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[Cancer Research 65, 7603-7611, September 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

The Distal Zinc Finger Domain of AML1/MDS1/EVI1 Is an Oligomerization Domain Involved in Induction of Hematopoietic Differentiation Defects in Primary Cells In vitro

Vitalyi Senyuk, Donglan Li, Alexander Zakharov, Fady M. Mikhail and Giuseppina Nucifora

Department of Pathology and The Cancer Center, University of Illinois at Chicago, Chicago, Illinois

Requests for reprints: Vitalyi Senyuk, Department of Pathology, University of Illinois at Chicago, Molecular Biology Research Building, Room 3312, M/C 737, 900 South Ashland Avenue, Chicago, IL 60607. Phone: 312-413-2875; E-mail: vsenyuk{at}uic.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
AML1/MDS1/EVI1 (AME) is a chimeric transcription factor produced by the (3;21)(q26;q22) translocation. This chromosomal translocation is associated with de novo and therapy-related acute myeloid leukemia and with the blast crisis of chronic myelogenous leukemia. AME is obtained by in-frame fusion of the AML1 and MDS1/EVI1 (ME) genes. The mechanisms by which AME induces a neoplastic transformation in bone marrow cells are unknown. AME interacts with the corepressors CtBP and HDAC1, and it was shown that AME is a repressor in contrast to the parent transcription factors AML1 and ME, which are transcription activators. Studies with murine bone marrow progenitors indicated that the introduction of a point mutation that destroys the CtBP-binding consensus impairs but does not abolish the disruption of cell differentiation and replication associated with AME expression, suggesting that additional events are required. Several chimeric proteins, such as AML1/ETO, BCR/ABL, and PML/RARa, are characterized by the presence of a self-interaction domain critical for transformation. We report that AME is also able to oligomerize and displays a complex pattern of self-interaction that involves at least three oligomerization regions, one of which is the distal zinc finger domain. Although the deletion of this short domain does not preclude the self-interaction of AME, it significantly reduces the differentiation defects caused in vitro by AME in primary murine bone marrow progenitors. The addition of a point mutation that inhibits CtBP binding completely abrogates the effects of AME on differentiation, suggesting that AME induces hematopoietic differentiation defects through at least two separate but cooperating pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AML1/MDS1/EVI1 (AME) is a product of a nonrandom reciprocal chromosomal translocation between the chromosome bands 3q26 and 21q22 (1). AME is associated with myelodysplastic syndrome, acute myeloid leukemia, and the blast crisis of chronic myelogenous leukemia (2, 3). This chimeric transcription factor is generated by the in-frame fusion of AML1 and MDS1/EVI1 (ME). The AML1 gene (also known as RUNX1, CBFA2, and PEBP2) encodes the DNA-binding subunit of the transcription factor CBF (4, 5). AML1 consists of a NH2-terminal DNA-binding domain, called Runt, with homology to the product of the Drosophila segmentation gene Runt and a COOH-terminal activation domain (6, 7). AML1 interacts with the methyltransferase SUV39H1 (8), with the coactivators CBP and p300 (9, 10), and with the corepressor TLE/Groucho (11). In addition, AML1 can be acetylated (12) and methylated (8) in the Runt domain. AML1 is normally expressed in hematopoietic lineages, is essential for definitive hematopoiesis, and regulates the expression of several genes playing a pivotal role in myeloid differentiation (13–15). AML1 is involved in chromosomal abnormalities associated with human leukemias (15–18). ME is a zinc finger transcription factor related to the leukemia-associated protein EVI1 (19, 20). ME contains a conserved NH2-terminal region, called PR domain, two sets of DNA-binding zinc finger domains, a proline-rich central domain, and an acidic COOH-terminal domain. AME consists of the DNA-binding domain Runt of AML1 fused to almost the entire ME and could potentially bind and regulate both AML1- and ME-regulated promoters (21). The mechanisms by which the inappropriate expression of AME induces leukemogenesis are largely unknown. It has been shown that in contrast to the parent transcription factors, which are transcription activators in vitro (20, 22), AME is a repressor (23). Indeed, we recently showed that AME interacts with CtBP and HDAC1 (24) and it is possible that the repression of genes activated by AML1 could contribute to the neoplastic transformation.

