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 - PubMed (original) (raw)

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 et al. Mol Cell Biol. 2006 May.

Abstract

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.

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Figures

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.

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.

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.

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.

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 Δ1-138 (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.

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 Hoxa9Δ1-138, 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 Δ138), 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 × 106 Vp16-Meis1 progenitors superinfected with retrovirus encoding Hoxa9 WT (n = 4) or Δ138 (n = 4).

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).

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.

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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 - PubMed (original) (raw)