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Molecular and Cellular Biology, May 2006, p. 3902-3916, Vol. 26, No. 10
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.10.3902-3916.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Persistent Transactivation by Meis1 Replaces Hox Function in Myeloid Leukemogenesis Models: Evidence for Co-Occupancy of Meis1-Pbx and Hox-Pbx Complexes on Promoters of Leukemia-Associated Genes

Gang G. Wang,1,2* Martina P. Pasillas,1 and Mark P. Kamps1

Department of Pathology and Molecular Pathology Graduate Program, University of California at San Diego School of Medicine, 9500 Gilman Drive, La Jolla, California 92093,1 Biomedical Sciences Graduate Program, University of California at San Diego School of Medicine, 9500 Gilman Drive, La Jolla, California 920932

Received 16 September 2005/ Returned for modification 16 November 2005/ Accepted 21 February 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Homeobox transcription factors Meis1 and Hoxa9 promote hematopoietic progenitor self-renewal and cooperate to cause acute myeloid leukemia (AML). While Hoxa9 alone blocks the differentiation of nonleukemogenic myeloid cell-committed progenitors, coexpression with Meis1 is required for the production of AML-initiating progenitors, which also transcribe a group of hematopoietic stem cell genes, including Cd34 and Flt3 (defined as Meis1-related leukemic signature genes). Here, we use dominant trans-activating (Vp16 fusion) or trans-repressing (engrailed fusion) forms of Meis1 to define its biochemical functions that contribute to leukemogenesis. Surprisingly, Vp16-Meis1 (but not engrailed-Meis1) functioned as an autonomous oncoprotein that mimicked combined activities of Meis1 plus Hoxa9, immortalizing early progenitors, inducing low-level expression of Meis1-related signature genes, and causing leukemia without coexpression of exogenous or endogenous Hox genes. Vp16-Meis1-mediated transformation required the Meis1 function of binding to Pbx and DNA but not its C-terminal domain (CTD). The absence of endogenous Hox gene expression in Vp16-Meis1-immortalized progenitors allowed us to investigate how Hox alters gene expression and cell biology in early hematopoietic progenitors. Strikingly, expression of Hoxa9 or Hoxa7 stimulated both leukemic aggressiveness and transcription of Meis1-related signature genes in Vp16-Meis1 progenitors. Interestingly, while the Hoxa9 N-terminal domain (NTD) is essential for cooperative transformation with wild-type Meis1, it was dispensable in Vp16-Meis1 progenitors. The fact that a dominant transactivation domain fused to Meis1 replaces the essential functions of both the Meis1 CTD and Hoxa9 NTD suggests that Meis-Pbx and Hox-Pbx (or Hox-Pbx-Meis) complexes co-occupy cellular promoters that drive leukemogenesis and that Meis1 CTD and Hox NTD cooperate in gene activation. Chromatin immunoprecipitation confirmed co-occupancy of Hoxa9 and Meis1 on the Flt3 promoter.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Meis1 (myeloid ecotrophic insertion site 1) encodes a homeodomain (HD) transcription factor that positively regulates the expansion and pool size of hematopoietic stem cells (HSCs) and is commandeered in leukemic transformation. Meis1 belongs to the three-amino-acid loop extension family of HD proteins that includes Pbx, which heterodimerizes with Meis1 (9). While Pbx genes are broadly expressed in hematopoiesis, Meis1 expression is high in Sca-I+ Lin– HSCs but down-regulated during transition to Lin+ lineages (4, 15, 37). A positive role of Meis1 in HSC expansion is suggested in Meis1–/– mice, which exhibit severe reduction in the number and colony-forming potential of HSCs (4, 15). Hoxa9, which is also expressed specifically in progenitors and required for the expansion and repopulating abilities of HSCs (29, 37, 47), binds both Meis1 and Pbx, suggesting that combinations of Meis-Pbx, Hox-Pbx, and Hox-Pbx-Meis complexes, such as those that regulate Hoxb1 and Hoxb2 expression (16, 21), may also target promoters that control HSC expansion.

The functions of Meis, Pbx, and Hox proteins are usurped in leukemogenesis. Originally, Meis1 was discovered as one of two genes activated by proviral integration in BXH2 murine acute myeloid leukemia (AML), the second being Hoxa9 or Hoxa7 (32, 33). Over 50% of human AML and virtually all acute lymphoblastic leukemias containing translocations of the mixed lineage leukemia (MLL) gene exhibit strong expression of MEIS1, HOXA9, and HOXA7 (2, 17, 30). In murine AML models, Meis1 dramatically accelerates leukemia initiated by Hoxa9, Hoxb6, or Hoxb3 (reduces latency from ~200 days to ~60 days) yet exhibits no independent ability to cause disease by itself (13, 28, 46). Dominant activating forms of the MEIS1-interacting protein PBX1 [E2A-PBX1, produced by the t(1;19) translocation in human pre-B leukemia (24)] and HOXA9 [NUP98-HOXA9, produced by the t(7;11) of human AML (8)] also induce AML in mouse models, suggesting that activation of the same subset of Meis1-Pbx-Hox target genes may underlie leukemic transformation.

Hox and Meis1 have distinct roles in progenitor immortalization and in regulating gene expression. Hoxa9 blocks differentiation of cultured myeloid progenitors that do not express endogenous Meis1/2/3 and do not cause leukemia when transplanted into syngeneic recipients (7, 47, 49). Coexpression of Meis1 plus Hoxa9 in cultured progenitors immortalizes an earlier myeloid progenitor that induces AML in syngeneic recipients. The leukemia stem cell (LSC) phenotype induced by Meis1 is paralleled expression of a set of leukemia-associated genes, including Cd34, Flt3, and Erg, which have been therefore defined as Meis1-related leukemic signature genes, and we hypothesized that these Meis1 signature genes are important for LSCs to survive and expand in marrow stem cell niches (49). Whether the expression of Meis1-related signature genes requires Hoxa9 function is unknown, but it is now strongly implicated by our results presented below.

Equally unclear is the type of transcriptional activity that Meis1-Pbx complexes use to induce the LSC phenotype. Whereas Pbx can mediate repression through its recruitment of corepressors such as histone deacetylases and mSin3, Meis1 contains a C-terminal domain (CTD) that can promote transactivation in response to cell signals such as those induced by protein kinase A (16, 40). By interacting with both Hox and non-Hox factors (e.g., MyoD [6] and Oct1 [38]), Meis1-Pbx complexes contribute to both gene activation (e.g., of Myogenin [6], Hoxb1 [16], Hoxb2 [21], and Pax6 [51]) and gene repression (e.g., of CYBB [5]). It is not clear whether activation, repression, or both functions mediate the role of Meis1 in leukemia.

