Identification of Zfp521/ZNF521 as a cooperative gene for E2A-HLF to develop acute B-lineage leukemia

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Abstract

E2A-hepatic leukemia factor (HLF) is a chimeric protein found in B-lineage acute lymphoblastic leukemia (ALL) with t(17;19). To analyze the leukemogenic process and to create model mice for t(17;19)-positive leukemia, we generated inducible knock-in (iKI) mice for E2A-HLF. Despite the induced expression of E2A-HLF in the hematopoietic tissues, no disease was developed during the long observation period, indicating that additional gene alterations are required to develop leukemia. To elucidate this process, E2A-HLF iKI and control littermates were subjected to retroviral insertional mutagenesis. Virus infection induced acute leukemias in E2A-HLF iKI mice with higher morbidity and mortality than in control mice. Inverse PCR detected three common integration sites specific for E2A-HLF iKI leukemic mice, which induced overexpression of zinc-finger transcription factors: g rowth f actor i ndependent 1 (Gfi1), zinc-finger protein subfamily 1A1 isoform a (Zfp1a1, also known as Ikaros) and zinc-finger protein 521 (Zfp521). Interestingly, tumors with Zfp521 integration exclusively showed B-lineage ALL, which corresponds to the phenotype of human t(17;19)-positive leukemia. In addition, ZNF521 (human counterpart of Zfp521) was found to be overexpressed in human leukemic cell lines harboring t(17;19). Moreover, both iKI for E2A-HLF and transgenic for Zfp521 mice frequently developed B-lineage ALL. These results indicate that a set of transcription factors promote leukemic transformation of E2A-HLF-expressing hematopoietic progenitors and suggest that aberrant expression of Zfp521/ZNF521 may be clinically relevant to t(17;19)-positive B-lineage ALL.

Introduction

The E2A gene, which encodes a basic helix-loop-helix transcription factor of E-box DNA-binding proteins on chromosome 19, is the target of subsets of B-lineage acute lymphoblastic leukemia (ALL) (Look, 1997). As a result of the t(17;19)(q22;p13), the E2A gene is fused to the HLF gene on chromosome 17 (Inaba et al., 1992). In the E2A-HLF chimeric gene product, the transactivation domain of E2A is fused to the basic region/leucine zipper domain of hepatic leukemia factor (HLF), which contributes to the DNA binding and dimerization (Inaba et al., 1992). Clinically, ALL with the E2A-HLF chimera is refractory to intensive therapy and is frequently associated with coagulopathy and hypercalcemia (Hunger, 1996).

The biological properties of E2A-HLF were initially analyzed using cultured cells. We showed that the expression of E2A-HLF in NIH 3T3 cells induced anchorage-independent cell growth in soft agar and rendered these cells tumorigenic in nude mice (Yoshihara et al., 1995; Inukai et al., 1997). In addition, using a zinc-inducible system, we showed that E2A-HLF expression protects interleukin 3-dependent hematopoietic cells from interleukin 3 deprivation-induced apoptosis (Inaba et al., 1996). Moreover, by a representational difference analysis, several downstream candidate genes of E2A-HLF were cloned, such as annexin II (Matsunaga et al., 2003), annexin VIII and s ushi- r epeat p rotein u pregulated in l eukemia (SRPUL; Kurosawa et al., 1999), two Groucho-related genes, Grg2 and Grg6 (Dang et al., 2001), and a gene encoding a zinc-finger transcription factor, Slug (Inukai et al., 1999).

The in vivo roles of E2A-HLF were analyzed by transgenic and bone marrow transplantation studies. We and others generated transgenic mice expressing E2A-HLF under the control of lymphoid-specific promoters (Honda et al., 1999; Smith et al., 1999). The transgenic mice showed increased thymocyte apoptosis, B-cell maturation arrest and eventual development of ALL, mainly with T-cell phenotype (Honda et al., 1999; Smith et al., 1999). On the other hand, bone marrow (BM) B-cell progenitors retrovirally co-transduced with E2A-HLF and Bcl-2 produced immortalized cells, which developed leukemia when transplanted into syngeneic recipients (Smith et al., 2002). These results showed that the expression of E2A-HLF perturbed normal lymphocyte development, rendered lymphocytes susceptible to malignant transformation and finally developed ALL. Interestingly, the phenotypes of the E2A-HLF transgenic mice closely resembled those of E2A-deficient mice, which also showed abnormal T-cell development, absence of B-cell precursors and rapid development of T-cell lymphomas (Bain et al., 1994, 1997; Zhuang et al., 1994). These results strongly suggested that E2A-HLF contributes to leukemogenesis by activating downstream target genes and/or by suppressing transcriptional activity of endogenous genes in a dominant-negative manner (Aspland et al., 2001; Seidel and Look, 2001).

We showed the in vivo oncogenecity of E2A-HLF by a transgenic approach (Honda et al., 1999). However, the transgenic system fundamentally differs from human disease in several ways. First, in the transgenic system, every cell contains the transgene and there are no normal cells, whereas the human disease originates from acquiredly transformed cells. Second, in the transgenic system, as the transgene-derived product is congenitally expressed, transgene-expressing cells are not eliminated by the immune system. In contrast, in human diseases, most transformed cells are ablated by immunocompetent cell and those that escape from this system proliferate and show a fully malignant phenotype. Therefore, the precise molecular mechanism(s) through which E2A-HLF contributes a growth advantage to hematopoietic cells and develops leukemia in vivo remains to be clarified.

In this study we report the generation and analysis of knock-in mice for E2A-HLF in which E2A-HLF was inducibly expressed under the control of the native regulatory elements of the E2A gene. Despite the induced E2A-HLF expression in the hematopoietic tissues, no disease was developed during the long-term observation period, indicating that secondary events are required for the development of leukemia. To elucidate this process, we applied retroviral insertional mutagenesis (RIM) using Moloney murine leukemia virus (MMLV), isolated common viral integration sites specific for E2A-HLF-expressing tumors, and identified Zfp521/ZNF521 as a cooperative gene for E2A-HLF to develop B-lineage ALL.

