeloid cells including KLSFs may be due to uncontrolled and abnormal differentiation of the myeloid lineage. Consistent with the latter hypothesis, analysis of the flow-sorted KLSF fraction indicated that KLSFs contained myeloid lineage cells showing less differentiation activity than GMPs (Supplementary Fig. S4). Increases in myeloid cells were also observed in conditional AML1-KO mice (Growney et al. 2005) and in hypomorphic PU.1 mice (Rosenbauer et al. 2004). Interestingly, KLSF cells were substantially elevated in mice with acute myeloid leukemia after transplantation of MOZ-TIF2-introduced bone marrow cells (Deguchi et al. 2003;Huntly et al. 2004). In addition, monocytic blasts are often elevated in human leukemia patients who exhibit rearrangement of MOZ. Taken together with previous results (Deguchi et al. 2003;Huntly et al. 2004), the present study suggests that MOZ fusion modulates normal MOZ function in the pathogenesis of leukemia, and that the KLSF fraction plays a role in the development of acute myeloid leukemia. Recent studies have indicated that mice carrying hypomorphic PU.1 alleles in which PU.1 expression is decreased to 20% of wild-type levels develop acute myeloid leukemia (Rosenbauer et al. 2004). The interaction between MOZ and 293T cells were cotransfected with PU.1 together with control vector, Flag-tagged p300, or MOZ. Expression of PU.1 in the lysates of transfectants was detected by immunoblotting using anti-PU.1 antibody. Flag-tagged p300 and MOZ were immunoprecipitated with anti-Flag (M2) antibody. (Bottom) Immunoprecipitates were analyzed by immunoblotting using anti-PU.1 antibody. (C) Interaction of endogenous PU.1 and MOZ. Cell lysates were prepared from E14.5 fetal liver cells (5 × 10 7 ) and PU.1 was immunoprecipitated with anti-PU.1 antibody. Cell lysates and immunoprecipitates were then analyzed by immunoblotting with anti-MOZ antibody (N). (D) MOZ activates PU.1-mediated transcription. SaOS2 cells were transfected with 100 ng of M-CSFR-luc, 50 ng of CMV-PU.1, indicated amounts (in micrograms) of LNCX-MOZ, and 2 ng of phRL-cmv. Cell lysates were prepared at 24 h after transfecton, and were analyzed for luciferase activity.
PU.1 suggests that MOZ fusion may affect function of PU.1 in the pathogenesis of leukemia.

Materials and methods
Generation of MOZ-deficient mice MOZ genomic clones were isolated from a mouse genomic BAC library by PCR screening. We constructed a targeting vector containing an MOZ genomic fragment interrupted in exon 2, which contains the first methionine ATG, using a neomycin resistance gene (neo) cassette with a flanking diphtheria toxin A fragment gene (DT-A) cassette. The construct was electroporated into ES cells, and G418-resistant clones were selected by Southern blot analysis using 5Ј and 3Ј external probes. Nine clones containing the MOZ-disrupted allele were obtained. Two independent clones were injected into day 3.5 C57BL/6J JcL embryos, which were subsequently transferred to CD-1 recipient females. All animals were maintained under specific pathogen-free, temperature-controlled conditions throughout this study, in accordance with Institutional Guidelines. Written approval for all animal experiments was obtained from the local Animal Experiments Committee of the National Cancer Center Research Institute.

Histological procedures
Embryos were isolated at E14.5 and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 48 h. Embryos were routinely processed for embedding in paraffin and 3-µm sections were cut for hematoxylin and eosin staining.

Immunofluorescent staining and flow cytometric analysis
Fetal liver cells were dissected from E12.5 and E14.5 embryos, and drawn through 21-and 25-gauge needles to generate a single-cell suspension. Erythrocytes were lysed in ammonium chloride potassium buffer. One-million cells were preincubated with 2.4G2 in order to prevent nonspecific binding of mAb via FcR interactions, and were then incubated on ice for 90 min with the appropriate staining reagents according to standard methods (Nakayama et al. 1990). Flow cytometry was performed on a FACScalibur (Becton Dickinson), and the results were analyzed using CELLQUEST software (Becton Dickinson). The reagents used in this study were as follows: FITC-conjugated streptavidin, PerCP-Cy5.5-conjugated streptavidin, anti- , and anti-Gr-1-APC (RB6-8C5-APC). All of the above reagents were purchased from PharMingen. Anti-CD45.2-APC (104-APC) was purchased from eBioscience.

Microarray analysis and RT-PCR
For gene expression analysis, total RNA was isolated using Isogen reagent (Nippon Gene) from livers of two each of E12.5 MOZ +/+ , MOZ +/− , and MOZ −/− embryos, and were purified with an RNeasy MinElute cleanup kit (Qiagen). The integrity of total RNA was confirmed using LabChip RNA 6000 Nano chips and a 2100 Bioanalyzer (Agilent Technologies). For gene expression profiling, a GeneChip Mouse Genome 430 2.0 oligonucleotide microarray (Affymetrix), which contained 45,037 probe sets, was used according to the manufacturer's instructions.
The expression value (Signal) of each gene was calculated and normalized using GeneChip Operating Software version 1.2 (Affymetrix). The change value (Signal Log Ratio) and change call (Increase, Marginal Increase, No Change, Marginal Decrease, or Decrease) for each gene was calculated by Comparison Analysis of the software. To identify genes that were significantly affected by MOZ knockout, we selected genes that showed a change call of Increase and a Signal Log Ratio of more than 1 (more than twofold increase) or genes that showed a change call of Decrease and a Signal Log Ratio of less than −1 (more than twofold decrease) in all four comparisons between MOZ −/− and MOZ +/+ fetal livers.

Luciferase assay
SaOS2 cells or 293T cells were transfected using the calcium phosphate precipitation method in 24-well plates, and luciferase activity was assayed after 24 h with a luminometer, Lumat LB9507 (Berthold), according to the manufacturer's protocol (Promega). Results of reporter assays represent the mean values for relative luciferase activity generated from three independent experiments normalized against the activity of the enzyme from phRL-CMV as an internal control.