Here, we ask whether activation or repression by Meis1 plays the more important role in myeloid leukemogenesis by fusing Meis1 to a dominant activation domain of Vp16 or a dominant repression domain of engrailed. This same strategy was used to identify transcriptional functions of the Drosophila melanogaster Meis1 homologue, Homothorax, that contribute to embryonic development (18). Strikingly, in the absence of coexpressed Hoxa9, Vp16-Meis1 alone was able to immortalize early hematopoietic progenitors in culture that caused myeloid leukemia in syngeneic mice. Neither the immortalized progenitors nor the leukemia cells derived from them expressed endogenous Hox genes. The transforming ability of Vp16-Meis1 required interaction with Pbx and binding to DNA but not the function of the Meis1 CTD, which is required for cooperative leukemogenesis by wild-type Meis1 with Hoxa9. Vp16-Meis1 progenitors expressed low to moderate levels of the same Meis1-related leukemic signature genes (e.g., Flt3, Cd34, Erg, and Msi2h). Surprisingly, retroviral expression of Hoxa9 or Hoxa7 in Vp16-Meis1-expressing progenitors strongly up-regulated these Meis1-associated signature genes, promoted cell division, and reduced disease latency to less than 30 days. This is the first evidence that Hox proteins contribute to transcriptional activation of Meis1-related signature genes. Hoxa7, which also cooperates with Meis1 to induce AML, induced the same cellular and genetic changes as Hoxa9. Strikingly, the N-terminal domain (NTD) of Hoxa9 (residues 1 to 138), which is essential for its transforming properties with Meis1, was unnecessary for promoting proliferation and transcription of Meis1 signature genes (such as Flt3) in Vp16-Meis1 progenitors. Taken together, these results indicate that transactivation represents the key function of Meis1 that contributes to leukemogenesis and that Meis, Hox, and Pbx proteins form a common regulatory complex that activates transcription through mechanisms dependent on the Hoxa9 NTD and the Meis1 CTD, functions that can be replaced by the Vp16 domain fused onto Meis1. Consistent with this hypothesis, chromatin immunoprecipitation (ChIP) revealed that Hoxa9 and Meis1 were both recruited to the Flt3 promoter.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid construction and retroviral expression system. The murine stem cell virus (MSCV) retroviral expression system for Meis1 and Nup98-Hoxa9 was described previously (8, 49). The NCBI accession numbers designating the nucleotide and protein sequences of the murine Meis1a isoform used are U33629 and AAA85508, respectively. Vp16-Meis1 and en-Meis1 were created by inserting sequences encoding the Vp16 activation domain or the engrailed repression domain into an MluI site made by mutating the first two codons of Meis1. Vp16-Meis1 mutants were generated as described elsewhere for Meis1 (49). Flag-Hoxa7 was generated by inserting the Flag tag at the N terminus of Hoxa7, and the bicistronic MSCV vector used for coexpression of Meis1 and Hoxa7 was constructed with Flag-Meis1 upstream of the internal ribosomal entry site (IRES) and Flag-Hoxa7 downstream of the IRES (49). Internal deletions in Hoxa9 were generated by excising cDNA regions flanked by two MluI sites created by site-directed mutagenesis in the MSCV-Neo vector encoding tagged Hoxa9 (7). Plasmids containing the Vp16 activation domain or engrailed repression domain were kindly provided by Adi Salzberg (18), and MLL-ENL was provided by Robert Slany. All constructs were verified by sequencing.

Infection and culture of hematopoietic progenitors. Protocols for retroviral infection and culture of primary hematopoietic progenitors in granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), or Flt3 ligand (FL) were described previously (7, 49). Briefly, 2 x 105 to 4 x 105 of enriched Sca+ Lin– bone marrow progenitors from BALB/c mice were subjected to a single round of spinoculation-infection using 1 ml of retroviral supernatant (2 x 105 to 4 x 105/ml), followed by 3 days of drug resistance selection (0.6 to 1 mg/ml of G418 for MSCV-Neo or 1 µg/ml of puromycin for MSCV-Puro [drug selection is optional]), and cultured in medium containing either GM-SCF, SCF, or FL. Proliferation kinetics were evaluated by plating 2 x 105 to 4 x 105 drug-resistant Lin– progenitors in a 12-well tissue culture plate (Corning, New York) and splitting every 3 to 4 days in fresh medium, using a protocol that keeps the progenitor number lower than 2 x 106 per well. For coexpression of Meis1 with Hoxa9 mutants, equal amounts of retrovirus encoding Flag-Meis1 (MSCV-Puro) and Hoxa9 (MSCV-Neo) were combined and used for infection, followed by dual drug selection and cultivation in SCF or FL. Wright-Giemsa staining and fluorescence-activated cell sorter (FACS) analysis were performed as described elsewhere (49).

Antibodies and immunoblotting. Ten microliters of total cellular protein (107 cells per ml) was used for immunoblotting. Anti-Flag, anti-Flt3, and anti-Hoxa9 were obtained and used as described previously (49). Anti-Pbx was obtained from Santa Cruz Biotechnology (C-20; sc-888).

Luciferase reporter assays. Cooperative transactivation by Vp16-Meis1 and Pbx1a was evaluated using 7xPRS-pGL3, a pGL3 luciferase derivative driven by seven tandem repeats of the Pbx1-Meis1-responsive sequence (PRS), TGATTGAT, which was first identified as an E2a-PBX1 binding site (27) and later demonstrated to be recognized by Meis1 plus Pbx1 (26). One microgram of 7xPRS-pGL3 was cotransfected with 1 µg of MSCV-Vp16-Meis1, 0.5 µg of pcDNA3-Pbx1a, and 0.2 µg of pRL-TK (internal control that expresses Renilla luciferase persistently) into 293T cells. At 48 h after transfection, cell lysate was prepared and quantified for firefly and Renilla luciferase activity using a dual luciferase reporter system (Promega) and LMaxII luminometer (Molecular Devices, California). Luciferase measurements were calculated as firefly luciferase units versus Renilla luciferase units. Transcriptional activation was compared to basal luciferase levels in cells cotransfected with 7xPRS-pGL3, empty MSCV, and Pbx1a.

Semiquantitative RT-PCR and real-time qPCR. Total RNA was extracted (49) from immortalized progenitors 6 to 8 weeks post-retroviral infection, when normal progenitors are no longer present and when immortalized progenitors exhibit stable proliferation, stable expression of surface markers, and little to no differentiation. First-strand total cDNA, used as a template for reverse transcription-PCR (RT-PCR) and quantitative PCR (qPCR), was generated with the SuperScript reverse transcription system (Invitrogen) by using 4 µg of total RNA and 50 ng of random hexamers. Real-time qPCR was performed using 4 µl of 1:10-diluted cDNA as a template, 400 nM of primers, and SYBR Green PCR master mix (Applied Biosystems) in a 15-µl reaction volume and an Mx3000P real-time PCR system (Stratagene). The housekeeping gene Gapdh was used as control for mass of cDNA. The specificity and relative signal intensity of real-time qPCR amplifications were analyzed according to the manufacturer's specifications (Stratagene). Primers covered at least one intron-exon boundary. Semiquantitative RT-PCR was performed as described elsewhere (49). The sequences of primers used for each murine gene were as follows: for Gapdh, 5'-ATGACATCAAGAAGGTGGTGAAG and 5'-TCCTTGGAGGCCATGTAGG; for Hoxa9, 5'-TGGTTCTCCTCCAGTTGATAG and 5'-AGAAACTCCTTCTCCAGTTCC; for Hoxa7, 5'-CGGGCTTATACAATGTCAACAG and 5'-AAATGGAATTCCTTCTCCAGTTC; for Hoxa5, 5'-GCAAGCTGCACATTAGTCAC and 5'-GCATGAGCTATTTCGATCCT; for Hoxa10, 5'-GGAAGGAGCGAGTCCTAGA and 5'-TTCACTTGTCTGTCCGTGAG; for Flt3, 5'-AAATCCCAGAAGGAGTTTGG and 5'-CGCATAGAAGGAGATGTTGTC; for Erg, 5'-GACCACAAATGAGCGCAGAG and 5'-CTTTGGACTGAGGGGTGAGG; for Cd34, 5'-ACAGCAGTAAGACCACACCAGC and 5'-AGACACTAGCACCAGCATCAGC; for Il7r, 5'-CTGACCTGAAAGTCGTTTATCGC and 5'-CATCCTCCTTGATTCTTGGGTTC; and for Msi2h, 5'-ACGATTGACCCAAAAGTTGC and 5'-ACAAAGCCAAACCCTCTGTG.

In vivo leukemogenesis assay. The leukemic potential of oncogenes was evaluated in sublethally irradiated syngeneic BALB/c mice (450-rad dosage) followed by tail vein injection with 2 x 106 immortalized progenitors cultured ex vivo for 4 to 8 weeks (49) or with 5 x 105 ScaI+ Lin– progenitors that were prestimulated in a cytokine cocktail containing murine SCF (1:100 dilution of culture supernatant of SCF-secreting cell lines), 10 ng/ml of murine interleukin-3 (IL-3; PeproTech Inc., New Jersey), 10 ng/ml of IL-6 (PeproTech), IL-7 (1:100 dilution of culture supernatant of IL-7-secreting cell lines), and 5 ng/ml FL (Sigma); infected with retrovirus; subjected to drug resistance selection for 2 days; and cultured for 2 more days in nonselection medium containing the same cytokine cocktail. SCF- or IL-7-containing supernatant from cytokine-secreting cell lines (SCF-secreting CHO cells and IL-7-secreting J558 cells) was harvested every 24 h as 50 ml of total medium from 15-cm plates of highly confluent cells (45). Mice exhibiting a leukemia phenotype (scruffy fur, lethargy, paralysis, or splenomegaly) were sacrificed, and cells isolated from leukemic tissues were subjected to further analysis, including flow cytometry, Wright-Giemsa staining, secondary injection, immunoblotting, retroviral integration site analysis by Southern blotting, and RT-PCR (45).