Results

Generation of inducible knock-in (iKI) mice for E2A-HLF and acquired expression of E2A-HLF in the hematopoietic tissues

To study the role of E2A-HLF in model animal systems that mimic human leukemogenesis, we planned to generate mice in which E2A-HLF could be inducibly expressed under the control of the native E2A promoter. For this purpose, we designed a knock-in vector in which a genomic region of the E2A gene (a 3′ part of exon 2, intron 2 and a 5′ part of exon 3) was replaced by a cassette containing the floxed neomycin resistance (Neo) gene, followed by E2A-HLF complementary DNA, IRES-GFP (IG) and an SV40 polyA signal (pA) (Figure 1a). Embryonic stem cell clones with homologous recombination were identified by Southern blot analysis (Figure 1b, upper panel) using a 5′ probe (Figure 1a) and by long-distance genomic PCR (Figure 1b, lower panel) using a 3′ primer set (P1 and P2, Figure 1a) and were used to create chimeric mice, which transmitted the mutant allele to the progeny and produced heterozygous mice (EHKINeo+). In the EHKINeo+ mice, the expression of the knock-in allele-derived message was detected by reverse transcriptase–PCR (RT–PCR) using a primer set, E2A-77 (derived from exon 1 of the E2A gene) and HLF-2 (derived from the HLF portion of the E2A-HLF fusion complementary DNA) (Figure 1a) in all tissues examined (indicated by Neo+ in Figure 1c, upper panel). However, because this message contains a floxed Neo gene and multiple in-frame stop codons, the E2A-HLF fusion protein cannot be translated. To confirm this, proteins extracted from tissues were immunoprecipitated with an anti-E2A antibody and immunoprecipitants were blotted with an anti-HLF antibody. As expected, no E2A-HLF protein (molecular weight 62 kDa) was detected in the hematopoietic tissues, such as the thymus or spleen of EHKINeo+ mice (the first and fourth lanes in Figure 1d).

Figure 1
figure1

Generation of inducible knock-in (iKI) mice for E2A-HLF and the acquired expression of E2A-HLF in the hematopoietic tissues. (a) Schematic illustration of the iKI strategy. Part of the non-coding region of exon 2, the coding region of exon 2, intron 2 and part of the coding region of exon 3 were replaced with the floxed neomycin resistance gene, followed by E2A-HLF fusion complementary (cDNA), IRES-GFP (IG) and a polyadenylation signal (pA). Restriction enzymes: H, HindIII; X, XbaI; N; NaeI; C;, ClaI. The positions of the 5′ probe for Southern blot analysis, P1 and P2 primers for genomic PCR and E2A-77 and HLF-2 for RT–PCR are shown. (b) Results of 5′ Southern blot analysis and 3′ genomic PCR to detect homologous recombination. Positions of germline (GL)- and KI-allele-derived bands determined by 5′ Southern blot analysis are indicated by arrows (upper panel) and the PCR product generated by 3′ genomic PCR is indicated by an arrowhead (lower panel). (c) Expression of the KI allele-derived mRNA. mRNAs extracted from tissues of EHKINeo+ and EHKIΔNeo mice were subjected to RT–PCR using E2A-77 and HLF-2 primers (seea). Positions of RT–PCR products with and without Neo are indicated by Neo+ and ΔNeo, respectively. (d) Acquired E2A-HLF protein expression in the lymphoid tissues of EHKIΔNeo mice. Proteins extracted from the thymus and spleen were immunoprecipitated with an anti-E2A antibody, and the immunoprecipitants were blotted with an anti-HLF antibody. The positions of E2A-HLF protein and immunoglobulin (Ig) are indicated by arrows. Total cell lysate (TCL) from a t(17;19)+ cell line, UOCB1, was used as a positive control.

We then mated EHKINeo+ mice with MxCre transgenic mice that express Cre under the control of the interferon-responsive Mx promoter (Kuhn et al., 1995). EHKINeo+/MxCre compound mice were injected with polyinosinic/polycytidylic acid (pIpC), which is a strong and transient inducer of interferon, to delete the floxed Neo gene from the knocked-in allele and to create Neo-deleted (EHKIΔNeo) mice (Figure 1a). In the pIpC-treated EHKINeo+/MxCre (that is, EHKIΔNeo) mice, a shorter message was amplified in various tissues, including the thymus, heart, liver and spleen, by RT–PCR using E2A-77 and HLF-2 primers (indicated by ΔNeo in Figure 1c, lower panel), indicating that the Neo gene was successfully deleted in these tissues. As a result, the induced expression of E2A-HLF protein was achieved, as shown by immunoprecipitation/western blot analysis in the thymus and spleen of the EHKIΔNeo mice (the second and fifth lanes in Figure 1d).

MMLV infection induced acute leukemias in EHKIΔNeo mice at a higher frequency and with a shorter latency than in EHKINeo+ mice

EHKINeo+ and EHKINeo+/MxCre mice treated with pIpC were continuously observed for any sign of illness, including routine examination of peripheral blood parameters. However, during the long-term observation period, no abnormality was detected in EHKINeo+ or EHKIΔNeo mice (Figure 2a, thin dotted and thin continuous lines). These results indicated that the induced E2A-HLF expression alone is not sufficient and additional genetic changes are required for the development of leukemia.

