The Leukemia-Associated Transcription Repressor AML1/MDS1/EVI1 Requires Ctbp to Induce Abnormal Growth and Di€Erentiation of Mu

The leukemia-associated transcription repressor AML1/MDS1/EVI1 requires CtBP to induce abnormal growth and dierentiation of murine hematopoietic cells

Vitalyi Senyuk1, Soumen Chakraborty1, Fady M Mikhail1,2, Rui Zhao1, Yiqing Chi1 and Giuseppina Nucifora*,1

1Department of Pathology and The Cancer Center, University of Illinois at Chicago, Chicago, Illinois, USA; 2Clinical Pathology Department, Faculty of Medicine, University of Alexandria, Alexandria, Egypt

The leukemia-associated fusion gene AML1/MDS1/ (ME) genes, is a product of the (3;21)(q26;q22) EVI1 (AME) encodes a chimeric transcription factor translocation that is associated with de novo and that results from the (3;21)(q26;q22) translocation. This therapy-related myelodysplastic syndrome/acute mye- translocation is observed in patients with therapy-related loid leukemia (t-MDS/AML) patients, and to a lesser myelodysplastic syndrome (MDS), with chronic myelo- extent with chronic myelogenous leukemia (CML) genous leukemia during the blast crisis (CML-BC), and patients during the blast crisis of their disease (Rubin with de novo or therapy-related acute myeloid leukemia et al., 1990; Nucifora et al., 1993). The AML1 gene (AML). AME is obtained by in-frame fusion of the (also known as CBFA2, PEBP2 or RUNX1) encodes AML1 and MDS1/EVI1 genes. We have previously the DNA-binding subunit of the core-binding tran- shown that AME is a transcriptional repressor that scription factor (CBF). AML1 interacts with the other induces leukemia in mice. In order to elucidate the role subunit of CBF, CBFb, which has no DNA-binding of AME in leukemic transformation, we investigated the domain. AML1 consists of a N-terminal DNA-binding interaction of AME with the transcription co-regulator domain with partial homology to the product of CtBP1 and with members of the histone deacetylase Drosophila segmentation gene runt, and a C-terminal (HDAC) family. In this report, we show that AME activation domain that interacts with transcription co- physically interacts in vivo with CtBP1 and HDAC1 and regulators (Ogawa et al., 1993a, b). AML1 is essential that these co-repressors require distinct regions of AME for de®nitive murine hematopoiesis (Wang et al., 1996; for interaction. By using reporter gene assays, we Okuda et al., 1996) and is involved in chromosomal demonstrate that AME represses gene transcription by abnormalities associated with human leukemias (Zent CtBP1-dependent and CtBP1-independent mechanisms. et al., 1997; Roulston et al., 1998) including T-cell Finally, we show that the interaction between AME and leukemia (Mikhail et al., 2002). ME is a DNA-binding CtBP1 is biologically important and is necessary for zinc-®nger transcription factor and a longer form of growth upregulation and abnormal dierentiation of the the leukemia-associated protein EVI1 (Fears et al., murine hematopoietic precursor cell line 32Dc13 and of 1996; Soderholm et al., 1997). ME contains a murine bone marrow progenitors. conserved N-terminal region, called PR domain, two Oncogene (2002) 21, 3232 ± 3240. DOI: 10.1038/sj/ sets of DNA-binding Cys2His2-type zinc-®nger do- onc/1205436 mains, a proline-rich central domain, and an acidic C- terminal domain. The evolutionarily conserved PR Keywords: AML1; MDS1; EVI1; t(3;21); CtBP1; domain was ®rst identi®ed