Affymetrix microarray analysis. Total RNA from 5 x 107 to 10 x 107 progenitors cultured in SCF was purified using a Maxi RNA isolation kit (QIAGEN). Gene expression profiling was examined by using the Affymetrix GeneChip Mouse Genome 430 2.0 array probed with total RNA (MicroArray Core Facility, UCSD-VA Medical Center). After normalization of overall signal intensities among different hybridization experiments, levels of gene expression were calculated using d-CHIP software, taking into consideration both the perfect match (PM) and mismatch (MM) values for all 11 sets of oligonucleotide probes representing each gene (49). At signal intensities of less than 50, the PM versus MM ratio approached unity; therefore, any signal below 50 units was designated background. Because computation analysis methods will include false positives selected due to high-level expression indicated by only one or a few of the 11 pairs of diagnostic oligonucleotides, all d-CHIP results were confirmed by visual inspection of normalized chip raw data, and a subset of genes was confirmed by semiquantitative or real-time PCR.

Two approaches were used to characterize leukemogenesis genes in Vp16-Meis1 progenitors: (i) identification of genes up-regulated threefold or greater in Vp16-Meis1 progenitors by comparison to nonleukemic Hoxa9 progenitors, and (ii) identification of genes up-regulated threefold or greater by Hoxa9 in Vp16-Meis1 progenitors. Among 39,000 genes represented on the array, only 24 genes were up-regulated >3-fold in three of the Vp16-Meis1 progenitor lines by comparison to five of the Hoxa9 progenitor lines, and only 77 genes (which included 22 of those 24 genes induced by Vp16-Meis1) were up-regulated >3-fold by expression of Hoxa9 in each of the Vp16-Meis1 progenitor lines, demonstrating that in both cases, the transcriptional impact of the oncoprotein is specific and limited. Among those 24 genes induced by Vp16-Meis1 were 16 of the original 25 genes we reported as Meis1-related signature genes that were expressed specifically in leukemogenic Hoxa9-plus-Meis1 progenitors but not in Hoxa9 progenitors (i.e., Cd34, Flt3, Il7r, Crlr, Erg, Gpr56, Tilz1b, Nrip1/RIP140, C1qr1, Tmsb10, Kcna3, Ngn, Ptprcap/CD45AP, Satb1, Ngn, and Ly86/MD-1) (see Table 3, below). The other nine of the original Meis1 signature genes, expressed at extremely low levels in our original analysis of Hoxa9-plus-Meis1 progenitors, have been eliminated as candidate leukemogenesis genes based on their inconsistent activation in additional cell lines immortalized by Hoxa9 plus Meis1, lack of their expression in Vp16-Meis1 progenitors, and lack of activation by Hoxa9 (Tspan2, CD244, Gem, PLZF, Cd27, Itgb7, Oaf, CXXC5, and Ebf). The eight new genes activated by Vp16Meis1 (Msi2h, Sox4, Dbx4, P2x, Bcl2, and Sesn3, plus two unknown expressed sequence tags) were also expressed at significantly higher levels in Hoxa9-plus-Meis1 progenitors than in Hoxa9 progenitors and were added to the genes shown below in Table 3. The remaining 54 genes that were activated by Hoxa9 in Vp16-Meis1 progenitors (except c-kit, listed below in Table 3) were not expressed in Hoxa9 progenitors or Hoxa9-plus-Meis1 progenitors; therefore, they may be artificial targets of Hoxa9 in the context of Vp16-fused Meis1 or they may be real targets under different cellular contexts that were mimicked/activated by Vp16-Meis1. However, their activation is physiologically irrelevant to leukemogenesis by Hoxa9 plus Meis1 (they are not included in Table 3; information is available upon request).


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  		TABLE 3. Gene expression profiling analysis of progenitors expressing Vp16-Meis1 or coexpressing Hox>

 
Retrovirus integration site analysis. Genomic DNA was purified, digested, resolved, and transferred to a membrane as previously described (49). EcoRI or BamHI was used to digest DNA from progenitors infected by retrovirus encoding Vp16-Meis1 or Meis1-IRES-Hoxa7, respectively, and cDNA fragments encoding the Vp16 activation domain and Hoxa7 were used for making probes.

EMSA. Heterodimerization of Hoxa9 with either Pbx1 or Meis1 on DNA was examined by electrophoretic mobility shift assay (EMSA), as described previously (7, 43).

ChIP analysis. ChIP analysis was performed according to a modified protocol based on ChIP protocols of Upstate Biotechnology (New York) and Active Motif (California). Briefly, 2 x 107 to 3 x 107 myeloid progenitors were collected and subjected to DNA-protein cross-linking in 10 ml of phosphate-buffered saline plus 1% formaldehyde for 10 to 15 min at room temperature, followed by a 5-minute treatment in 5 ml of 0.125 M glycine (supplemented with 1x Complete protease inhibitor cocktail [PIC; Roche]) to stop the cross-linking reaction. After washing with cold phosphate-buffered saline-1x PIC, the cell pellet was suspended in 200 µl of sodium dodecyl sulfate lysis buffer (Upstate; 1x PIC added), incubated on ice for 15 min, and then subjected to three repeats of 12- to 13-second sonication (Sonifier 450 [VMR Scientific]). Sheared DNA was 400 bp to 1.5 kbp. The sheared sample was centrifuged at 14,000 rpm for 15 min at 4°C, and the supernatant was diluted 1:10 with ChIP dilution buffer (Upstate; 1x PIC added). The chromatin was immuno-precleared with 60 µl salmon sperm DNA-protein A-agarose (Upstate) per 2 ml chromatin, followed by a 1-h rotation (4°C) and removal of the agarose by 2 min of centrifugation at 1,500 rpm. Five hundred microliters of chromatin was used for overnight immunoprecipitation by control antibody (immunoglobulin G [IgG]) or specific antibody with rotation at 4°C. Sixty microliters of protein A-agarose was added to each tube followed by rotation for 2 h and sequential washing with low-salt wash buffer, high-salt wash buffer, LiCl wash buffer, and Tris-EDTA buffer as described elsewhere (Upstate; 1x PIC added to all buffers). Chromatin was eluted twice with 100 µl of freshly made 1% sodium dodecyl sulfate plus 0.1 M NaHCO3 with 15 min of vigorous shaking on a vortex mixer. A 200-µl aliquot of eluted chromatin-protein was subjected to reverse cross-linking, removal of RNA (by addition of 8 µl of 5 M NaCl and 1 µl of RNase A [10 µg/µl] and incubation at 65°C for 6 to 16 h), and then removal of protein (by subsequent addition of 4 µl of 0.5 M EDTA, 8 µl of 1 M Tris-HCl [pH 6.5], and 1 µl of 20-mg/ml protease K, with incubation at 45°C for 1 to 2 h). DNA was recovered using a QIAQuick spin column and suspended in 100 µl of elution buffer (QIAGEN). PCR amplification was performed using 1 U of Platinum Taq polymerase (Invitrogen) and 2 to 4 µl of eluted DNA.

Antibodies for ChIP (amount added per 500 µl of chromatin) included M2 anti-Flag (8 µg; Sigma), anti-Hoxa9 HD (10 µg), anti-Meis1 HD (10 µg), anti-Meis1 N terminal (10 µg; gift of Mark Featherstone), anti-acetyl-histone H3 (1 µg; Upstate 06-599) and anti-dimethylated histone H3 Lys4 (anti-H3K4Me2 [1 µg]; Upstate 07-030).