Figure 2
figure2

Survival curves of EHKINeo+ and EHKIΔNeo mice with or without MMLV infection, and flow cytometric and gene rearrangement analyses of leukemic tissues of EHKINeo+ and EHKIΔNeo mice infected with MMLV (EHKINeo+/MMLV and EHKIΔNeo/MMLV). (a) Survival curves of EHKINeo+, EHKIΔNeo, EHKINeo+/MMLV and EHKIΔNeo/MMLV mice. No disease was observed in EHKINeo+ or EHKIΔNeo mice (indicated by thin dotted and thin continuous lines, respectively). MMLV infection induced acute leukemias in both EHKINeo+/MMLV and EHKIΔNeo/MMLV mice (indicated by thick dotted and thick continuous lines, respectively) and the EHKIΔNeo/MMLV mice showed higher morbidity and mortality than EHKINeo+/MMLV mice. The diseased EHKIΔNeo/MMLV and EHKINeo+/MMLV mice are numbered and the time points of MMLV injection and E2A-HLF induction by pIpC are indicated by arrows. (b) Representative results of a flow cytometric analysis. Leukemic cells in EHKINeo+/MMLV and EHKIΔNeo/MMLV mice were stained with anti-Thy1.2, anti-B220, anti-Gr1 and anti-Mac1 antibodies and analyzed using a FACSCalibur. No. 1 of EHKINeo+/MMLV leukemic mice that was positive for Thy1.2 but negative for other antigens and no. 2 of EHKIΔNeo/MMLV leukemic mice that was positive for B220 but was negative for other antigens are shown in the left and right panels, respectively. (c) Results of gene rearrangement analysis. DNAs extracted from leukemic tissues of EHKINeo+/MMLV and EHKIΔNeo/MMLV mice were digested with EcoRI and blotted with JH (upper panels) and TCRJ-β (lower panels) probes. Germline (GL) and rearranged bands are indicated by arrows and arrowheads, respectively.

To address this possibility, mice were subjected to retroviral insertional mutagenesis. Neonatal EHKINeo+ and EHKINeo+/MxCre mice were infected with MMLV and were then injected with pIpC. Both types of mice developed leukemias, but MMLV-infected EHKIΔNeo (EHKIΔNeo/MMLV) mice showed higher morbidity and mortality than virus-infected EHKINeo+ (EHKINeo+/MMLV) littermates (Figure 2a, thick dotted and thick continuous lines). EHKIΔNeo/MMLV mice began to develop acute leukemias at as early as 2.6 months of age, and all died by 6 months of age. In contrast, EHKINeo+/MMLV mice developed leukemias at approximately 4–6 months of age and 6 out of 11 mice died within 1 year. The difference in the survival curves between EHKIΔNeo/MMLV and EHKINeo+/MMLV mice was statistically significant (P<0.01).

EHKINeo+/MMLV mice mainly developed T-cell leukemia but EHKIΔNeo/MMLV mice showed B-progenitor and lineage marker-negative leukemias

The leukemic mice were hematologically and macroscopically examined, and the leukemic cells were immunophenotypically and molecularly analyzed. Interestingly, macroscopic appearances of EHKINeo+/MMLV leukemic mice were different from those of EHKIΔNeo/MMLV leukemic mice.

Most of EHKINeo+/MMLV leukemic mice (four of six samples) showed thymic enlargement, associated with splenomegaly and lymph node swelling, except two samples that showed splenomegaly and lymph node swelling. In contrast, EHKIΔNeo/MMLV leukemic mice did not show thymic enlargement but showed splenomegaly, frequently associated with lymph node swelling. To determine the lineage of the leukemic cells, disaggregated cells were subjected to flow cytometric analysis. In EHKINeo+/MMLV mice, samples with thymic enlargement (nos. 1 and 3–5) were positive for T-cell (Thy1.2) antigen, but negative for B-cell (B220), myeloid (Gr1) and macrophage (Mac1) antigens, whereas the other two samples lacking thymic enlargement (nos. 2 and 6) did not express any of Thy1.2, B220, Gr1 or Mac1 antigen. In EHKIΔNeo/MMLV mice, two samples (nos. 2 and 4) were positive for B220 but negative for other antigens, whereas the remaining 9 samples (nos. 1, 3 and 5–11) did not express any of Thy1.2, B220, Gr1 or Mac1 antigen. Representative results of flow cytometric analysis of Thy1.2-positive EHKINeo+/MMLV leukemic samples and B220-positive EHKIΔNeo/MMLV leukemic samples are shown in Figure 2b. As B-lineage leukemia is rarely developed in MMLV-infected mice, B-cell commitment of the two B220-positive samples (nos. 2 and 4 of EHKIΔNeo/MMLV mice) was further analyzed by using antibodies against CD19, BP1, CD20, CD43 and immunoglobulin M. The result showed that both samples were positive for CD19, BP1, CD20 and CD43 but negative for immunoglobulin M, showing that they were B-progenitor leukemias (Supplementary Figure 1).

The leukemic samples were then subjected to gene rearrangement analysis using JH and TCRJ-β probes. As expected from the results of flow cytometric analyses, Thy1.2-positive samples (nos. 1 and 3–5 of EHKINeo+/MMLV group) showed rearranged bands in the TCR-β locus (indicated by arrowheads in the left lower panel of Figure 2c), and B220-positive samples (No. 2 and 4 of EHKIΔNeo/MMLV group) showed rearranged bands in the IgH locus (indicated by arrowheads in the right upper panel of Figure 2c), whereas other samples lacking lineage markers (nos. 2 and 6 of EHKINeo+/MMLV mice and nos. 1, 3, and 5–11 of EHKIΔNeo/MMLV mice) showed germline patterns in both IgH and TCR-β regions. These results indicated that four EHKINeo+/MMLV leukemias (nos. 1 and 35) were T-cell ALL and two EHKIΔNeo/MMLV leukemias (nos. 2 and 4) were B-lineage ALL, but others were lineage marker-negative leukemias that were derived from immature cells not yet committed to a specific cell lineage. The characteristics of EHKINeo+/MMLV and EHKIΔNeo/MMLV leukemic mice are summarized in Table 1.