in the RIZ1 protein and in HDAC1 the PRDI-BF1/BLIMP1 transcriptional repressor (Huang, 1994; Fears et al., 1996). ME is expressed in several tissues but is not detected in normal hematopoietic cells (Fears et al., 1996). Both AML1 and ME Introduction are transcriptional activators (Zhang et al., 1996; Soderholm et al., 1997), whereas AME is a transcrip- The AML1/MDS1/EVI1 (AME) fusion gene, obtained tional repressor (Zent et al., 1996). AME consists of by in frame fusion of the AML1 and MDS1/EVI1 the DNA-binding domain of AML1 fused to almost the entire ME including the PR domain (Nucifora et al., 1994). AME potentially possesses the ability to bind both the AML1- and ME-target promoters. *Correspondence: G Nucifora, Department of Pathology and The Recently, it was shown that AME induces leukemia Cancer Center, Molecular Biology Research Building, M/C 737, in mice transplanted with syngeneic bone marrow cells University of Illinois at Chicago, 900 South Ashland Avenue, that express AME (Cuenco et al., 2000). The pathways Chicago, Illinois, IL 60607, USA; E-mail: [email protected] Received 21 November 2001; revised 15 February 2002; accepted through which AME induces cell transformation and 21 February 2002 leukemia are still unknown. It is possible that AME AME de-regulates growth and differentiation via CtBP V Senyuk et al 3233 could inappropriately repress the expression of genes to a much tighter chromatin structure which is that are primary targets of AML1 and ME by presumably inaccessible to transcription factors (Gro- inappropriate recruitment of transcription repressors zinger et al., 1999). Two distinct classes of HDACs at the promoter site, as it was proposed for AML1/ have been identi®ed in mammalian cells. HDAC1, ETO and PML/RARa (Lin and Evans, 2000; Minucci HDAC2, HDAC3, and HDAC8 belong to class I and et al., 2000). It was shown recently that EVI1 interacts are homologous to yeast Rpd3 and Xenopus HDACm with the co-repressor CtBP (Izutsu et al., 2001; Palmer proteins, with a single deacetylase domain at the N- et al., 2001; Chakraborty et al., 2001). CtBP was ®rst terminus. HDAC4, HDAC5, HDAC6, HDAC7, and identi®ed as a protein that binds to the C-terminal HDAC9 are members of class II and are similar to region of the adenovirus E1A protein (Schaeper et al., yeast Hda1 with one or two catalytic domains in their 1995) and it is a well-characterized transcriptional co- C-terminal regions (Bertos et al., 2001). In order to repressor (Turner and Crossley, 2001). CtBP also binds elucidate the potential role of HDACs in AME- to BKLF, FOG, AREB6 (Turner and Crossley, 1998), mediated transcription repression, we tested the Knirps, Kruppel, Snail (Nibu et al., 1998; Keller et al., interaction between HA-AME and Flag-tagged 2000), ZEB (Postigo and Dean, 1999), polycomb HDAC1, 74, 75, and 76. The results are shown in proteins (Sewalt et al., 1999) and other transcription Figure 3. We found that AME strongly interacts with co-regulators with the consensus motif PXDLX to HDAC1 (Figure 3, lane 2), but has a weak or no enhance transcription repression. In this study, we interaction with HDAC4, HDAC5, and HDAC6 investigate the interaction of AME with the transcrip- (Figure 3, lanes 3 to 5). tion co-regulator CtBP1 and with members of the To identify the region of AME that is necessary for histone deacetylase (HDAC) family. We show that the interaction with HDAC1, we used the AME AME interacts