The murine Flt3 genomic structure was deduced using information provided by the UCSC Genome Browser (http://genome.ucsc.edu), and the putative transcription start site was defined using in silico mapping of common 5' expressed sequence tag sequences onto the genomic sequence. The ChIP primers used to amplify regions of the Flt3 locus were P3 (5'-GGACAAATGGCTCAGGAGGG and 5'-CATCTGGTAGATCTGCATTGTGG), P4 (5'-CTAGATGTGGCTCTGGGAACC and 5'-AGACAGATAGACAGAGTGCCAGC), P5 (5'-AGTCAGAAGGGACTGGCTCC and 5'-GAGTGCTGCTTAGCAGATTACC), and P6 (5'-CCTCAGAAGTGAACTCAGTTTCC and 5'-CTGCATCCAGACCATGAAGG).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hoxa7 mimics Hoxa9 in its independent and Meis1-dependent leukemic properties. Hoxa7 is also coactivated with Meis1 in BXH-2 murine AML (32); however, its leukemic functions have not been described in experimental models. Therefore, Hoxa7 was cloned into the MSCV retroviral vector, and its independent and Meis1-dependent transforming functions were assessed. Like Hoxa9, Hoxa7 immortalized GM-CSF-dependent (Fig. 1A and E) or SCF-dependent (Fig. 1B) progenitors in the absence of coexpressed Meis1. Like Hoxa9-immortalized progenitors, Hoxa7-immortalized progenitors were 100% MacI+ (data not shown), differentiated into mature neutrophils when shifted into medium containing G-CSF (Fig. 1E), and did not cause leukemia when injected into sublethally irradiated mice (Fig. 1F). Similar to the effect of coexpressing Meis1 with Hoxa9, coexpression of Meis1 with Hoxa7 immortalized a distinct SCF-dependent progenitor (Fig. 1B and E) that expressed Flt3 (Fig. 1C and D), proliferated more rapidly (Fig. 1B), underwent self-renewal in FL (Fig. 1C and E) or IL-7, GM-CSF, or SCF plus G-CSF (data not shown), and induced myeloblastic leukemia (Fig. 1F and G) (Mac-I+ B220– Cd19–) that exhibited the same pathophysiology as that induced by Hoxa9 plus Meis1 (Table 1). Leukemic cells contained the same retroviral integration sites as injected progenitors (Fig. 1H) and expressed retroviral Flag-Hoxa7 and Flag-Meis1 and endogenous Flt3 (Fig. 1I), confirming that they arose from injected cells.


Figure 1
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  		FIG. 1. Hoxa7 mimics Hoxa9 in terms of its independent and Meis1-dependent transforming functions. (A) Growth kinetics of GM-CSF-dependent marrow progenitors infected with MSCV retrovirus encoding vector only, wild-type (WT) Hoxa7, or a DNA-binding-defective Hoxa7 (N51S), followed by drug selection. Error bars indicate the standard deviations among three repeated experiments. (B and C) Growth kinetics of primary marrow progenitors infected with retrovirus encoding vector only, Meis1, Hoxa7, or Meis1 plus Hoxa7, followed by culture in SCF (B) or in FL (C). (D) Immunoblot analysis of Hoxa7, Meis1, and Flt3 in cultures shown in panel B. (E) Wright-Giemsa stain of GM-CSF-dependent progenitors immortalized by Hoxa7 in GM-CSF or when shifted to G-CSF and those immortalized by Meis1 plus Hoxa7 in SCF or FL. (F) Survival curves of mice injected with two progenitor lines immortalized by Hoxa7 in SCF (four for each), progenitors immortalized by coexpressed Meis1 and Hoxa7 (four for one line in SCF and four for one line in FL), and Sca+ Lin– marrow progenitors freshly transduced with retrovirus encoding Hoxa7 (four mice) or Meis1 plus Hoxa7 (six mice). (G) Representative Wright-Giemsa stains of tissue samples from mice bearing AML induced by Meis1 plus Hoxa7. BM, bone marrow; SP, spleen; LN, lymph node; PB, peripheral blood. (H) Southern blot of retroviral integration sites, using BamHI-digested genomic DNA from control Hoxa9-immortalized progenitors (C), injected parental progenitors immortalized by Meis1 plus Hoxa7 (P), and leukemic cells extracted from bone marrow (M) or spleen (S). The arrowhead indicates a band that represents the proviral Hoxa7 integration site. Stars indicate nonspecific bands. ID, identification. (I) Immunoblot analysis of expression of retrovirus-encoded genes, Flag-tagged Hoxa7 and Meis1, and Flt3 in control Hoxa9-immortalized progenitors (C); in parental progenitors immortalized by Meis1 plus Hoxa7 that were injected into mice (P); in leukemia cells from bone marrow (M), spleen (S), and lymph node (L); and in purified white blood cells (B). The arrowhead indicates a specific band, and the star indicates a nonspecific band.

 

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  		TABLE 1. Phenotypes of leukemias induced by coexpressed Hoxa7 and Meis1c

 
In contrast to the use of stably immortalized progenitors to initiate leukemia, we also reconstituted mice with Sca+ Lin–-enriched marrow progenitors following an immediate infection with Hoxa7 or Hoxa7 plus Meis1 retrovirus. Mice transduced with Hoxa7-infected progenitors developed myeloid leukemia after a long latency (170 days), while those transduced with Hoxa7 plus Meis1 acquired AML rapidly (74 days) (Fig. 1F) with leukemic myeloblasts that evidenced the same phenotypes as those resulting from injections of immortalized progenitor lines (Table 1). Therefore, Hoxa7 behaved the same as Hoxa9, as both immortalized a nonleukemic myeloid progenitor when expressed without Meis1 and immortalized an Flt3+, leukemia-initiating progenitor when coexpressed with Meis1 (49).

Vp16-Meis1 immortalizes hematopoietic progenitors in the absence of coexpressed Hox genes. To identify the transcriptional function of Meis1 that is important for cooperation with Hoxa9 and Hoxa7 in establishing the leukemic stem cell phenotype, dominant transactivating or transrepressing forms of Meis1 were produced by fusing it to the activation domain of herpes simplex viral protein Vp16 or to the repression domain of Drosophila engrailed (en) (Fig. 2A). Sca1+ Lin–-enriched marrow progenitors were infected with retrovirus encoding empty vector, Meis1, Vp16-Meis1, or en-Meis1 and cultured in medium containing SCF or FL as the sole cytokine. With either cytokine, expression of Vp16-Meis1 itself was able to immortalize progenitors, while cultures infected with Meis1 or en-Meis1 retrovirus followed the same decline in progenitors as uninfected cultures (Fig. 2B). Vp16-Meis1 had no effect on NIH 3T3 fibroblasts, indicating that the differentiation arrest phenotype was not accompanied by a cell proliferation phenotype, as is the case with E2A-PBX1, a transcriptionally activated form of the MEIS1 partner PBX1 (25). To test whether immortalization by Vp16-Meis1 might be mediated by expression of endogenous Hox genes, the expression of Hoxa5, Hoxa7, Hoxa9, and Hoxa10 was measured by semiquantitative RT-PCR (Fig. 2C) and quantitative real-time PCR (data not shown, except for Hoxa9 in Fig. 4D, below). None of these four Hox genes was expressed in two of the Vp16-Meis1-immortalized progenitors, while they were all expressed strongly in progenitors immortalized by the Hox locus transactivators MLL-ENL (50) or Nup98-Hoxa9 (8). A global view of Hox gene expression assessed by using Affymetrix mouse gene arrays demonstrated no expression of any of 31 Hox genes present on the array chip (Hoxa1 to -5, -a7, -a10, -a11, -a13, -b1, -b3 to -9, -b13, -c4 to -6, -c8, -c9, -c13, -d1, -d3, -d4, or -d10 to -13) among five independent Vp16-Meis1 progenitor lines (19), while each of two Nup98-Hoxa9-immortalized progenitor lines exhibited strong expression of eight Hox genes (Hoxa5, -a7, -a9, -a10, -c4 to -6, and -c8). Therefore, Hox genes are not expressed in Vp16-Meis1 progenitors. Affymetrix arrays also revealed strong expression of Pbx1a, weak expression of Pbx2a, and no expression of Pbx1b, Pbx2b, or Pbx3 in Vp16-Meis1 progenitors (Fig. 2E). Pbx1a expression was verified by Western blotting (Fig. 2D). Vp16-Meis1 progenitors were cytokine dependent, proliferating as progenitors in SCF or FL, and a subset (5 to 25%) proliferated as undifferentiated progenitors in IL-7, GM-CSF, or G-CSF (Fig. 2F and G), a behavior similar to progenitors coexpressing Hoxa9 plus Meis1 (49). Thus, Vp16-Meis1 mimics the combined activities of Hoxa9 plus Meis1, immortalizing progenitors capable of responding to both lymphoid and myeloid cytokines. The ability of a dominant activating form of Meis1 to replace the immortalizing function of Hox proteins raises the possibility that Meis1 binds the same promoters as Hoxa9 and Hoxa7 and that the histone acetyltransferase (HAT) activity recruited by Vp16 (31) replaces an intrinsic Hox-dependent activation function.