Table 1 Characteristics of EHKINeo+/MMLV and EHKIΔNeo/MMLV leukemic samples

Identification of Gfi1, Ikaros and Zfp521 as common integration sites (CISs) in leukemias developed in EHKIΔNeo/MMLV mice

To identify gene(s) whose altered expression cooperated with E2A-HLF, genomic DNAs extracted from leukemic samples of EHKIΔNeo/MMLV mice were subjected to inverse PCR (iPCR). DNAs from leukemias of EHKINeo+/MMLV mice were also analyzed as controls. Genes identified by iPCR in EHKIΔNeo/MMLV and EHKINeo+/MMLV leukemic mice are listed in Supplementary Tables 1 and 2, respectively. In the iPCR products of EHKIΔNeo/MMLV mice, we found three CISs (shown by asterisks and bold type in Supplementary Table 1), all of which encode zinc-finger transcription factors.

First, in four leukemic samples (nos. 1, 6, 8 and 11), viruses were integrated in an 10-kb upstream region (nos. 1, 8 and 11) or in the 3′ untranslated region (no. 6) of g rowth f actor i ndependent 1 (Gfi1) gene (upper panel of Figure 3a). Southern blot analysis using genomic fragments adjacent to the integration sites showed rearrangement bands in all the tumors (indicated by arrowheads in the lower left panel of Figure 3a), indicating that cells with these integration sites were predominant in the related tumors. In addition, northern blot analysis revealed that Gfi1 mRNA expression levels were significantly enhanced in nos. 1, 8 and 11, and moderately increased in no. 6 when compared with those in a control spleen (C) and Gfi1-non-integrated samples (nos. 7 and 10, see Table 1 and Supplementary Table 1) (indicated by an arrow in the lower right panel of Figure 3a).

Figure 3
figure3

Retroviral integration sites, gene rearrangements and altered expression patterns in CISs detected in EHKIΔNeo/MMLV leukemic mice (a) Gfi1 gene. Upper panel: schematic illustrations of viral integration sites in the Gfi1 gene. Exons are boxed, and the coding and non-coding regions are indicated by black and white boxes, respectively. Viral integration sites are indicated by vertical arrows with the related mouse identification numbers (nos. 1, 11, 6 and 8). Positions of probes used for Southern blot analyses are also shown. Lower left panel: Southern blot analysis for gene rearrangements. DNAs extracted from a control spleen (C) and Gfi1-integrated EHKIΔNeo/MMLV mice (nos. 1, 11, 6 and 8) were digested with BamHI and probed with the adjacent genomic fragment shown in (a) (probe A for nos. 1, 11 and 6, and probe B for no. 8). Germline (GL) and rearranged bands are indicated by arrows and arrowheads, respectively. Lower right panel: Northern blot analysis for Gfi1 mRNA expression. mRNAs of a control spleen (C) and Gfi1-integrated EHKIΔNeo/MMLV mice (nos. 1, 11, 6 and 8) were probed with the Gfi1 coding region. Gfi1-non-integrated tumors (nos. 7 and 10) were also used as controls. The position of Gfi1 mRNA is indicated by an arrow and β-actin hybridization served as the internal control. (b) Ikaros gene. Upper panel: schematic illustrations of virus integration sites in the Ikaros gene. Exons are boxed, and the coding and noncoding regions are indicated by black and white boxes, respectively. Viral integration sites are indicated by vertical arrows with the related mouse identification numbers (no. 3, 5 and 9). Positions of probes used for Southern blot analyses are also shown. Lower left panel: Southern blot analysis for gene rearrangements. DNAs extracted from a control spleen (C) and Ikaros-integrated EHKIΔNeo/MMLV mice (nos. 3, 5 and 9) were digested with BamHI and probed with the adjacent genomic fragment shown in (a) (probe C for nos. 3 and 5, probe D for no. 9). Germline (GL) and rearranged bands are indicated by arrows and arrowheads, respectively. Lower middle panel: Northern blot analysis for Ikaros mRNA expression. mRNAs of a control spleen (C) and Ikaros-integrated EHKIΔNeo/MMLV mice (nos. 3, 5 and 9) were probed with the Ikaros coding region. Ikaros-non-integrated tumors (nos. 7 and 10) were also used as controls. The position of Ikaros mRNA is indicated by an arrow and β-actin hybridization served as the internal control. Lower right panel: RT–PCR for Ikaros mRNA isoforms. mRNAs of a control spleen (C) and Ikaros-integrated EHKIΔNeo/MMLV mice (nos. 3, 5 and 9) were subjected to RT–PCR to detect Ikaros mRNA isoforms. The positions of isoforms Ik1, Ik2, Ik3, Ik4 and Ik6 are indicated. A human CML BC cell line, BV173, was used to show the position of Ik-6 (Nakayama et al., 1999). (c) Zfp521 gene. Upper panel: Schematic illustrations of viral integration sites in the Zfp521 gene. Exons are boxed, and the coding and noncoding regions are indicated by black and white boxes, respectively. Virus integration sites are indicated by vertical arrows with the related mouse identification numbers (nos. 2 and 4). Position of a probe used for Southern blot analyses is also shown. Lower left panel: Southern blot analysis of gene rearrangements. DNAs extracted from a control spleen (C) and Zfp521-integrated EHKIΔNeo/MMLV mice (nos. 2 and 4) were digested with BamHI and probed with the adjacent genomic fragment shown in (a) (probe E). Germline (GL) and rearranged bands are indicated by an arrow and arrowheads, respectively. Lower right panel: Northern blot analysis for Zfp521 mRNA expression. mRNAs of a control spleen (C) and Zfp521-integrated EHKIΔNeo/MMLV mice (nos. 2 and 4) were probed with the Zfp521coding region. Zfp521-non-integrated tumors (nos. 7 and 10) were also used as controls. Two alternatively spliced forms of Zfp521 mRNA are indicated by arrows and β-actin hybridization served as the internal control.