It has been shown that several chimeric transcription factors generated by chromosomal translocations contain protein-protein interaction motifs and are able to oligomerize. For example, AML1/ETO (25, 26), PML/RARa (27), and BCR/ABL (28, 29) form multimers through the coiled-coil motif of ETO, PML, and BCR, whereas TEL/ABL (30, 31) and TEL/PDGFRß (32) multimerize through the HLH domain of TEL. Therefore, it was suggested that oligomerization plays an inappropriate role in the transforming potential of chimeric proteins by allowing the formation of constitutively activated kinases (BCR/ABL and TEL/PDGFRß) or high-order transcription complexes that inappropriately recruit coregulators at specific promoter sites leading to gene deregulation (AML1/ETO and PML/RARa). In many cases, the loss of the oligomerization domain correlates with the loss of transforming properties by the chimeric proteins (32–36). Furthermore, the substitution of the self-interaction domain with a heterologous oligomerization module recapitulates the transforming potential of the primary chimeric protein (33) and inactivates known target genes (37, 38). Therefore, it is thought that oligomerization of chimeric proteins is required for their oncogenic potential.

Here, we report that AME is capable to oligomerize in a very complex way. The distal zinc finger domain, which was thought to be a DNA-binding domain, is involved in AME oligomerization by interaction with Runt and the proximal zinc finger region. In addition, both latter domains are able to self-interact. We found that although the deletion of the distal zinc finger domain is unable to prevent oligomerization, it strongly attenuates the defects caused by AME in differentiation and cell renewal potential of primary murine bone marrow progenitors. Previously, we showed that AME interacts with the corepressors CtBP (24). Here, we show that a double mutant that has lost the distal zinc finger domain and the ability to interact with CtBP does not affect the normal pattern of differentiation and self-renewal capacity of the hematopoietic cells. These findings suggest that AME induces neoplastic transformation in vivo through at least two separate but cooperating pathways and that oligomerization of a chimeric protein is not always the major transforming requirement as generally proposed for chimeric transcription factors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
DNA constructs. The cloning vectors used in this work were pCMV-myc-nuc (Invitrogen/Life Technologies, Carlsbad, CA), pFlag-CMV2 (Sigma, St. Louis, MO), and pMSCV-neo (Clontech, Palo Alto, CA). The full-length hemagglutinin (HA)–tagged AME-1499 and the AME deletion mutants were described (24) and are shown in Fig. 1A (lines 3-19). All the AME-related cDNAs were obtained with PCR technique and standard cloning and were confirmed by DNA sequencing. Each deletion mutant cDNA was HA- or Flag-tagged at the NH2 terminus and included a nuclear localization signal derived from pCMV-myc-nuc plasmid (Invitrogen/Life Technologies) at the COOH terminus to insure proper nuclear homing. AML1 and EVI1 were cloned downstream of the glutathione S-transferase (GST) open reading frame in the pGEX-2T vector (Amersham, Piscataway, NJ).1



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  		Figure 1. AME interacts with itself. A, diagram of AME mutants. The first three lines show the diagram of the parental proteins ME and AML1 and of the chimeric protein AME. The DNA-binding domain of AML1, Runt, is maintained in the chimeric protein. The PR domain and two zinc finger domains (ZnF) of ME are also shown. The remaining lines show the deletion mutants. The point mutants AME-C and AME-C-{Delta}(1184-1246) containing the substitution DL to AS (asterisk) are unable to interact with CtBP. The numbers mark the amino acid boundary of the deletion. HA-AME contains 1,499 amino acids, including the HA-tag. B, 293T cells were cotransfected with HA-AME and Flag-AME (lanes 2 and 5) or HA-AME alone (lanes 3 and 6; top) or Flag-AME alone (lanes 3 and 6; bottom). Two days after the transfection, the cell lysates were collected and incubated with anti-Flag antibody (lanes 1-3; top) or anti-HA antibody (lanes 1-3; bottom) followed by immunoprecipitation. The immunoprecipitated proteins (lanes 1-3) and cell lysates (lanes 4-6) were separated by electrophoresis, transferred to a polyvinylidene difluoride (PVDF) membrane, and probed as marked in the figure. Lanes 1 and 4, mock-transfected cells. C, in vitro translated AME was mixed with purified recombinant Flag-AME (lanes 2 and 4) or Flag-eluted solution from the mock-transfected cells (lanes 1 and 3) and incubated with anti-Flag antibody (lanes 1 and 2) followed by immunoprecipitation.