Figure 2
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  		FIG. 2. Vp16-Meis1 immortalizes early hematopoietic progenitors in the absence of coexpressed Hox genes. (A) Depiction of Flag-tagged Meis1, Vp16-Meis1, and en-Meis1. (B) Proliferation kinetics of progenitors cultured in SCF, after infection with retrovirus encoding Vp16-Meis1, Meis1, or en-Meis1 followed by 3-day puromycin resistance selection. A total of 200,000 puromycin-resistant progenitors were plated at day 0, and culture medium was changed every 3 days. Error bars indicate standard deviations from three repeat experiments. The inserted panel indicates Western blot analysis of Meis1 (lane 1), Vp16-Meis1 (lane 2), and en-Meis1 (lane 3) expressed in NIH 3T3 fibroblasts with anti-Flag antibody. (C) Semiquantitative RT-PCR demonstrating that Vp16-Meis1 progenitors do not express Hoxa5, Hoxa7, Hoxa9, or Hoxa10, while progenitors immortalized by Nup98-Hoxa9 or MLL-ENL express these Hox genes. RNA was prepared from immortalized progenitor cultures 5 to 7 weeks after retroviral infection, when progenitor phenotypes were stabilized. Gapdh served as the cDNA internal control. Triangles indicate a 1:10 serial dilution of cDNA template from right to left. (D) Western blot analysis of Pbx1a protein levels in progenitors immortalized by Hoxa9 alone or with coexpressed Meis1 and by Vp16-Meis1 in the absence or presence of coexpressed Hoxa9/a7; positive and negative controls were 1 µl of in vitro transcription-translation (TNT) reaction mixture added with Pbx1a or empty expression vector. (E) mRNA expression level of Pbx isoforms quantified by Affymetrix arrays in progenitors immortalized by Hoxa9, Hoxa9 plus Meis1, or Vp16-Meis1. (F and G) Vp16-Meis1 progenitors proliferate in SCF or FL as the sole supporting cytokine. All four tested lines proliferated well in SCF or FL, two lines proliferated well in IL-7, and 5 to 20% of each cell line proliferated slowly in G-CSF or GM-CSF. The morphology of immortalized progenitors following Wright-Giemsa stain is illustrated in panel F, and their relative expansion in various cytokines is quantified in panel G.

 

Figure 4
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  		FIG. 4. Vp16-Meis1 causes AML and MPD by a mechanism independent of Hoxa7 or Hoxa9. (A) Survival curves for mice injected with Lin– marrow progenitors 48 h following infection with retrovirus expressing Vp16-Meis1 (VM, fresh marrow), a VM mutant defective in binding Pbx (M2 LRF/LELL), or Meis1, mice injected with progenitor cell lines immortalized by VM (VM progenitor) or by VM plus Hoxa9 (VM-Ires-Hoxa9), or mice injected with VM progenitors expressing retroviral Hoxa9 (VM+Hoxa9). The second injection was performed using bone marrow cells freshly extracted from a VM leukemic mouse. The n value indicates the cohort size (injected mice). (B) Wright-Giemsa stains of cells extracted from tissues of mice with AML or MPD induced by Vp16-Meis1 or by Vp16-Meis1 plus Hoxa9. (C) Western blot analysis of Vp16-Meis1, Flt3, and Hox in tissues from mice with leukemia caused by Vp16-Meis1. Arrowheads indicate specific bands. NS, nonspecific bands; N, Hoxa9 progenitors as a control; P, injected parental VM progenitors; M, marrow; S, spleen; L, lymph nodes; B, peripheral blood white cells; T, thymus; C, cell lines derived from marrow leukemic progenitors. (D) Real-time qPCR analysis of Hoxa9 mRNA levels in immortalized cell lines and in leukemias induced by Vp16-Meis1. Cell lines were the same as those used in Fig. 2C. Samples of leukemia induced by Vp16-Meis1 were derived from the experiment shown in panel A from either the fresh marrow group or the progenitor group. The amplicon of Hoxa9 targets sequences encoding the N-terminal domain, which does not exist in Nup98-Hoxa9. Values to the right of the bars represent relative values of Hoxa9 transcript intensity, after verification of PCR specificity, threshold setup, relative transcript intensity calculated from the cycle threshold value, and normalization against Gapdh. BM, bone marrow; SP, spleen; BML, leukemic cell lines from bone marrow. (E) Southern blot analysis of Vp16-Meis1-retrovirus integration sites among leukemia tissues, as indicated by arrowheads. C, a different VM progenitor line as a negative control; P, injected parental progenitors; M, bone marrow; L, lymph node. (F) FACS analysis of leukemia induced by Vp16-Meis1 alone or with coexpressed Hoxa9.

 
Vp16-Meis1-mediated immortalization requires binding to Pbx and to DNA but not the function of the Meis1 CTD, an essential domain for cooperation of wild-type Meis1 with Hoxa9. The immortalization potential of Vp16-Meis1 corresponded to its ability to induce cooperative transactivation with Pbx1. A mutation that eliminated the entire M1-M2 Pbx interaction domain ({Delta}64-202), a five-residue neutral mutation in the M2 domain (M2{Delta}LRF/LELL) that disrupts heterodimerization with Pbx1 (49), or an HD mutation (HDN51S/{Delta}RRR) that virtually eliminates binding of Meis1-Pbx1 heterodimers to DNA (Fig. 3A) each reduced activation of a reporter construct driven by Pbx1-Meis1 binding motifs to levels 20% or less of those induced by wild-type Vp16-Meis1 (Fig. 3C), and each of these mutants was incapable of immortalizing progenitors in either SCF or FL (Fig. 3B). By contrast, an N-terminal deletion preceding the Pbx interaction domain ({Delta}1-64), a three-residue neutral mutation in M2 (M2{Delta}LELL), and an internal deletion between the M2 domain and HD ({Delta}202-260), all of which retained active Pbx-Meis1 heterodimerization potential on DNA (49), maintained more robust activation of the reporter construct and immortalized FL-responsive progenitors actively (Fig. 3B and C). The Meis1 CTD, which is essential for the ability of wild-type Meis1 to immortalize Flt3+ progenitors in cooperation with Hoxa9, was dispensable for transactivation by Vp16-Meis1 and was also unnecessary for Vp16-Meis1-mediated immortalization of Flt3+ progenitors. Immunoblot analysis confirmed expression of all retrovirus-encoded Vp16-Meis1 proteins in infected progenitors subjected to drug selection (Fig. 3D). The fact that the Vp16 transactivation domain can replace the function of the Meis1 CTD in immortalization of FL-responsive progenitors (16) supports the notion that gene activation is the essential biochemical function performed by the Meis1 CTD in establishing leukemia with Hoxa9.


Figure 3
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  		FIG. 3. Vp16 replaces the essential function of the Meis1 CTD and circumvents Hox cofactor dependence. (A) Depiction of Vp16-Meis1 wild-type and mutant constructs (a) and point mutations in M2 or in the HD (b and c). (B) Mutational analysis of domains required for immortalization by Vp16-Meis1 in SCF or FL. Plotted are the cumulative progenitor numbers after 26 days of culture following plating of 200,000 retrovirus-infected and puromycin-resistant Lin– progenitors. Error bars indicate standard deviations from three duplicate cultures. (C) Transactivation of a Pbx-Meis-responsive luciferase reporter (7xPRS-luc) by Vp16-Meis1 wild type and mutants in the presence of Pbx1a in 293T cells: increase of normalized luciferase activity. Error bars represent the standard deviations from two duplicate experiments. (D) Immunoblot using anti-Flag antibody to examine retroviral expression of Vp16-Meis1, 1 week after infection of Lin– progenitors followed by drug resistance selection shown in panel B (SCF). NS, nonspecific band. (E) Immunoblot demonstrating that Vp16-Meis1 activates a low level of Flt3 expression in the absence of Hoxa9 (lanes 4 and 6), which is augmented by subsequent coexpression of Hoxa9 (lane 3). (F) Typical presentation of FACS analysis of Vp16-Meis1-immortalized progenitors without (left panels) and with sequential retroviral infection of Hoxa7 or Hoxa9 (middle and right panels). Vp16-Meis1-immortalized progenitors (6 weeks after initial Vp16-Meis1 infection) were used for a sequential Hox infection, and cells were subjected to FACS analysis after cultivation for another 4 weeks.