Second, z inc- f inger p rotein subfamily 1A1 isoform a (Zfp1a1, also known as Ikaros, hereafter referred to as Ikaros) gene was the retroviral target in three samples (nos. 3, 5 and 9). Integrations occurred in intron 1 (nos. 3 and 5) and intron 3 (no. 9) (upper panel of Figure 3b). All these three samples (nos. 3, 5 and 9) carried rearranged bands (indicated by arrowheads in the lower left panel of Figure 3b) and showed enhanced Ikaros mRNA expression when compared with a control spleen (C) and Ikaros-non-integrated samples (nos. 7 and 10) (indicated by an arrow in the lower middle panel of Figure 3b). Previous reports showed that Ikaros contributes to leukemogenesis by an isoform change from normally expressed forms (Ikaros (Ik)-1, Ik-2, Ik-3 and Ik-4) to a shorter splicing variant, Ik-6, which suppresses downstream gene expressions in a dominant-negative manner (Nakayama et al., 1999; Beverly and Capobianco, 2003). To analyze whether Ik-6 mRNA was expressed in the three Ikaros-integrated samples, we performed RT–PCR to detect alternatively spliced mRNA isoforms. All three samples expressed Ik-1, -2, -3 and -4 mRNAs but did not express Ik-6 mRNA (lower right panel of Figure 3b), indicating that the viral integrations simply upregulated Ikaros gene expression without affecting splicing.

Finally, z inc- f inger p rotein 521) (Zfp521, also known as Evi3) gene was integrated by retroviruses in two B-lineage leukemia mice (nos. 2 and 4). One integration site was in the 5′ upstream region and the other was in the 5′ untranslated region of exon 1 (upper panel of Figure 3c). Both samples showed rearranged gene patterns (indicated by arrowheads in the lower left panel of Figure 3c) and showed enhanced Zfp521 mRNA expression when compared with a control spleen (C) and Zfp521-non-integrated samples (nos. 7 and 10) (indicated by arrows in the lower right panel of Figure 3c).

On the other hand, among the iPCR products of EHKINeo+/MMLV leukemic mice, we detected one CIS, which was A belson h elper i ntegration site 1 (Ahi1) gene (shown by asterisks and bold type in Supplementary Table 2). This CIS was found in samples 3 and 4, in which retroviruses were integrated in introns 9 and 23, respectively (upper panel of Supplementary Figure 2). Southern blot analyses using genomic fragments adjacent to the integration sites detected rearranged bands, indicating that these are the major integration sites in the related tumors (indicated by arrowheads in the lower panel of Supplementary Figure 2).

Taken together, the iPCR analysis revealed that the virus integrations in three transcription factors, Gfi1, Ikaros and Zfp521, were preferentially associated with EHKIΔNeo/MMLV leukemias and strongly suggested that overexpression and/or aberrant expression of these gene products would have a cooperative role with E2A-HLF in the leukemogenic process.

Enhanced expression of ZNF521 in human leukemic cell lines with t(17;19)

To analyze the clinical significance of the three transcription factors identified in EHKIΔNeo/MMLV leukemias (Gfi1, Ikaros and Zfp521) in human leukemia with t(17;19), we examined mRNA expression levels of the three genes in t(17;19)-positive (t(17;19)+) ALL lines and in control B-lineage ALL lines without t(17;19). Cell lines were used instead of primary patient samples, because t(17;19)+ ALL constitutes only a small subset of B-precursor leukemias (Look, 1997).

The results obtained using quantitative RT–PCR are shown in Figure 4. Gfi1 mRNA levels were mostly constant in control and t(17;19)+ cell lines, but the overall Gfi1 expression in t(17;19)+ lines was lower than that in control lines (Figure 4, left panel). As for Ikaros, mRNA expression levels were relatively stable in control lines but were varied among t(17;19)+ lines, and the mean Ikaros expression in t(17;19)+ lines was slightly lower than that in control lines (Figure 4, middle panel). These results indicated that the expression levels of Gfi1 and Ikaros were not enhanced in t(17;19)+ cell lines.

Figure 4
figure4

Quantitative mRNA expression of Gfi1, Ikaros and ZNF521 in human leukemic cell lines with or without t(17;19). The mRNA expression levels in five control B-progenitor cell lines (control) and three t(17;19)-positive cell lines (t(17;19)+) relative to the mean of the control cell lines (white bar) are indicated by white and black circles, respectively. The mean of t(17;19)+ cell lines is indicated by a black bar. The relative expression ratio (vertical bar) is shown on a logarithmic scale. The high expression patterns of ZNF521 in t(17;19)+ cell lines are indicated by arrows and an arrowhead (right panel).

In contrast, the expression levels of ZNF521, the human homolog of Zfp521 (also known as early hematopoietic zinc-finger protein (EHZF)), were found to be consistently higher in t(17;19)+ lines than in control lines. Two lines showed approximately 10-fold upregulation and one line showed more than >50-fold upregulation (indicated by arrows and an arrowhead in the right panel of Figure 4). These results strongly suggest that the overexpression of ZNF521 would be clinically relevant to t(17;19)-positive B-lineage ALL.

Expression of E2A-HLF and Zfp521 conferred a growth advantage on B-progenitor cells, and both knocked-in for E2A-HLF and transgenic for Zfp521mice developed B-lineage ALL

We finally analyzed the in vivo cooperative role of Zfp521 with E2A-HLF. For this purpose, we generated transgenic mice for Zfp521 and crossed them with EHKIΔNeo mice. To express Zfp521 in lymphoid cells, Zfp521 complementary DNA with an HA tag (Zfp521HA) was subcloned into EμSV vector, which has been successfully used to express target genes in the lymphoid lineage (Rosenbaum et al., 1990) (Figure 5a). Among several transgenic lines established (EμSV/Zfp521), mice of a line that expresses Zfp521HA at a high level in lymphoid cells (data not shown) were chosen and crossed with EHKIΔNeo mice.