 
Cell culture. Adherent cell lines 293, 293T, NIH3T3, and Phoenix (American Type Culture Collection, Manassas, VA) were maintained in DMEM supplemented with 10% newborn calf serum. Murine bone marrow progenitor cells were isolated and cultured in methylcellulose medium as described (39).

Transfection. DNA transfection of adherent cells was done by the calcium phosphate precipitation method (40) or with NovaFector reagent (Venn Nova, Inc., Pompano Beach, FL) according to the manufacturer's instructions. We used 10 µg of plasmid per 10 cm plate for each transfection. Bone marrow hematopoietic cells were infected and selected as described (39).

Differentiation assay. The in vitro differentiation of murine bone marrow progenitors was carried out in methylcellulose as described (39). The assays were repeated thrice and the results represent the average of all independent experiments.

Western blot analysis and coimmunoprecipitation assays. Cells were harvested 48 hours after transfection and the assays were carried out as described (41). We used commercially available monoclonal mouse antibody M2 to the Flag epitope (Sigma) and monoclonal rat antibody to the HA epitope (Roche, Indianapolis, IN).

Recombinant proteins. 293T cells were transiently transfected with the plasmid encoding Flag-AME. Cells were harvested 48 hours after transfection and immunoprecipitated by Flag beads (Sigma). After washing thrice, the beads were resuspended in 100 µL washing buffer with 100 µg/mL Flag peptide (Sigma) and placed on ice for 10 minutes with occasional mixing. The supernatant was collected after 1-minute centrifugation and passed through the Micro Bio-Spin 6 chromatography column (Bio-Rad, Hercules, CA) to replace the buffer.

In vitro translated AME was generated by using TNT Lysate Coupled Reticulocyte Lysate System (Promega, Madison, WI).

Reporter gene studies. We used the AML1-dependent minimal MCSF-R-Luc promoter driving the luciferase gene (42). NIH3T3 cells were transiently cotransfected with the MCSF-R-Luc reporter gene in the presence of the effector plasmids. We used 4 µg of each plasmid per plate. For normalization of the efficiency of transient transfections, we used Renilla luciferase control pRL-TK vector (Promega). All measurements were done in triplicate and the experiments were repeated at least thrice.

GST-fusion pull-down assays. The assays were carried out as described (32).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AML1/MDS1/EVI1 interacts with itself. It was shown that chimeric proteins generated by chromosomal translocations involving AML1, such as AML1/ETO and TEL/AML1, acquire the ability to oligomerize through a homodimerization domain present in the partner protein. To determine whether AME is also capable of self-interaction, we used coimmunoprecipitation assays of 293T cells transiently cotransfected with HA- and Flag-tagged AME. Two days after transfection, the cells were harvested and lysed and the total cellular extracts were coimmunoprecipitated with anti-Flag antibody. The immunoprecipitated proteins were separated by electrophoresis, transferred to a membrane, and analyzed with anti-HA antibody. The results show that HA-AME (Fig. 1B, lane 2, top) coprecipitates with Flag-AME, indicating that AME interacts with itself in vivo. Anti-Flag antibody alone did not precipitate HA-AME (lane 3, top). Lanes 5 and 6 show the expression of HA-AME and Flag-AME in the transfected cells. Lanes 1 and 4 show the results of coimmunoprecipitation in mock-transfected cells. The reciprocal coimmunoprecipitation assay in which HA-AME was used to coprecipitate Flag-AME gave identical results (Fig. 1B, lane 2, bottom).

To further confirm AME self-interaction, we tested purified recombinant Flag-AME and in vitro translated AME in coimmunoprecipitation assay. The results show that AME interacts in vitro with itself (Fig. 1C).