 
Vp16-Meis1 induces basal transcription of Meis1-related signature genes. Previously, we demonstrated that the AML-initiating progenitors produced by coexpressed Meis1 plus Hoxa9 exhibited a genetic signature that includes stem cell-specific genes (i.e., Meis1-related signature genes, such as Cd34, Flt3, Erg, Msi2h, etc.) which were not expressed in nonleukemic progenitors immortalized by Hoxa9 alone (49). Because Vp16-Meis1 progenitors expressed Flt3, we evaluated the expression of other Meis1-related signature genes using FACS, immunoblotting, and microarray analysis. FACS demonstrated that Vp16-Meis1-immortalized progenitors were Cd34low Flt3low c-Kitlow/medium MacIvariable B220– Cd19– Cd27– Thy1.2–/low (Fig. 3F), a profile similar to that of progenitors immortalized by Meis1 plus Hoxa9 (49), with the major difference being a lower level of Cd34 and Flt3. Immunoblot analysis confirmed a low level of Flt3 protein in Vp16-Meis1 progenitors (Fig. 3E). Analysis of Affymetrix gene expression arrays revealed that many of the previously identified Meis1 signature genes were expressed at low to moderate levels (see Fig. 5D and E and Table 3, below). The additional genes (identified as described in Materials and Methods), Msi2h, Sox4, Dbx4, P2rx3, Bcl2, and Sesn3, which were expressed in Vp16-Meis1 progenitors, were also found expressed at significantly higher levels in Hoxa9-plus-Meis1 progenitors than in Hoxa9 progenitors and have been added to the list of Meis1-related signature genes (see Table 3, below). The expression of Cd34, Flt3, Erg, Msi2h, Il7r, Grp56, and Tizl1b was further confirmed by semiquantitative RT-PCR (data not shown) and real-time PCR (see Fig. 5E).


Figure 5
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  		FIG. 5. Both Hoxa9 and Hoxa7 activate expression of Meis1-related signature genes in Vp16-Meis1 progenitors. (A) Immunoblot analysis of Flt3 in two Vp16-Meis1 progenitor lines (VM.1 and VM.2) before and after coexpression of Hoxa9, analyzed subsequently by Affymetrix gene arrays. (B) Proliferation rates of Vp16-Meis1 progenitors after infection with MSCV-neo retrovirus encoding empty vector (open triangles), wild-type Hoxa9 (filled triangles), or Hoxa9 {Delta}1-138 (A9del38; filled squares). Three different lines of Vp16-Meis1 progenitors were used for infection, and 400,000 G418-selected progenitors were plated in SCF-containing medium at day zero. Error bars represent standard deviations. (C) Southern blot analysis of Vp16-Meis1-retrovirus integration sites demonstrated the clonality of Vp16-Meis1 progenitors before (lane 1) and after expression of Hoxa9 (lane 2) or Hoxa7 (lane 3). Lane C, control progenitor line containing a different retroviral integration site. (D) Overall expression pattern of the 25 Meis1-related signature gene listed in Table 3, quantified using Affymetrix gene arrays in progenitors immortalized by Hoxa9 alone or with coexpressed Meis1 or by Vp16-Meis1 alone or superinfected with Hoxa9/a7 retrovirus. The y axis indicates relative mRNA intensity. (E) Comparison of expression levels of five representative Meis1-related signature genes shown in panel D and Table 3 among different progenitors, using real-time PCR. The template was first-strand cDNA produced by reverse transcription using pooled mRNA purified from two progenitor lines. Error bars represent the standard deviations from two repeated experiments.

 
Vp16-Meis1 induces AML and MPD in vivo. Activation of Meis1-related signature genes suggested that Vp16-Meis1 progenitors would initiate leukemia. Indeed, AML arose in each of 12 mice injected with three different Vp16-Meis1-immortalized progenitor lines (2 x 106 cells per mouse), as well as in each of 8 mice injected with Lin– marrow progenitors freshly infected with Vp16-Meis1 retrovirus (5 x 105 cells per mouse) with a latency of 74 and 134 days, respectively (Fig. 4A and Table 2). Mice injected with progenitors transduced with a Pbx interaction-defective mutant, M2{Delta}LRF/LELL, did not develop leukemia over 370 days. Leukemia cells were factor dependent, expressed retroviral Vp16-Meis1 (Fig. 4C), and contained the same retroviral integration sites as those within the oligoclonal parental progenitors (Fig. 4E), indicating that they arose from injected cells. FACS analysis demonstrated that the majority of leukemic cells were myeloid (Mac-I+ B220– Cd19– Thy1.2– c-Kit+) (Fig. 4F and Table 2). Mice exhibited two forms of disease. The first (10 of 18 examined animals) was AML, in which 30 to 95% of leukemic cells stained as immature myeloblasts. The second (in 8 out of 18) was a myeloproliferative disease (MPD) in which 70 to 90% of leukemia cells exhibited significant neutrophil maturation (Table 2 and Fig. 4B). In both diseases, mice presented with 3- to 12-fold enlarged spleens, 10- to 50-fold enlarged lymph nodes, and 4- to 40-fold increases in circulating myeloid cells (Table 2). Comparable amounts of Vp16-Meis1 protein were present in myeloid cells from both diseases (data not shown), discounting the possibility of a reactive granulocytosis. Over 50% of leukemic progenitors expressed Flt3 (FACS in Fig. 4F and Table 2 and immunoblot in Fig. 4C). Thus, Vp16-Meis1 causes myeloid leukemia with 100% penetrance, and leukemic myeloblasts retain a low level of Flt3 and Cd34 expression, similar to the parental cells.


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  		TABLE 2. Phenotypic characterization of Vp16-Meis1-induced leukemia in micea

 
Secondary recipients of leukemic marrow acquired leukemia with a much shorter latency, averaging 26 days (Fig. 4A), indicating that additional genetic change accelerates leukemogenesis initiated by Vp16-Meis1. Because Hoxa9 and Hoxa7 are coactivated with Meis1 in BXH2 mouse AML, we evaluated whether Hoxa9 or Hoxa7 might be activated as a cooperating event during progression of Vp16-Meis-induced leukemia. Immunoblot analysis using a polyclonal antibody against the homeodomain of Hoxa9, which also detects Hoxa7 less efficiently, revealed no apparent up-regulation of these two proteins in 10 independent leukemias, regardless of whether AML arose from progenitor lines or from freshly infected marrow (Fig. 4C). This result was confirmed by real-time qPCR (Fig. 4D): in seven of eight leukemias examined (three from the fresh marrow group and the other five leukemias from the progenitor group [as in Fig. 4A]), Hoxa5, Hoxa7, Hoxa9, and Hoxa10 transcripts were absent or were present at levels lower than 0.01% of that expressed in progenitors immortalized by Nup98-Hoxa9 or MLL-ENL (results for Hoxa9 are exemplified in Fig. 4D). A single exception, exhibiting a very low level of Hoxa9 (~3% of the Hoxa9 level expressed in MLL-ENL progenitors) (sample sc1.2), also expressed Hoxa5, Hoxa7, and Hoxa10 at levels 1 to 5% of that expressed in progenitors immortalized by MLL-ENL. Thus, the large majority of myeloid leukemias induced by Vp16-Meis1 did not acquire activation of endogenous Hoxa7 or Hoxa9 genes as secondary cooperating events.

Expression of Hoxa9 or Hoxa7 in Vp16-Meis1 progenitors induces further activation of Meis1 signature genes, stimulates proliferation, and accelerates disease progression to the level of bona fide AML myeloblasts. An important unresolved question concerning the mechanism of cooperativity by Hox plus Meis1 is whether Hoxa7 or Hoxa9 assists Meis1 in the activation of Meis1-related signature genes or Meis1 functions independent of coexpressed Hox proteins. Because Vp16-Meis1 progenitors expressed low levels of Meis1-related signature genes and expressed no endogenous Hox genes, they represented a good cell model to test whether Hoxa9 or Hoxa7 could further activate expression of Meis1-related signature genes. Strikingly, retroviral expression of Hoxa9 or Hoxa7 in Vp16-Meis1 progenitors induced strong proliferation (Fig. 5B), strong up-regulation of Flt3 and Cd34 proteins (Fig. 3F and 5A), and 3- to 20-fold activation of other Meis1 signature genes (Table 3 and Fig. 5D). The reliability of the Affymetrix array analysis was verified using semiquantitative RT-PCR (data not shown) and real-time qPCR (Fig. 5E). Six newly identified genes (Msi2h, Sox4, Dbx4, and P2rx3, plus two unknown genes) were activated by Hoxa9 in Vp16-Meis1 progenitors and expressed at much higher levels in Hoxa9-plus-Meis1 progenitors than in Hoxa9 progenitors, and they have been added to our list of Meis1 signature genes (Table 3).