Figure 5
figure5

Cooperative oncogenecity of Zfp521 with E2A-HLF and increased proliferative ability of B-cell precursors in EHKIΔNeo knock-in and EμSV/Zfp521HA transgenic mice. (a) Schematic structure of the transgene for generating EμSV/Zfp521HA transgenic mice. EμSV enhancer/promoter, Zfp521HA complementary (c)DNA, SV40 splicing and polyA signals are shown as gray, black and shaded boxes, respectively. The positions of the splicing and primers encompassing the splicing signal (SV40-1 and SV40-2) are indicated. (b) Survival curves of EHKIΔNeo mice, EμSV/Zfp521HA mice and EHKIΔNeo × EμSV/Zfp521HA mice. During the observation period of 6 months, whereas no disease was observed in EHKIΔNeo and EμSV/Zfp521HA mice (thin continuous and thin dotted lines), half of EHKIΔNeo × EμSV/Zfp521HA mice died of leukemia (thick continuous line). The time point of E2A-HLF induction by pIpC is indicated by an arrow and the diseased EHKIΔNeo × EμSV/Zfp521HA mice are numbered. (c) Gene rearrangement analysis of leukemias developed in EHKIΔNeo × EμSV/Zfp521HA mice. DNAs extracted from a control spleen (C), an EHKIΔNeo mouse spleen, an EμSV/Zfp521HA mouse spleen and five tumors developed in EHKIΔNeo × EμSV/Zfp521HA mice were digested with EcoRI and blotted with the JH probe. Germline (GL) and rearranged bands are indicated by an arrow and arrowheads, respectively. (d) Expression of E2A-HLF and Zfp521HA in leukemias developed in EHKIΔNeo × EμSV/Zfp521HA mice. RNAs extracted from a control spleen (C), an EHKIΔNeo mouse spleen, an EμSV/Zfp521HA mouse spleen and five tumors developed in EHKIΔNeo × EμSV/Zfp521HA mice were subjected to RT–PCR for E2A-HLF (upper panel) and Zfp521HA (middle panel). All the tumors expressed both iKI allele- and transgenic allele-derived products. HPRT RT–PCR served as the internal control (lower panel). (e) Results of flow cytometric analysis (upper panels) and B-cell colony formation assay (lower panels). Upper panels: BM cells extracted from EHKINeo+ and EHKIΔNeo mice (left panels) and wild-type (WT) and EμSV/Zfp521HA mice (right panels) were analyzed by flow cytometry using anti-B220 and anti-Thy1.2 antibodies. The mean percentages of B220-positive cells of three independent mice are shown. Lower panels: BM cells extracted from the same types of mice were subjected to B-cell colony formation assay. The mean colony numbers of three independent mice are shown.

The survival curves of the offspring are shown in Figure 5b. During about 6 months of observation period, half of the compound mice developed acute leukemia (thick continuous line), whereas none of EHKIΔNeo or EμSV/Zfp521 alone showed hematological disease (thin continuous and thin dotted lines). All the leukemic cells were positive for B220 but negative for Thy1.2, Mac1 or Gr1 (data not shown) and showed rearrangement patterns in the IgH region (Figure 5c). The expression of E2A-HLF and Zfp521HA in the tumor tissues was confirmed by RT–PCR using primer sets specific for transcripts from E2A-HLF knocked-in allele (E2A-77+HLF-2, see Figure 1c) and EμSV/Zfp521 transgene (SV40-1+SV40-2, see Figure 5a), respectively (Figure 5d).

As EHKIΔNeo mice with Zfp521 overexpression exclusively developed B-lineage leukemia (nos. 1–5 of EHKIΔNeo × EμSV/Zfp521HA mice and nos. 2 and 4 of EHKIΔNeo/MMLV mice), we analyzed the proliferative potential of B-progenitor cells in EHKIΔNeo mice and EμSV/Zfp521 mice. For this purpose, BM cells extracted from both types of mice and their controls were subjected to flow cytometric analysis and B-cell colony formation assay. As shown in Figure 5e, both of EHKIΔNeo knock-in and EμSV/Zfp521 transgenic BM cells contained increased number of B-cell precursors and possessed an enhanced B-cell colony formation ability when compared with those of control EHKINeo+ and wild-type mice (as for the results of flow cytometry, see also Supplementary Figure 3). These results indicated that expression of E2A-HLF and Zfp521 rendered a proliferative ability to B-progenitor cells and suggest that their coexpression synergizes and contributes to the development of B-lineage leukemia.

Discussion

In earlier studies, we analyzed the role of E2A-HLF by a transgenic approach and showed that the expression of E2A-HLF under the control of lymphocyte-specific promoters perturbs normal lymphocyte development and contributes to the development of ALL (Honda et al., 1999). However, in contrast with the fact that human leukemia harboring t(17;19) exclusively shows a B-cell phenotype, all the E2A-HLF transgenic mice developed T-cell ALL (Honda et al., 1999). In this work, to circumvent this problem and to create a mouse model that further mimics human t(17;19)-positive ALL, we generated mice in which E2A-HLF was inducibly expressed under the control of the native E2A promoter.

Stimulation of EHKINeo+/MxCre mice with pIpC produced EHKIΔNeo mice, in which the deletion of the floxed Neo gene induced the expression of the E2A-HLF chimeric gene product in the hematopoietic tissues (Figures 1c and d). However, no disease was developed in EHKIΔNeo mice during the long-term observation period (Figure 2a, thin lines), indicating that the acquired expression of E2A-HLF per se is insufficient for the development of leukemia. This finding is in line with previous reports showing that iKI mice of other leukemogenic transcription factor chimeras, such as AML1-ETO and MLL-CBP, did not show hematopoietic disorders, and secondary mutations induced by N-methyl-N-nitrosourea or irradiation were required to induce a fully malignant phenotype (Higuchi et al., 2002; Wang et al., 2005). In this study, to introduce additional gene alterations, we used RIM, as it not only successfully induces mutations in the mouse genome but also has the advantage that the mutated genes can be detected by iPCR using the tumor genome and virus-specific primers (Jonkers and Berns, 1996; Mikkers and Berns, 2003; Nakamura, 2005).