with CtBP1 and HDAC1 through deletion mutants shown in Figure 1 in co-IP assays. dierent domains and represses gene transcription by The results show that whereas the full length AME and CtBP1-dependent and CtBP1-independent mechanisms. the deletion mutants AME-1184, AME-994, and AME- More importantly, we also show that the interaction 704 were co-immunoprecipitated by anti-Flag anti- between AME and CtBP1 is functionally important bodies that recognize the Flag-tagged HDAC1 (Figure and is required for maximum cell growth upregulation 4, lanes 2 to 5), the shortest deletion mutant AME-374 by AME and for abnormal dierentiation and was not co-immunoprecipitated by anti-Flag antibodies immortalization of murine bone marrow progenitors. (Figure 4, lane 11). These results suggest that the interaction between AME and HDAC1 requires the aa region 374 to 704. This region includes the entire proximal zinc-®nger domain and a small part of the Results PR domain from ME. It was reported that HDAC1 interacts with CtBP1 in vivo (Sundqvist et al., 1998). To AME interacts with the co-repressor CtBP1 con®rm that AME does not require CtBP1 for CtBP1 binds to the amino acid (aa) pentapeptide motif HDAC1 interaction, we tested the ability of AME C- PXDLX. The AME fusion protein has two potential mutant to co-IP with HDAC1. The results, shown in CtBP1 binding sites: PFDLT (aa 1036 to 1040) and Figure 5, indicate that full length AME (lane 2 and 4) PLDLS (aa 1067 to 1071). To determine whether AME and AME-C (lanes 3 and 5) interact with HDAC1, and associates with CtBP1 at these consensus motifs we therefore further con®rm that the interaction between tested the full length AME and the AME point AME and HDAC1 does not require CtBP1. However, mutants A, B, and C (Figure 1) in co-immunoprecipi- our data do not indicate whether the interaction is tation (co-IP) assays. The A-mutant has a DL to AS direct. substitution in the proximal site (PFDLT to PFAST); the B-mutant has the same substitution in the distal AME co-localizes in the nucleus with CtBP1 or HDAC1 site (PLDLS to PLASS); the C-mutant has both sites mutated. As shown in Figure 2, the wild type AME The interaction between AME and either CtBP1 or (lane 2) and the A-mutant (lane 3) interact with CtBP1. HDAC1 was also con®rmed by confocal microscopy In contrast, the B- and C-mutants, which have the DL analysis of 293 cells that were co-transfected with HA- to AS substitution in the distal site (PLDLS, aa 1067 AME and either Flag-CtBP1 or Flag-HDAC1. As to 1071), were not co-immunoprecipitated with CtBP1 shown in Figure 6, AME (Figure 6, top panels in red (Figure 2, lanes 4 and 5). These results indicate that the color) co-localizes with either CtBP1 or HDAC1 distal PLDLS site has a very strong anity for CtBP1 (Figure 6, middle panels in green color) in the cell and that the mutations DL to AS in this site abrogate nucleus. To determine the role of CtBP1 in the the interaction between AME and CtBP1 in co-IP assembly of the speckles, we used confocal microscopy assays. to analyse the pattern of AME-C (unable to interact with CtBP1) and CtBP1. Double staining of the co- transfected cells showed a diused nuclear staining in AME interacts with HDAC1 most of the nuclei (data not shown) con®rming that The HDAC enzymes regulate gene expression by CtBP1 must interact with AME to induce the deacetylation of nucleosomal histone proteins leading formation of the speckles.