Vp16-Meis1 progenitors expressing Hoxa9 or Hoxa7 contained the same Vp16-Meis1 retroviral integration site as parental progenitors (Fig. 5C), ruling out the possibility that these dramatic transcriptional changes arose from selective expansion of an unrelated Cd34+ Flt3+ stem cell subpopulation that might still exist in cultures. Expression of Hoxa9 in Vp16-Meis1 progenitors reduced AML latency to an average of 24 days (Fig. 4A) and generated AML that was more similar to the myeloblastic phenotype (Fig. 4B, bottom panels), comprised of Lin– and MacI+ B220– subpopulations with high levels of Flt3 and Cd34 (Fig. 4F, bottom panels). This suggests that Hoxa9 and Hoxa7 participate in gene coactivation with Meis1.

The Hoxa9 N-terminal domain, which is essential for all transforming functions of Hoxa9, is dispensable for activating Flt3 expression and transforming properties in Vp16-Meis1-expressing progenitors, suggesting that it performs an activation function replaced by Vp16 on promoters co-occupied by Vp16-Meis1 and Hoxa9. The fact that fusion of Vp16 to Meis1 replaces Hox function, combined with the fact that both Meis1/Vp16-Meis1 and Hoxa9/a7 activate transcription of the same Meis1-related signature genes, favors a model in which Meis1, Hox, and Pbx directly bind these signature gene promoters that mediate leukemogenesis. If this model were correct, then Hoxa9 transactivation domains that are essential for activation of the Meis1 signature genes in the context of wild-type Meis1 might be dispensable in the context of Vp16-Meis1. Indeed, the Hoxa9 NTD (residues 1 to 138), which has been reported to contain a transactivation domain and Meis1 interaction motif (41), which is essential for immortalization by Hoxa9 alone in GM-CSF (Fig. 6A) or in SCF (not shown), is essential for immortalization of SCF- or FL-dependent progenitors with Meis1 (Fig. 6B), and is essential for Meis1-dependent production of Flt3 in infected ScaI+ Lin– marrow progenitors (Fig. 6C, lanes 2 to 5 versus lane 1), was dispensable for Hoxa9-induced proliferation (Fig. 5B), for Flt3 up-regulation (Fig. 6F, lane 4 versus lane 2) in Vp16-Meis1 progenitors, or for increasing the aggressiveness of leukemias produced by Vp16-Meis1 progenitors (Fig. 6G). By contrast, the DNA binding and Pbx interaction mutants Hoxa9-HDN51S and Hoxa9-PIMAAAA (7) exhibited no activation or minimal activation (25% of wild type) of Flt3 transcription (Fig. 6F, lanes 7 and 8 versus lane 2). Hoxa9{Delta}1-138 retained its ability to interact with Pbx1 on DNA (Fig. 6D, lane 3 versus lane 4) and lost its heterodimerization abilities with Meis1 on DNA (Fig. 6E, lane 4 versus lane 8). The fact that the NTD of Hoxa9 is dispensable for Hoxa9 transforming functions in Vp16-Meis1 progenitors suggests that its transactivation function is replaced by the persistent transactivation function of a Vp16-Meis1 complex bound to an adjacent DNA element.


Figure 6
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  		FIG. 6. The N-terminal residues 1 to 138 of Hoxa9 are dispensable for activation of Flt3 and transformation in Vp16-Meis1-expressing progenitors but are required for Hoxa9-mediated immortalization and Meis1-cooperating functions in terms of Flt3 activation and transformation. (A) Growth kinetics of primary bone marrow progenitors infected with retrovirus encoding either wild-type (WT) Hoxa9 or Hoxa9{Delta}1-138, followed by selection for 3 days in G418. The number of progenitors cultured in GM-CSF was counted on days 1 (solid bars), 15 (gray bars), and 30 (open bars) after drug selection. Error bars represent standard deviations of three duplicates. (B) Growth kinetics of primary ScaI+ Lin–-enriched progenitors infected with a 1:1 mixture of retrovirus encoding Meis1 and that encoding Hoxa9 (WT or {Delta}138), followed by dual drug resistance selection and cultivation in SCF or FL. Duplicate cultures were evaluated, and error bars indicate standard deviations. (C) Expression of Flt3, Hoxa9, and Flag-Meis1, 2 weeks after coinfection (as for panel B, with SCF and dual selection) of ScaI+ Lin–-enriched marrow progenitors with retrovirus encoding Flag-Meis1 and Hoxa9. (D) Heterodimer formation between Pbx1 and Hoxa9 examined by EMSA on the DNA element TGATTTAT. The identity of proteins added to the binding reaction mixture is indicated above each lane. (E) Interaction between Meis1a and wild-type or mutant Hoxa9 examined by EMSA to the DNA probe TGACAGTTTTAT. Recombinant proteins produced by coupled transcription-translation were lysate only (lane 1), Flag-Meis1 (lane 2), Hoxa9 (lane 3), and Flag-Meis1 plus Hoxa9 (lanes 4 to 8). Antibody against Flag-Meis1 (lane 5) or Hoxa9 (lane 6) was added to supershift protein-DNA complexes. (F) Expression levels of Flt3 and Hoxa9 in Vp16-Meis1 progenitors 3 weeks after retroviral expression of wild-type or mutant forms of Hoxa9 followed by drug selection in SCF-containing medium. (G) Survival curves of mice intravenously injected with 1 x 106 Vp16-Meis1 progenitors superinfected with retrovirus encoding Hoxa9 WT (n = 4) or {Delta}138 (n = 4).

 
Hoxa9 and Meis1 directly bind the Flt3 proximal promoter and 5' enhancer regions. ChIP was used to examine whether Hoxa9 and Meis1 bind the promoter of Flt3, the prototypic Meis1 signature gene whose transcription is further augmented by either Hoxa9 or Hoxa7. Primers were designed to randomly cover 10-kb upstream Flt3 promoter/enhancer sequences (Fig. 7A). ChIP demonstrated that Hoxa9 was recruited to the proximal promoter (P3) and 5' enhancer regions (P5 and P6) in progenitors expressing either Hoxa9 alone or coexpressing Meis1 plus Hoxa9 (Fig. 7B) and that Meis1 (Flag tagged; anti-Flag used for ChIP) was recruited to loci P3, P5, and P6 only in progenitors immortalized by Hoxa9 plus Meis1. The same ChIP results were observed using antibodies against the N terminus or homeodomain of Meis1 (data not shown). Interestingly, recruitment of Meis1 was accompanied by acetylation of histone H3 along Flt3 promoter and enhancer regions, while both cell lines exhibited dimethylation of histone H3 Lys 4 (H3K4) within the same regions. All signals were specific, based on the cell type specificity and precipitation control results using nonspecific IgG. These results indicate that Meis1 may induce histone acetylation prior to activation of Flt3 transcription.


Figure 7
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  		FIG. 7. Meis1 and Hoxa9 bind to the Flt3 proximal and distal promoter regions. (A) Schematic of the murine Flt3 locus. Black boxes represent exons, and the arrowhead represents the putative transcription start site. Black bars P3 to -6 designate positions of the amplicons used in ChIP analysis; ATG indicates the position of the initiation methionine. (B) ChIP analysis examining the recruitment of Hoxa9 and Flag-Meis1 and the status of histone modification on Flt3 promoters (loci P3 to P6) in myeloid progenitors immortalized by Hoxa9 alone or by both Hoxa9 and Flag-Meis1. Lanes were loaded with products of PCR amplification using template prepared from either 1% sheared chromatin (input control) or immunoprecipitated chromatin using nonspecific antibodies (IgG) or specific antibodies against the Flag tag, the homoedomain of Hoxa9, acetylated histone H3, or dimethylated histone 3 Lys 4 (H3K4).