The AML1 part of AML1/MDS1/EVI1 mediates AML1/MDS1/EVI1 oligomerization. To map the interaction domain, we tested the full-length Flag-AME with the HA-AME deletion mutants shown in Fig. 1A (lines 4-8). The analysis was carried out by transient transfection of 293T cells and coimmunoprecipitation assays as described earlier. As shown in Fig. 2A, the wild-type AME and all five AME deletion mutants (lanes 2-7) interact with full-length AME, indicating that the shortest deletion mutant (AME-273) containing only the AML1 region of the chimeric protein is itself an oligomerization region. Lanes 8 to 13 show the expression of the wild-type and AME deletion mutants (Fig. 2A, top) and full-length Flag-AME (bottom) in the transfected cells. Lanes 1 and 14 show the results with mock-transfected cells.



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  		Figure 2. The NH2 and COOH termini of AME are essential for oligomerization. A, mapping the NH2-terminal domain of AME. 293T cells were transiently cotransfected with full-length Flag-AME and HA-AME or each one of the HA-tagged deletion mutants shown in Fig. 1A (lines 4-8). Two days after transfection, Flag-AME was immunoprecipitated with anti-Flag antibody and the immunoprecipitated proteins were separated by electrophoresis. After transfer to a PVDF membrane, the HA-AME deletion mutants (lanes 2-7) were detected with anti-HA antibody in coimmunoprecipitation reactions. Lanes 8 to 13, expression level of the AME deletion mutants (top) and Flag-AME (bottom) in the transfected cells; lanes 1 and 14, mock-transfected cells. B, mapping the COOH-terminal domain of AME. 293T cells were transiently cotransfected with full-length HA-AME and each one of the Flag-tagged fragments shown in Fig. 1A (lines 13-16) and analyzed as described in A. Lanes 6 to 10, expression level of HA-AME (top) and mutants (bottom) in the transfected cells.

 
The COOH terminus of AML1/MDS1/EVI1 is also an oligomerization region. Coimmunoprecipitation assays with large regions of AME indicated that the COOH terminus, including the distal zinc finger domain (amino acids 1,184-1,499), contains an interaction domain (data not shown). To better analyze this region, we generated sequentially smaller fragments distal to amino acid 1,184 (Fig. 1A, lines 13-16). Each fragment was Flag-tagged at the NH2 terminus and included a nuclear localization signal at the COOH terminus. We tested these fragments with full-length HA-AME in coimmunoprecipitation assays after their coexpression in 293T cells. As shown in Fig. 2B, fragment AME-1184-1499 (lane 1) and fragment AME-1184-1306 (lane 4), both of which contain the distal zinc finger domain, efficiently coprecipitate HA-AME. In contrast, the fragments AME-1247-1499 (lane 2) and AME-1306-1499 (lane 3), which do not include the distal zinc finger domain, interacted very weakly with HA-AME. Lanes 6 to 10 in Fig. 2B show the expression of full-length HA-AME (top) and the Flag-tagged fragments (bottom) in the transfected cells.

To determine whether the distal zinc finger domain is essential for interaction, we tested the AME-{Delta}(1184-1246) mutant in coimmunoprecipitation assay. This mutant contains a small internal deletion of only 64 amino acids that correspond to the distal zinc finger domain (Fig. 1A, line 17). We found that the 1184-1246 deletion does not abrogate AME self-interaction (Fig. 3A, lane 3). Lanes 5 and 6 of Fig. 3A report the expression after Western blot analysis of the HA-tagged (top) or Flag-tagged (bottom) AME and AME-{Delta}(1184-1246) in the transfected cells. Lanes 1 and 4 represent mock-transfected cells. These results indicate that AME contains additional oligomerization region(s).