Oncogene AME de-regulates growth and differentiation via CtBP V Senyuk et al 3234

Figure 1 Diagram of AME mutants. AML1, ME, and the fusion protein AME are shown in the ®rst three lines of the diagram. The vertical dashed line indicates the breakpoint fusion. AME contains 1499 aa including the HA-tag. The DNA-binding domain of AML1, the Runt domain, is maintained in the fusion protein. The two zinc ®nger domains of ME (ZnF), the PFDLT site (*, aa 1036 ± 1040), and the PLDLS site (8, aa 1067 ± 1071) are also shown. The remaining lines show the deletion mutants and the three point mutants AME-A (containing only the PLDLS site), AME-B (containing only the PFDLT site), and AME-C. The size of the proteins in aa residues is also shown

Figure 2 The interaction between AME and CtBP1 requires the PLDLS site. 293T cells were co-transfected with Flag-CtBP1 and Figure 3 AME physically interacts with HDAC1. 293T cells the full length HA-AME or the HA-AME point mutants (lanes 2 were co-transfected with wild type HA-AME and Flag-tagged to 5). Lane 1, mock-transfected cells. Flag-tagged CtBP1 was HDAC 1, 4, 5 or 6. The Flag-HDAC proteins were immunopre- immunoprecipitated with anti-Flag antibodies and the immuno- cipitated with anti-Flag antibodies and the immunoprecipitated precipitated proteins were separated by electrophoresis. After proteins were analysed as described in the legend of Figure 2. transfer to a PVDF membrane, the HA-tagged AME (lane 2) or Only Flag-HDAC1 (lane 2) but not Flag-HDAC4, 5, or 6 (lanes the HA-tagged point mutants (lanes 3, 4, and 5) were analysed 3, 4, and 5) interacts with HA-AME. Lane 1, mock-transfected with anti-HA antibodies. Only the full length HA-AME (lane 2) cells. The right side of the ®gure shows that HA-AME (top) and or the HA-AME-A mutant (lane 3), but not HA-AME-B (lane 4) the Flag-HDAC proteins (bottom) were expressed in the cells or HA-AME-C (lane 5), are capable of interaction with Flag- CtBP1. Lanes 6 to 10, top and bottom, show that the HA-AME and HA-AME-mutant proteins (top) and the Flag-CtBP1 protein (bottom) were expressed at comparable levels in the transfected cells Figure 7a, AME represses the M-CSFR promoter. Repression by the wild type AME and the AME-C mutant was not signi®cantly dierent, suggesting that AME represses transcription by CtBP1-dependent and the repression of this promoter by AME could be CtBP1-independent mechanisms CtBP1-independent. AME also strongly repressed the AME contains the DNA-binding motifs of AML1 and pTAK150 promoter and the repression level was about ME. Therefore, potentially AME is a bifunctional 70% (Figure 7b). However in contrast to the results transcription factor that can regulate two dierent obtained with the M-CSFR promoter, AME-C was types of promoters. By using reporter gene assays we able to repress this promoter although to a lesser tested both the AML1-dependent minimal M-CSFR extent than the full length AME. The addition of promoter (Rhoades et al., 1996) and the ME-dependent exogenous CtBP1 or HDAC1 or treatment of the cells pTAK150 promoter (Kreider et al., 1993). As we with TSA did not signi®cantly change the level of reported before (Zent et al., 1996) and as shown in repression (data not shown).

Oncogene AME de-regulates growth and differentiation via CtBP V Senyuk et al 3235

Figure 4 The interaction between HDAC1 and AME requires the proximal zinc-®nger domain of AME. 293T cells were co- transfected with Flag-HDAC1 and the full length or deletion mutants of HA-AME. The proteins were immunoprecipitated with anti-Flag antibody, separated by electrophoresis, and analysed with anti-HA antibody. Lanes 1 and 6, mock-transfected cells. Lanes 2 to 5, the full length HA-AME and three deletion mutants interact with Flag-HDAC1 and are co-immunoprecipi- Figure 6 AME co-localizes in the nucleus with HDAC1 and tated with Flag antibody. However, the shortest mutant HA- CtBP1. 293 cells were cultured on glass cover slips in 3.5 cm AME-374 (lane 12) has lost the interaction domain and is not plates and were co-transfected with HDAC1, CtBP1, and AME. detected in the immunoprecipitated proteins (lane 11). Lanes 7 to After treatment of the slides as described in Materials and 10 and lane 12 show the level of expression of the proteins by methods, the proteins were visualized with confocal microscopy Western blot analyses analysis. Top panels, localization pattern of AME. Middle panels, localization patterns of HDAC1 and CtBP1. Bottom panels show the overlap of the images

with CtBP1 is necessary for the upregulation of cell growth. To con®rm that the steady-state level of expression of the transgenic proteins was comparable, the 32Dc13 cells were lysed and 100 mg of proteins were separated by gel electrophoresis and analysed by Western blot. The results (Figure 8b) indicate that the level of proteins in the samples is comparable.