MMLV infection induced acute leukemias in EHKIΔNeo mice at a higher frequency and with a shorter latency than in control EHKINeo± mice (Figure 2a, thick lines). This finding indicates that E2A-HLF possesses an oncogenic potential in hematopoietic cells, which was accelerated by viral integrations. In addition, it is to be noted that the phenotypes of the leukemias were different between EHKIΔNeo/MMLV and EHKINeo+/MMLV mice. In contrast with the fact that EHKINeo+/MMLV mice mainly developed T-cell ALL (four of six samples), EHKIΔNeo/MMLV mice showed B-progenitor ALL (two samples) and lineage marker-negative leukemias (other nine samples; Figures 2b and c and Table 1).

Previous studies showed that that MMLV induces T-cell leukemia in wild-type mice very efficiently, at almost 100% penetrance (Jonkers and Berns, 1996; Mikkers and Berns, 2003). Thus, the reason why all the EHKINeo+/MMLV mice did not develop T-cell ALL is unclear. One possibility is the low copy number of the virus. This idea is supported by our previous RIM study, in which only 60% of the MMLV-infected wild-type mice developed T-cell ALL (Mizuno et al., 2008). In addition, it also remains to be clarified why leukemias of EHKIΔNeo/MMLV mice showed B-progenitor and lineage marker-negative phenotypes. A previous report showed that transgenic background affected the disease phenotype of MMLV-induced leukemia. MMLV-infected wild-type mice exclusively developed T-ALL, whereas virus-infected /bcl2 transgenic mice mainly succumbed to B-lineage leukemia (Shinto et al., 1995). Therefore, it could be postulated that induced expression of E2A-HLF might exert its oncogenic potential in hematopoietic cells differentiating from a very early to the B-cell committed stage. This idea is in line with the finding that B-cell precursors in the EHKIΔNeo BM possessed a proliferative ability (Figure 5e) and is also in good agreement with the result that t(17;19)-positive human leukemia is exclusively of early B-progenitor phenotype (Inaba et al., 1992).

Intriguingly, pathological analysis revealed that microthrombi, a clinical feature of coagulopathy, were observed in the lung of three EHKIΔNeo/MMLV leukemic mice with relatively low platelet count (nos. 3, 6 and 8, indicated by arrows in Supplementary Figure 4, and see also Table 1). Microthrombus formation was not observed in control EHKINeo+/MMLV leukemic tissues and has not been detected in our previous RIM studies (Mizuno et al., 2008; Miyazaki et al., 2009), strongly suggesting that this pathological abnormality is specific for E2A-HLF-expressing leukemic mice. Taken together, our mouse model would not only reflect the oncogenicity of E2A-HLF in hematopoietic progenitor cells differentiating to the B-cell lineage (Inaba et al., 1992), but also represent the coagulopathic property of t(17;19)-positive leukemic cells (Hunger, 1996).

iPCR of EHKIΔNeo/MMLV leukemic mice identified Gfi1, Ikaros and Zfp521 as CISs, whereas that of EHKINeo+/MMLV leukemic mice detected Ahi1 as a CIS (Supplementary Tables 1 and 2). Major contribution of the CISs to tumor formation was confirmed using Southern blot analysis (Figure 3 and Supplementary Figure 2), and aberrant expression of the gene products in the related leukemic tissues in EHKIΔNeo/MMLV mice was shown using northern blot analysis (Figure 3). These results indicated that the three transcription factors, Gfi1, Ikaros and Zfp521, would have a cooperative role preferentially in E2A-HLF-mediated leukemogenesis.

Gfi1 was originally cloned as a gene whose activation in T-cells by MMLV insertion leads to IL-2 independence (Gilks et al., 1993) and was subsequently found as a target in tumors that developed in MMLV-infected transgenic mice (Zörnig et al., 1996; Scheijen et al., 1997). Transgenic studies showed that the aberrant Gfi1 expression itself does not efficiently induce leukemia, but exerts its oncogenic potential when coexpressed with other genes such as Myc or Pim. Thus, our results suggested that E2A-HLF might be a new candidate gene that cooperates with Gfi1.

The frequent retroviral integration in the Ikaros gene (3 of 11 samples, see Figure 3b and Table 1) is to be noted, as in a world-wide RIM screen (http://RTCGD.ncifcrf.gov), only four Ikaros-integrated samples were reported among more than several hundred CISs. A previous study using MMLV-infected lck/NotchIC (the active form of Notch1) transgenic mice identified Ikaros as a CIS (Beverly and Capobianco, 2003), in which MMLV was preferentially integrated in intron 2 and induced the expression of the dominant interfering Ik-6. However, in this study, the integration of MMLV in introns 1 or 3 increased expression of normal Ikaros isoforms (Ik-1 to Ik-4) but did not induce Ik-6 expression (Figure 3b). These results suggested that Ikaros might contribute to leukemogenesis through different mechanisms, depending on the partner genes.

Identification of Zfp521 as a CIS is particularly interesting, as both Zfp521-integrated mice (nos. 2 and 4) developed B-progenitor ALL (Figure 2 and Table 1), which corresponds to the phenotype of human t(17;19)-positive leukemia. Therefore, it could be strongly postulated that ZNF521, the human counterpart of Zfp521, has an important role in the leukemogenic process of ALL with t(17;19). Indeed, among three zinc-finger proteins isolated as CISs in EHKIΔNeo/MMLV leukemic mice, we found that only ZNF521 was consistently overexpressed in human ALL cell lines harboring t(17;19) (Figure 4). In addition, both knocked-in for E2A-HLF and transgenic for Zfp521 mice frequently developed B-lineage ALL (Figure 5), which showed the in vivo cooperative oncogenecity of Zfp521 with E2A-HLF.