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  		Figure 3. Mapping the oligomerization mechanism of AME. A, 293T cells were transiently cotransfected with HA- and Flag-tagged full-length AME (lanes 2 and 5) or HA- and Flag-tagged AME-{Delta}(1184-1246) (lanes 3 and 6) and analyzed as described in Fig. 2A. Lanes 5 and 6, expression level of the HA-tagged (top) or Flag-tagged proteins (bottom) in the transfected cells; lanes 1 and 4, mock-transfected cells. B, 293T cells were transiently cotransfected with the distal zinc finger fragment (Flag-AME-1184-1306) and each one of the HA-tagged constructs shown in Fig. 1A (lines 9-12) and analyzed as described in Fig. 2A. Lanes 8 to 12, expression of the AME fragments (top) and the distal zinc finger fragment (bottom) in the transfected cells; lanes 1 and 7, mock-transfected cells. C, T-antigen–negative 293 cells were stably transfected with the empty expression vector or the HA-AME expression vector and selected. Single clones were transiently transfected with Flag-tagged AME (lanes 1-4) or the fragments AME-80-206 (lanes 5-8), AME-469-691 (lanes 9-12), and AME-1184-1306 (lanes 13-16). Two days after the transfection, the cell lysates were collected and incubated with anti-HA antibody (lanes 1, 2, 5, 6, 9, 10, 13, and 14) followed by immunoprecipitation. Lanes 3, 4, 7, 8, 11, 12, 15, and 16, expression level of AME and mutants in the transfected cells; lanes 13 and 14, the band corresponding to the IgG. D, 293T cells were cotransfected with HA-AME-469-691 and Flag-AME-469-691 (lanes 3 and 6), HA-AME-469-691 alone (lanes 2 and 5), HA-AME-80-206 and Flag-AME-80-206 (lanes 9 and 12), or HA-AME-80-206 alone (lanes 8 and 11) and analyzed as described in Fig. 2A. Lanes 5 and 6, expression of HA-AME-469-691 and Flag-AME-469-691; lanes 11 and 12, expression of HA-AME-80-206 and Flag-AME-80-206; lanes 1, 4, 7, and 10, mock-transfected cells. E, a model of AME oligomerization. Solid double arrows, stronger interaction between the proximal zinc finger domain and Runt compared with others (dashed arrows). F, purified GST, GST-AML1, and GST-EVI1 conjugated to glutathione-Sepharose beads were incubated with in vitro translated AME. After extensive washing, the beads were subjected to SDS-PAGE and the separated proteins were analyzed by autoradiography (top) or Western blot with anti-GST (lane 1; expected protein size, 29 kDa), anti-AML1 (lane 2; expected protein size, 80 kDa), or anti-EVI1 (lane 3; expected protein size, 180 kDa) antibodies (bottom).

 
Runt strongly interacts with the distal zinc finger domain. To understand how AME interacts with itself, we tested the distal zinc finger fragment (Flag-AME-1184-1306) with other fragments, including Runt (AME-80-206), the PR domain (AME-274-468), the proximal zinc finger domain (AME-469-691), and the COOH-terminal domain (AME-1263-1499) (Fig. 1A, lines 9-12). As shown in Fig. 3B, the Runt domain very strongly interacts with the distal zinc finger domain (lane 2). The proximal zinc finger and full-length AME interact with the distal zinc finger domain (lanes 4 and 6, respectively). No interaction was detected for the PR domain (lane 3) and the COOH-terminal domain (lane 5). Lanes 8 to 12 in Fig. 3B show the expression of the HA-tagged fragments (top) or Flag-AME-1184-1306 (bottom). Lanes 1 and 7 are mock-transfected cells.

In some cases, the massive overexpression of recombinant proteins in 293T-transfected cells can result in artificial protein-protein interactions. Therefore, to confirm our findings in cells that express low levels of AME, we generated a 293 cell line, which stably expresses HA-AME, and used these cells in coimmunoprecipitation assays as described earlier. The results (Fig. 3C) show that all three fragments, Runt and the proximal and distal zinc finger domains, as well as the full-length AME interact with AME stably expressed from an integrated promoter.

Thus, AME has at least three oligomerization domains, the Runt domain and the proximal and distal zinc finger domains. Next, we tested the possibility of self-interaction for each one of these domains. As shown in Fig. 3D, both the proximal zinc finger (lane 3) and Runt (lane 9) domains are able to self-interact. In contrast, neither the PR nor the distal zinc finger domains seem to self-interact (data not shown). Table 1 summarizes the interaction results and Fig. 3E presents the results graphically. Taken together, our data indicate that AME interacts with itself in a very complex way. The model we propose (Fig. 3E) takes into account the results of the coimmunoprecipitation assays and predicts that the deletion of any single domain is unable to prevent the self-interaction of the protein.