AME requires CtBP1-binding for immortalization of Figure 5 HDAC1 interacts with AME-C point mutant. 293T normal murine bone marrow cells cells were co-transfected with Flag-HDAC1 and wild type HA- AME or HA-AME-C point mutant. Lane 1, mock-transfected Earlier, we reported that AME induces myeloid cells. Flag-HDAC1 was immunoprecipitated with anti-Flag leukemia in mice (Cuenco et al., 2000). To determine antibodies and HA-AME was detected with anti-HA-antibodies whether CtBP1 has a dominant role in the abnormal (lanes 2 and 3). Lanes 4 and 5 show the level of expression of the HA-AME proteins (top) and of HDAC1 (bottom) dierentiation of AME-expressing murine bone marrow cells, we used AME and AME-C to infect murine bone marrow cells for in vitro colony assays. Lineage-depleted bone marrow cells prepared as described in Materials and methods, were infected with recombinant retro- AME accelerates the cell growth viruses expressing AME or AME-C, or with the The forced expression of AME in 32Dc13 cells retroviral vector MSCV. The cells were cultured in upregulates cell growth and blocks the granulocytic methylcellulose-based medium in the presence of IL-3, dierentiation of the cells (Sood et al., 1999). In the IL-6, SCF, and GM ± CSF, a combination of cytokines present work we investigated the role of CtBP1 in that stimulates myeloid cell growth and dierentiation. regulation of cell growth. We generated 32Dc13 cell Five days after infection, the cells were plated and, as lines with stable expression of wild type AME or expected (Du et al., 1999), they produced granulocyte AME-C. As shown in Figure 8a, AME strongly and/or macrophage colonies. The colonies were har- accelerates the growth rate of 32Dc13 cells. However vested and the disaggregated cells were plated in fresh AME-C reduces cell growth to the level observed for semisolid medium at day 13 (2nd plating), day 23 (3rd the naive 32Dc13 cells, indicating that the interaction plating), day 31 (4th plating), day 39 (5th plating), and

Oncogene AME de-regulates growth and differentiation via CtBP V Senyuk et al 3236

Figure 7 AME represses the activation of promoters by CtBP1-dependent and CtBP1-independent mechanisms. NIH3T3 cells were co-transfected with AME or AME-C and either with the reporter gene luciferase regulated by AML1 (a) or the reporter gene CAT regulated by ME (b). A, full length AME or the point mutant AME-C have similar repressive eect on the promoter. (b) the point mutation that abolishes the ability of AME to interact with CtBP1 reduces the repression ability of AME by about 70%

infected cells was the highest, indicating that as for the 32Dc13 cells (Figure 8a) AME has the ability to confer a proliferation advantage to the hematopoietic cells. However, in contrast to results obtained from the 32Dc13 cell line, AME-C-cells had a proliferative potential intermediate between that of the wild type AME and of the vector-infected control cells (Table 1). These results would suggest that normal hematopoietic murine progenitor cells that express AME require not only CtBP1 for maximum growth upregulation but also another element as yet unidenti®ed. Based on the morphology of the cells (examined at each plating), we calculated the relative percentage of blast cells, granulocytes/macrophages, and cells in mitosis. Morphological analysis showed a clear delay in myeloid dierentiation in the AME-infected BM cells compared to AME-C and empty vector cells (Table 2 and Figure 9). Manual counting of the cell types indicted that after 13 days in culture, 52% of the AME-cells were blasts cells, compared to 15 and 13% of the AME-C and the vector cells respectively. On the 3rd and 4th platings, there was Figure 8 AME requires CtBP1 for cell replication upregulation. still evident delay in the dierentiation of AME cells. (a) Stably transfected 32Dc13 clones were used in this assay. The Furthermore, the per cent of cells in mitosis was higher in cells were cultured in IL-3 and manually counted every 24 h. Wild the AME cells than in the other two groups of cells type AME (AME-1499, triangles) accelerates cell replication. In throughout the experiment. Finally, the AME-infected contrast, the point mutant AME-C (squares), which lost the cells demonstrated evidence of dysplastic features in the ability to bind CtBP1, grows at a rate similar to that of empty vector-cells (circles) and naive 32Dc13 cells (diamonds). (b) granulocytic lineage consisting of cytoplasmic hypo- Expression of transgenic proteins in 32Dc13 clones. One hundred granulation and a relatively higher percentage of cells mg of cell proteins were separated by electrophoresis, transferred with ring-shaped nuclei compared to the corresponding to a PVDF membrane, and hybridized to anti-HA antibodies. stage of granulocytic dierentiation in both the control Lane 1, empty vector; lane 2, AME; lane 3, AME-C cells and the AME-C cells (Figure 9). Both these features were described as characteristics of MDS cells (Langen- huijsen, 1984; Hast et al., 1989). In contrast, the AME-C and the empty vector cells showed normal cytoplasmic granulation and nuclear segmentation patterns (Figure day 47 (6th plating) from infection. To assess the 9), suggesting that CtBP1 is required and sucient for proliferative potential of the cells grown in semisolid abnormal dierentiation of murine hematopoietic pre- medium, we counted the total number of cells and cursors. colonies in each plate. We did not include the ®rst plating After the ®rst plating, about 10 000 cells of each type in our analysis because the number of the hematopoietic were placed separately in liquid culture and maintained colonies generated in the ®rst plating depends in large in RPMI medium supplemented with 10% of WEHI- part on the infection eciency of each recombinant 3B-conditioned medium as a source of IL-3. As retrovirus. As shown in Table 1, the number of AME- expected, the cells that had been infected with the