Zfp521 was originally identified as a retroviral integration site in AKXD mice with B-lineage lymphomas, which encodes a transcription factor with multiple zinc-fingers (Warming et al., 2003). Although the molecular mechanisms by which aberrant expression of Zfp521 contributes to leukemogenesis are not fully understood, one possibility is that Zfp521 impairs normal B-cell development by inhibiting the function of EBF1 (Hentges et al., 2005), a transcription factor required for B-cell development (Lin and Grosschedl, 1995). Another possibility is that Zfp521 itself functions as a trans-repressor and perturbs normal hematopoietic cell development through a N-terminal conserved domain that recruits and interacts with the nucleosome remodeling and deacetylase corepressor complex (Bond et al., 2008).

Zfp521 was found to be widely associated with B-cell leukemia/lymphoma in mouse, whereas aberrant expression of ZNF521 is rarely found in B-progenitor ALL in human (Bond et al., 2008). Considering that t(17;19)-positive leukemia is found in a small portion of human ALL (Look, 1997), it might be postulated that Zfp521/ZNF521 is a preferential partner of E2A-HLF and the cooperative oncogenicity of these two genes constitutes a small subset of human B-lineage ALL.

In this report, we applied retrovirus insertional mutagenesis to E2A-HLF iKI mice, isolated Gfi1, Ikaros and Zfp521 as cooperative genes with E2A-HLF and identified Zfp521/ZNF521 to be a cooperative gene for E2A-HLF in t(17;19)-positive B-lineage leukemia. These results provide evidence that multi-step gene alterations are required for leukemogenesis and prove that the iKI system in conjugation with RIM is a valuable tool for identifying genes whose aberrant expression contributes to the malignant transformation of hematopoietic cells.

Materials and methods

Construction of iKI and transgenic vectors and generation of knock-in and transgenic mice

Detailed procedures for construction of iKI and transgenic vectors and for generation of iKI and transgenic mice are described in Supplementary Table 3.

Primer sequences

All the primer sequences used in this study are shown in Supplementary Table 4.

RT–PCR

To detect E2A-HLF mRNA, RT–PCR was performed using E2A-77 and HLF-2 primers that were derived from E2A exon 1 and the HLF portion of E2A-HLF complementary DNA as previously described (Miyazaki et al., 2002). Zfp521 mRNA expression was examined by RT–PCR using SV40-1 and SV40-2 primers that encompass the SV40 splicing signal as described (Honda et al., 1995). To detect Ikaros mRNA isoforms, RT–PCR was performed as described elsewhere (Nakayama et al., 1999). To quantitate mRNA expression in human cell lines and mouse tissues, quantitative RT–PCR was performed using primers listed in Supplementary Table 4 as previously described (Miyazaki et al., 2002).

Immunoprecipitation and western blot

Tissues were homogenized in 1% Triton lysis buffer and immunoprecipitation and western blot were performed as previously described (Honda et al., 1999). Positive signals were visualized using enhanced chemiluminescence.

MMLV infection and identification of retroviral integration sites

Preparation and infection of retroviruses were performed as previously described (Wolff et al., 2003a, 2003b). Identification of retroviral integration sites was performed essentially as described elsewhere (Yamashita et al., 2005). Position mapping on the mouse chromosome was performed with a Basic Local Alignment Search Tool (BLAST) search using the University of California Santa Cruz Genome Bioinformatics database (http://genome.ucsc.edu) and the definition of a CIS was the same as in the retrovirus tagged cancer gene database (http://RTCGD.ncifcrf.gov) (Akagi et al., 2004).

Pathological and flow cytometric analyses

Smears and stamp specimens of leukemic tissues were examined as described (Honda et al., 1999). Flow cytometric analysis were performed as previously described (Miyazaki et al., 2009).

Colony assays

Colony assays were performed as previously described (Miyazaki et al., 2009). In brief, 1 × 105 BM cells were subjected for a B-cell colony formation assay using MethoCult M3630 (StemCell Technologies, Inc., Vancouver, Canada), which contains 10 ng/ml rhIL-7. After 12–14 days of incubation, colony numbers were counted.

Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

We thank Yuki Sakai, Kayoko Hashimoto, Yuko Tsukawaki and Rika Tai, for the care of the mice and technical assistance, Dr Nobuaki Yoshida for E14 ES cells, Dr Søren Warming, Dr Neal G Copeland and Dr Nancy A Jenkins for mouse Zfp521 cDNA, Dr Koichi Ikuta for mouse TCRJβ probe, Dr Hirotaka Matsui for statistical analysis and Dr Takuro Nakamura for helpful discussion. This work was in part supported by a grant-in-aid from the Ministry of Education, Science and Culture of Japan, a grant-in-aid for Cancer Research from the Ministry of Health, Labour and Welfare of Japan (13-2), Takeda Science Foundation, Astellas Foundation for Research on Metabolic Disorders, the Japan Leukaemia Research Fund and Tsuchiya Foundation.

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Correspondence to H Honda.

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Authorship

Contribution: NY, Z-iH, TI and HH designed and performed the research and wrote the paper; HO centralized the pathological analysis; RK and LW generated the retrovirus; KM, MM and TS participated in the flow cytometric analysis; AN performed colony assays. All the authors checked the final version of the paper.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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Yamasaki, N., Miyazaki, K., Nagamachi, A. et al. Identification of Zfp521/ZNF521 as a cooperative gene for E2A-HLF to develop acute B-lineage leukemia. Oncogene 29, 1963–1975 (2010) doi:10.1038/onc.2009.475

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Keywords

  • E2A-HLF
  • inducible knock-in mice
  • retrovirus insertional mutagenesis
  • Zfp521/ZNF521

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