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  		Table 1. Interaction regions of AME

 
To determine whether AME interacts with itself directly or requires intermediate mammalian proteins, we used GST-fusion pull-down of in vitro translated AME. Because GST-AME is very unstable in bacteria and is degraded rapidly and to confirm that both major interaction domains (Runt and the distal zinc finger domain) interact directly, we separately cloned AML1 and EVI1 in GST, obtaining GST-AML1 and GST-EVI1, respectively. These two fusion proteins were expressed in bacteria, purified, and separately tested with in vitro translated full-length AME. As shown in Fig. 3F, both GST-AML1 (lane 2) and GST-EVI1 (lane 3) efficiently interact with in vitro translated AME, suggesting that the interaction is direct. In contrast, GST alone (lane 1) was unable to efficiently interact with AME.

Work by us and by others showed that two of the AME domains, Runt and the proximal zinc finger region, have biological functions that have been characterized independently of their role in AME oligomerization. For example, it was shown that Runt interacts with DNA and several factors (GATA1, C/EBPA, PU.1, SUV39H1, etc.). Similarly, it was shown that the proximal zinc finger domain specifically binds to DNA, mediates transforming growth factor-ß signaling, and recruits coregulators. Therefore, it is expected that the deletion of either one of these large domains would dramatically alter the functions of AME by removing the DNA-binding property or by altering the signaling response mediated by AME. For these reasons and because our data indicate that the interaction between the distal zinc finger domain and Runt is stronger than any other interaction involved in AME oligomerization, we focused on the potential role that the distal, smaller, and less characterized zinc finger domain could play in transactivation and hematopoietic differentiation disruption by AME.

The distal zinc finger domain of AML1/MDS1/EVI1 is required to repress the MCSF-R promoter. We reported earlier that AME is a repressor and that the binding of CtBP is necessary to fully repress a reporter gene. To determine the role the distal zinc finger domain in the response of an AML1-dependent promoter, we tested full-length AME and the AME-{Delta}(1184-1246), AME-C, and AME-C-{Delta}(1184-1246) mutants in reporter gene assay. The AME-C mutant, described previously (24), has the CtBP-binding site mutated resulting in the inability to recruit CtBP. The AME-C-{Delta}(1184-1246) is a double mutant that contains both mutations. As a reporter promoter, we used again the AML1-dependent MCSF-R promoter (43). NIH3T3 cells were transiently cotransfected with the MCSF-R-Luc reporter gene and the effector plasmid [either AME or AME-{Delta}(1184-1246) or AME-C or AME-C-{Delta}(1184-1246)] as shown in Fig. 4. As reported previously (42), AML1 alone activates the MCSF-R promoter (Fig. 4, column 1). However, addition of AME destroys AML1-mediated activation of this promoter (column 2). The substitution of AME with the AME-{Delta}(1184-1246) mutant (column 3) or AME-C (column 5) decreases the repression. However, the double mutant AME-C-{Delta}(1184-1246), which lost both the distal zinc finger domain and CtBP-binding site, completely restores promoter activation by AML1 (column 4).



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  		Figure 4. The distal zinc finger domain of AME cooperates with CtBP in the repression of the MCSF-R promoter. A, NIH3T3 cells were transiently cotransfected with a MCSF-R reporter gene in presence of the effector plasmids. AML1 alone activates the MCSF-R promoter (column 1), but in the presence of AME, the activation of this promoter was abrogated (column 2). The substitution of AME with the mutant AME-{Delta}(1184-1246) (column 3) or the CtBP binding–defective AME-C (column 5) decreased but did not abolish the AME-mediated repression. In contrast, the double mutant AME-C-{Delta}(1184-1246) completely restores the activation by AML1 (column 4). The background has been subtracted. B, expression of transgenic proteins in NIH3T3 cells. Nuclear proteins (100 µg) were separated by electrophoresis, transferred to a PVDF membrane, and hybridized to anti-Flag (top) or anti-HA (bottom) antibody to compare the relative levels of effectors' expression.