Oncogene AME de-regulates growth and differentiation via CtBP V Senyuk et al 3237 Table 1 Number of colonies and cells in the bone marrow cultures Vector AME AME-C Days after Cells Cells Cells infection (6103) Colonies (6103) Colonies (6103) Colonies

13 178 198 846 514 580 309 23 23 1 809 492 244 177 31 ± ± 821 485 187 92

Each plate was seeded with 15 000 cells. The cell count was not performed after the fourth plating

Table 2 Morphological analysis of bone marrow cultures Days after Vector AME AME-C infection BC* G/M* CM* BC* G/M* CM* BC* G/M* CM*

13 13 86 1 52 44 4 15 84 1 23 5 94 1 36 61 3 8 90 2 31 All cells are dead 11 87 2 1 98 1

*The numbers represent the percentage of the total number of cells. BC, Blast cells. G/M, Granulocytes and macrophages. CM, Cells in mitosis

empty retroviral vector died after 10 ± 15 days in liquid Among the less frequent translocations involving culture. We found that the AME-C-cells also died in AML1, the t(3;21) is the only one that has been cloned this culture condition. However, the AME-infected and characterized at the molecular level (Nucifora et al., cells have been slowly growing in culture for over 60 1994). In contrast to the others, this translocation, days. After this time, the cells were frozen and leading to the expression of AME, is observed in very maintained in liquid nitrogen. aggressive myeloid leukemia that are characterized by a very poor prognosis. Recently, it was reported that AME is sucient to generate leukemia in mice that have been Discussion transplanted with AME-infected syngeneic bone marrow (Cuenco et al., 2000). We earlier described that AME is a AML1 and CBFb are the two subunits of the transcription repressor that blocks the granulocytic transcription factor CBF and are involved in about dierentiation of the murine 32Dc13 hematopoietic cell 30% of myeloid and lymphoid leukemia. The most line (Sood et al., 1999). In this work, we have identi®ed frequent chromosomal translocations where these two two co-repressors that interact with AME: CtBP1 and genes are rearranged are the inv(16), leading to the HDAC1. By using co-IP assays, we found that AME fusion between CBFB and the myosin heavy chain separately interact with both CtBP1 and HDAC1 MYH11, the t(8;21) leading to the fusion gene AML1/ through non-overlapping domains and that only the ETO, and the t(12;21) associated with childhood distal PLDLS site in AME is essential for CtBP1 binding. leukemia and leading to the fusion gene TEL/AML1. This ®nding is similar to what reported for EVI1 In general, the identi®cation of these translocations in (Chakraborty et al., 2001; Palmer et al., 2001; Izutsu et the leukemic bone marrow of the patients is considered al., 2001). At this time, we have no information about the a favorable prognostic factor compared to similar interaction of endogenous proteins. Because the t(3;21) is patients without that speci®c translocation. During