 

   DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we demonstrated that in the absence of coexpressed Hox genes, a dominant activating form of Meis1 (Vp16-Meis1) sustains self-renewal of undifferentiated hematopoietic progenitors that exhibit leukemic potential in vivo, functions that are not possessed by either wild-type Meis1 or the dominant repressive form of Meis1, en-Meis1. We then demonstrated that Vp16-Meis1 mimics the combined functions of Hoxa9 and Meis1 in terms of activating expression of Meis1-related signature genes, albeit at low levels, which are characteristically expressed in leukemic progenitors immortalized by Hoxa9 plus Meis1 but not in nonleukemic progenitors immortalized by Hoxa9 alone. Next, we demonstrated that Hoxa9 and Hoxa7 further augment transcription of most of these Meis1 signature genes (Hox/Meis1 signature genes, as a more proper definition), indicating that their transcription is driven by the combined functions of Meis1 and Hoxa7/a9 functions in AML. The mechanism of cooperativity is implicated as promoter co-occupancy by Hoxa7/a9 and Pbx1/2-Meis1 complexes, based on the result that fusion of the Vp16 domain to Meis1 replaced both the essential function of the Hoxa9 NTD and the Meis1 CTD and was verified as such for the Flt3 promoter, using ChIP analysis. Collectively, our data support a model in which a subset of leukemogenesis-related promoters are directly bound and activated by complexes containing Hoxa7/a9, Meis1, and Pbx1/2.

Four transcriptional principles related to Meis1 and its interacting cofactors in hematopoietic progenitors are suggested by our analysis. First, the fact that Vp16-Meis1-Pbx complexes enforce self-renewal of leukemic stem cells in vivo suggests that Meis1-Pbx complexes regulate progenitor expansion during normal hematopoiesis by activating target gene transcription. Second, the fact that Hoxa7 and Hoxa9 augment expression of Meis1 signature genes in Vp16-Meis1 progenitors suggests that Hox proteins actively participate in transactivation of Meis1-related signature genes in normal hematopoiesis. Prior to this report, there was no evidence that Hoxa7 or Hoxa9 controlled expression of Meis1 target genes in leukemia. Third, the mutational analysis of Hoxa9 and Meis1 domains critical for their functions in Vp16-Meis1 progenitors suggests a model in which all three homeodomain proteins—Pbx1/2, Meis1, and Hoxa7/a9—cooperate on single promoters critical for establishing the leukemogenic stem cell phenotype (Fig. 8). Specifically, the Meis1 CTD and the Hoxa9 NTD may recruit cofactors harboring HAT activity (Fig. 8), functions replaced by the Vp16 domain in Vp16-Meis1. Fourth, the ability of the C-terminal half of Hoxa9 to activate transcription of Flt3 in Vp16-Meis1 progenitors in a manner as robust as that of wild-type Hoxa9 suggests that this region possesses an additional transcriptional function which might be, for example, recruiting histone methyltransferase or chromatin remodeling factors (Fig. 8) that complements those provided by the Meis1 CTD and Hoxa9 NTD. The biochemical activities responsible for the N-terminal and C-terminal transactivating functions of Hoxa9 remain to be determined. This hypothesis, which is based on the genetic and cellular properties of leukemic progenitors, is consistent with physical interaction studies, which also found Meis1, Hoxa9, and Pbx2 in single nuclear complexes in myeloid cells (44), and reporter studies, which delineated cooperating functions of Meis1, Pbx1, and Hoxa1 proteins on regulation of the Hoxb1/b2 enhancer (16, 21).


Figure 8
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  		FIG. 8. Model of Pbx, Meis (Vp16-Meis1), and Hox complexes binding to a subset of promoters required for expression of leukemogenesis. Numbers near the arrowheads indicate the following transcriptional activities: 1, a signal-induced transactivation mediated by the Meis1 CTD; 2, Hoxa9-NTD-mediated transactivation; 3, transactivation function mediated by the Hoxa9 C-terminal half, residues 139 to 271; 4, transactivation by the Vp16 domain fused onto Meis1, by recruiting HAT. The Vp16 domain replaces the function mediated by the Meis1 CTD and Hoxa9 NTD, suggesting that the Meis1 CTD and Hoxa9 CTD might recruit cofactors containing HAT activity and the Hoxa9 C-terminal half might utilize a different transactivation mechanism, such as recruiting factors harboring chromatin remodeling factor. AD, activation domain; RD, repression domain.

 
We also demonstrated that Hoxa7 is functionally redundant with Hoxa9 in terms of its Meis1-independent ability to arrest differentiation of nonleukemic progenitors and its Meis1-dependent function of targeting SCF- or FL-dependent leukemia-initiating progenitors that express Flt3. Hoxa7 was originally identified as a locus coactivated with Meis1 in BHX2 mouse leukemia (32) and activated in leukemia induced by large-scale retroviral insertional mutagenesis (23). Hoxa7 was also suggested to be a crucial downstream mediator of MLL fusion oncoproteins (3, 50). Our results imply that Hoxa7, like Hoxa9, controls HSC expansion in cooperation with Meis1 and is likely to form interacting complexes. While Hoxa7 does not bind Meis1 in gel shift analysis (43), our data (as suggested by Hoxa9{Delta}1-138) suggest that it interacts with Pbx-Meis1 complexes via Pbx on target promoters as others have hypothesized previously (21). The promoter co-occupancy model would also account for leukemic cooperativity between Meis1 and Hoxb6/b3, which are also not predicted to bind Meis1 directly. We predict that coexpression of Meis1 and Hoxb6/b3 in SCF-dependent immortalized progenitors would also result in transcriptional activation of Flt3 and other Meis1 signature genes.

The Hox/Meis1-related signature genes that are coactivated by Meis1 plus Hox are involved in normal HSC biology and implicated in leukemogenesis. Cd34 and Flt3 are prominent HSC markers (1). Flt3 catalytic function is activated by mutations in over one-third of human AML, and we propose that Flt3 expression permits expansion of LSCs in response to FL in stem cell niches. Gpr56 (20), Msi2h (34), Erg (48), C1qR1 (10, 48), and Wbscr5 (48) are enriched in HSC or other tissue-type stem cells. The Msi2h-related gene Msi1 is crucial for maintenance of neuronal stem cells (39). The Ets-related transcription factor Erg is fused to TLS/FUS and EWS in leukemia and Ewing sarcoma, respectively (14, 35). Il7r is important for lymphoid progenitor expansion, and Gpr56 is overexpressed in brain tumors and mutated in a genetic disorder involving brain cortical malformation (36, 42). It is possible that up-regulation of the Grb2-interacting adaptor Wbscr5/LAB (22) contributes to specific signaling pathways in HSCs. Understanding functions of these genes will cast additional light on how they contribute to the LSC phenotype.

Previous studies reported over 200 genes regulated by Hoxa9 in leukemia cell lines or in CD34+ HSCs (11, 12), but with the exception of Erg, these genes do not include the Hox/Meis1-activated signature genes examined herein. It is possible that studies using leukemia cell lines do not identify these signature genes because such genes are already expressed at high basal levels in the control parental progenitors. Alternatively, these Hox/Meis1 signature genes may not be activated by introduction of exogenous Hoxa9 if progenitors do not express essential cofactors, such as Meis1. Indeed, Hoxa9 alone does not activate any of the signature genes, as exhibited by Hoxa9-immortalized progenitors. When exogenous Hoxa9 is expressed in normal CD34+ HSCs, the expression of endogenous Hox and Meis1 may preclude significant further activation of these Hox/Meis1 signature genes. The heterogeneity of this population may also dilute the strong response of responsive subsets that express required cofactors. By contrast, because Vp16Meis1 progenitors lack Hox gene expression, they may be particularly sensitive to transcriptional regulatory mechanisms induced by Hox and, because they are uniform populations, the impact of Hox genes is maximized. Thus, Vp16-Meis1 progenitors may represent a unique tool for determining both the specificity and biochemical mechanisms of Hoxa7 and Hoxa9 gene activation in AML.


   ACKNOWLEDGMENTS
 
This work is supported by Public Health Service grant NIH CA56876 awarded to M.P.K.

We thank Dennis Young for assistance with FACS analysis and Joe Aguilera for assistance with mouse radiation (UCSD Cancer Center Shared Resources). We give special thanks to Mark Featherstone, Corey Largman, Robert Slany, and Adi Salzberg for providing reagents for work related to this paper.


   FOOTNOTES
 
* Corresponding author. Mailing address: Leichtag 249B, UCSD Medical School, 9500 Gilman Dr., La Jolla, CA 92093. Phone: (858) 534-5822. Fax: (858) 534-4715. E-mail: gawang{at}ucsd.edu. Back


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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