 
Up-regulation of self-renewal capacity and abnormal differentiation of murine bone marrow progenitors require the distal zinc finger domain of AML1/MDS1/EVI1. In our previous work, we reported that the interaction between AME and CtBP is necessary for self-renewal and abnormal differentiation of primary murine bone marrow cells (24). In that study, we showed that cells infected with the AME-C point mutant unable to recruit CtBP had a proliferative potential lower than the AME-cells but higher than control cells, suggesting that AME requires not only CtBP binding for growth up-regulation but also another element (24). To determine whether the distal zinc finger domain of AME also has a role in the disruption of hematopoietic differentiation, we used in vitro colony formation assays (39). Murine bone marrow cells were isolated from the femurs of 5-week-old mice and depleted of lineage-positive cells by negative selection. The cells were infected in vitro with a recombinant MSCV retrovirus that expressed either the wild-type AME, AME-{Delta}(1184-1246), AME-C-{Delta}(1184-1246), AME-C, or the empty vector used as control. After 5 days in culture with G418 to eliminate the uninfected cells, the colonies were isolated and disaggregated. Fifteen thousand G418-selected cells of each type were cultured in methylcellulose-based medium in presence of interleukin (IL)-3, IL-6, SCF, and granulocyte macrophage colony-stimulating factor to stimulate differentiation. After 7 days, the colonies were manually counted, isolated, and disaggregated, and 15,000 cells were replated. This procedure was repeated until the cells reached terminal differentiation and were unable to induce colony formation. The assay was repeated thrice and the results represent the average of all independent experiments. As shown in Table 2, the expression of wild-type AME in primary bone marrow progenitors induces the highest number of colonies compared with the cells infected with empty vector, AME-C, AME-{Delta}(1184-1246), or AME-C-{Delta}(1184-1246). All three AME mutants increase the colony formation potential of the hematopoietic progenitors compared with the vector-infected controls. However, the number of colonies generated is intermediate between the vector-infected cells and the AME-infected cells. Among the three AME mutants, the number of colonies generated by the AME-C-{Delta}(1184-1246) mutant was closer to the control (Table 2). As we reported previously (24), we confirmed that the ability to delay terminal differentiation was markedly high for AME-cells and low (although still higher than the control cells) for AME-C cells (Table 2). We also found that the internal deletion mutant AME-{Delta}(1184-1246) conferred to the cells the ability to delay the differentiation by ~7 days (one plating) compared with the empty vector. This delay was similar to that found for AME-C. In contrast, progenitor cells that expressed the double mutant AME-C-{Delta}(1184-1246) were essentially indistinguishable from the control cells (Table 2). Morphologic analysis of cytospin preparations of the cells reproduced very closely the finding with the wild-type AME or AME mutants (Fig. 5). We confirmed that AME had the highest number of blast and mitotic cells reflecting the ability of these cells to be immortalized by AME as we reported (data not shown). In contrast, the deletion of either the CtBP-binding site or the distal zinc finger domain completely destroyed the potential for self-renewal. As determined with the colony formation assay (Table 2), we found that the differentiation of cells that expressed the double AME-C-{Delta}(1184-1246) mutant was essentially identical to the control cells. Taken together, these data indicate that the aggressive property of AME in the disruption of hematopoietic differentiation and regulated self-renewal requires the distal zinc finger domain and CtBP recruitment.


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  		Table 2. Number of colonies in the bone marrow cultures

 


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  		Figure 5. Disruption of hematopoietic differentiation by AME requires the distal zinc finger domain and the intact CtBP-binding site. A, cytospin preparations of murine bone marrow progenitor cells infected with the empty pMSCV vector, the full-length AME, the AME-C point mutant, the AME-{Delta}(1184-1246) deletion mutant, and the AME-C-{Delta}(1184-1246) double mutant were stained with Wright-Giemsa stain at the fourth replating (26 days after infection). At this time, cells infected with the empty vector and the double mutant were completely differentiated and unable to further induce colony formation. In contrast, bone marrow cells that expressed the full-length AME showed evidence of numerous cells in mitosis (white arrowheads). Mutation of the CtBP-binding site (AME-C) or deletion of the distal zinc finger domain [AME-{Delta}(1184-1246)] impaired the ability of AME to disrupt differentiation but did not reestablish a normal pattern. B, expression of transgenic proteins in murine bone marrow progenitor cells. Cell proteins (100 µg) were separated by electrophoresis, transferred to a PVDF membrane, and hybridized to anti-HA antibody.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Among the less frequent translocations involving AML1, the t(3;21) is the only one that has been cloned and characterized at the molecular level (21). This translocation, leading to the expression of AME, is observed in the most aggressive myeloid leukemias (1, 2, 44). Recently, it was shown that AME induces leukemia in mice transplanted with syngeneic bone marrow cells that express AME alone (45) or in cooperation with BCR/ABL (46). To date, the pathways through which AME induces cell transformation and leukemia are still unknown. By analogy to other chimeric proteins, we suggested that AME could inappropriately repress the expression of genes that are primary targets of AML1 and ME.