the a rare translocation, we did not have the opportunity to last several years, several investigators have tried to analyse the patients' leukemic cells. In addition, no generate murine models of human leukemia with t(3;21) cell line is currently available. However, by CBFB/MYH11, AML1/ETO and TEL/AML1 by bone analogy to other fusion proteins, it is likely that the marrow infection and transplantation. However, the interaction we observe with transfected proteins is also results have not been very encouraging and there is no true for endogenous proteins. The interaction with report proving that these fusion genes can induce CtBP1 is necessary for repression of a promoter leukemia in the mouse. These fusion proteins have regulated by AME. Thus, AME belongs to a growing been characterized biochemically. We and others have group of transcription factors that interact with CtBP shown that they inappropriately interact with the and require active CtBP for eective repression (Turner transcription co-repressors Sin3A, HDAC, SMRT, and Crossley, 1998; Nibu et al., 1998; Postigo and Dean, and N-CoR (Chakrabarti and Nucifora, 1999; Amann 1999; Sewalt et al., 1999; Keller et al., 2000 and others). et al., 2001), and it was suggested that the oncogenic Several fusion proteins that result from chromoso- property of the fusion proteins could be due to their mal translocations associated with leukemia are inappropriate ability to recruit transcription repressors inappropriate transcription repressors. In general, (Lin and Evans, 2000; Minucci et al., 2000). Sin3, N-CoR, and SMRT are co-repressors inappro-

Oncogene AME de-regulates growth and differentiation via CtBP V Senyuk et al 3238 entiation. Therefore, the disruption of the AME-CtBP1 binding could be sucient to restore the normal dierentiation pattern in leukemic cells with a t(3;21). However, our data also suggest that disruption of the CtBP1 binding is not sucient to restore proper growth rate to the cells, and they indicate that AME probably aect the normal dierentiation/growth patterns at several levels.

Materials and methods

DNA constructs The cloning vectors used in this work were pCMV-myc-nuc (Invitrogen/Gibco) and pMSCV-neo (Clontech). HA or Flag epitopes were ampli®ed by PCR from appropriate templates and were cloned into the NcoI/PstI sites of the pCMV vector. The entire reading frame of AME (about 4.4 Kb) was cloned in HA-pCMV or Flag-pCMV using standard techniques. AME deletion and point mutants (Figure 1) were prepared by using PCR cloning methods. All PCR reactions were performed with high ®delity Pfu-DNA polymerase (Strata- gene). All cloning junctions, PCR ampli®ed regions, and point mutations were veri®ed by DNA sequencing. Plasmid CtBP1-Flag-pCMV was a gift of Dr R Baer, Columbia University. Plasmids encoding several members of the HDAC family (HDACs-Flag- pBJ5.1 plasmids) were a gift of Dr SL Schreiber, Harvard University.

Cell culture Adherent cell lines 293T, 293, NIH3T3, and Phoenix (ATCC) were maintained in DMEM supplemented with 10% new- born calf serum (CS). Suspension cells 32Dcl3 were Figure 9 AME delays the in vitro dierentiation of murine BM maintained in RPMI 1640 medium supplemented with 10% progenitor cells. Cytospin preparations of the murine BM CS and 10% WEHI-3B conditioned medium as a source of progenitor cells infected with the empty MSCV vector, the full IL-3. Murine bone marrow progenitor cells were isolated and length AME, and the point mutant AME-C, stained with cultured in methylcellulose medium as described (Lavau et Wright ± Giemsa stain. After approximately 30 days in culture, al., 2000). the empty vector cells (a and b) were dead, whereas cells that expressed AME-C were able to grow and dierentiate until day 47 (g, h and i). In contrast, the bone marrow cells that express the Transfection full length AME showed an evident delay in dierentiation with a relatively high percentage of undierentiated blast cells in all DNA-transfection of adherent cells was performed by the platings (c, d, e and f). In addition, the AME cells showed some calcium phosphate precipitation method (Sambrook et al., dysplastic ®gures characteristic of MDS in the form of hypo- 1989) or with NovaFector reagent (Venn Nova, Inc.) granulation of the granulocytes (black arrow) and a relatively according to the manufacturer's instructions. We used 10 mg large number of granulocytes with ring-shaped nuclei (black of plasmid/10 cm plate for each transfection, unless otherwise arrowhead). After the 6th plating, the AME cells have been indicated. Murine 32Dcl3 and murine bone marrow maintained in liquid culture supplemented with IL-3 hematopoietic cells were infected and selected as described (Lavau et al., 2000; Sood et al., 1999).

Culture and differentiation of 32Dcl3 and bone marrow cells priately recruited to promoter of target genes by these fusion proteins. Studies carried out with the fusion 32Dcl3 cells were plated at a density of 175 000 cells/ml in a proteins PML/RARa and AML1/ETO support the total volume of 4.5 ml. Manual cell count was performed hypothesis that the recruitment of co-repressors and every 24 h and the cell density was adjusted to 500 000 cells/ the inappropriate repression has a causative role in ml by addition of fresh medium. The dierentiation of 32Dcl3 and murine bone marrow progenitors was carried out promotion of leukemia (Lin and Evans, 2000; Minucci as described (Lavau et al., 2000; Sood et al., 1999). et al., 2000). These ®ndings are clinically very relevant in that they can provide new targets for therapeutic tests. Our studies indicate that when inappropriately Immunofluorescence analyses recruited to a promoter site, CtBP1 is also important in 293 cells were cultured on glass cover-slips and transfected with leading to abnormal hematopoietic growth and dier- NovaFector reagent (Venn Nova, Inc.) according to the

Oncogene AME de-regulates growth and differentiation via CtBP V Senyuk et al 3239 manufacturer's instructions. Forty-eight hours later, the cells Reporter gene studies were washed three times with PBS and ®xed in 4% paraformaldehyde in PBS for 20 min. The cell membrane was For these assays, we used the ME-dependent pTAK150 permeabilized by treatment with acetone at 7208C for 3 min. synthetic promoter and the AML1-dependent minimal The cells were washed with PBS, and non-speci®c antibody- Macrophage-Colony-Stimulating Factor Receptor (M-CSFR) binding was blocked with 5% normal goat serum (Santa Cruz). promoter. The promoters and the assays have been described The cells were treated with mouse anti-Flag monoclonal (Kreider et al., 1993; Rhoades et al., 1996; Zent et al., 1996). antibody (Sigma) and rat anti-HA monoclonal antibody (Santa Cruz) at a dilution 1 : 200 for 1 h. After washing with PBS, the cells were stained with donkey anti-mouse FITC 488 and goat anti-rabbit Texas 594 (Molecular Probe) at a dilution 1 : 2000 for Acknowledgments 1 h. The cells were washed, treated with prolonged anti-fading We thank Ms S Sitailo for technical assistance. We thank media (Molecular Probe) and the proteins were visualized using Dr R Baer (Columbia University) for the CtBP1-Flag- an immuno¯uorescence microscope (Zeiss Inc.). pCMV plasmid and Dr SL Schreiber (Harvard University) for the HDACs-Flag-pBJ5.1 plasmids. This work was supported by NIH-NCI grants CA67189 and CA72675 Western blot analyses and co-IP assays (G Nucifora), and by a translation research award from Cells were harvested 24 to 48 h after transfection, and the the Leukemia and Lymphoma Society (G Nucifora). G assays were carried out as described (Chakraborty et al., Nucifora is a Scholar of the Leukemia and Lymphoma 2001). Society.

References

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Oncogene