We have reported in this study that AME is able to oligomerize in vivo and in vitro and most probably the interaction with itself is direct. In contrast to many other chimeric proteins, AME displays a very complex pattern of self-interaction and contains at least three oligomerization domains. The deletion of any single domain is unable to prevent the self-interaction of the chimeric protein. Therefore, it is difficult to correlate directly AME oligomerization with the functional consequence of its disruption. Our results indicate that CtBP recruitment does not affect AME oligomerization (data not shown). The distal zinc finger domain has a special role in our finding because it is able to interact with both the other domains. In addition, our data indicate that the interaction between the distal zinc finger domain and Runt is stronger than any other interaction involved in AME oligomerization (Table 1). Based on our data, we can predict for all AME-related proteins (AML1, ME, and EVI1) their capability to form multimeric complexes between themselves or with each other. Of particular interest is the interaction of Runt with itself, because thus far AML1 has been thought to form only heterodimers containing a single AML1 subunit. However, for the sake of clarity, the characterization of AML1-AML1 interaction will be reported in detail in a separate communication.

Although the aggressive nature of AME as a leukemia oncogene has long been known, there are very few reports that address the potential role of each domain in cell transformation. Here, we show the importance of the distal zinc finger domain in deregulation of the hematopoietic self-renewal and differentiation programs of normal murine bone marrow progenitor cells. Whereas several years ago it was shown that the immortalized cell line Rat1 can be induced to form macroscopic colonies in soft agar when AME was introduced (47), our data indicate for the first time that this domain is essential for the disruption of differentiation and regulated self-renewal but that is not sufficient for transformation and other elements are required. As judged from colony formation assay, this domain efficiently cooperates with the CtBP corepressor recruitment in the disruption of normal hematopoietic pathways.

To date, the functions of the distal zinc finger domain of AME are still unclear. It has been reported that this domain binds to the consensus DNA sequence GAAGATGAG in vitro (48). However, there are no reports confirming that the distal zinc fingers are a real DNA-binding domain in vivo. Employing reporter-based assays, it was shown that AME raises activator protein (AP-1) activity and that the deletion of the distal zinc finger domain abrogates AP-1 activation (47, 49). However, the mechanism of AP-1 deregulation by AME is still unknown and it is not clear what the role of each domain could be in activation/repression of AP-1. The distal domain contains three zinc finger motifs and each one of them can carry out different functions, including oligomerization. Taken together, our data predict that the distal zinc finger domain is also involved in intramolecular interactions operating as a conformational switch, which may underline the diversity of AME functions, including AP-1 activation.

It has been proposed that oligomerization has a critical role in the oncogenic progression induced by many chimeric proteins. A few years ago, a model was presented (33, 34) in which the chimeric transcription factors inappropriately maintain high local concentration of corepressors, such as NCo-R, Sin3A, and HDACs at promoter sites because of their ability to oligomerize, resulting in the deregulation of genes involved in differentiation, apoptosis, and proliferation. AME is also able to oligomerize and recruit corepressors; however, our data suggest that other pathways activated by the distal zinc finger domain are involved in hematopoietic transformation by AME. Although the t(3;21) is a rare translocation, the prognosis of patients with this abnormality is extremely poor. Hopefully, our finding will provide new energy for the search of a treatment for this selected group of patients.


   Acknowledgments
 
Grant support: NIH grants HL72691, CA67189, and CA96448 (G. Nucifora) and American Cancer Society grant IRG 99-224 (V. Senyuk).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank S. Buonamici for her skillful help with experiments of bone marrow cells infection and in vitro differentiation and Dr. K.K. Sinha for the GST-AML1 and GST-EVI1 plasmids.


   Footnotes
 
1 K.K. Sinha, unpublished data. Back

Received 2/ 7/05. Revised 6/22/05. Accepted 6/24/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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