INTRODUCTION

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Dysregulated expression of transcription factors with key roles in cell proliferation or differentiation can lead to gain-of-function abnormalities that give rise to diverse types of human leukemia and lymphoma (34, 35, 49). An additional mechanism, interference with biochemical pathways that control apoptosis, is attracting increased attention because of recognition that the BCL-2 oncogene in human B-cell lymphoma functions by inhibiting programmed cell death (31, 45, 57). Originally identified as a result of its translocation into the immunoglobulin (Ig) heavy-chain locus (6, 11, 55), BCL-2 encodes a membrane-associated protein that acts as a dominant repressor of multiple independent signal transduction pathways culminating in cell death (53, 59). Thus, inappropriate expression of BCL-2 in mature lymphocytes extends their longevity, permitting the accumulation of transforming mutations, including those that otherwise would result in cell death (57, 59).

We have published data indicating that the product of the E2A-HLF fusion gene in acute pro-B-cell leukemia also functions as an inhibitor of apoptosis (26). Human leukemia cells carrying the translocation t(17;19) rapidly died by apoptosis when programmed to express a dominant-negative suppressor of the chimeric E2A-HLF protein. Moreover, E2A-HLF blocks apoptosis in growth factor-deprived murine pro-B cells, suggesting that the chimeric protein contributes to leukemic transformation of immature lymphoid cells by preventing their death (26). Because of the close sequence identity between the basic leucine zipper (bZIP) DNA-binding and protein dimerization domain of HLF (hepatic leukemic factor) and that of CES-2 (39), a cell death specification protein in the nematode Caenorhabditis elegans (39), we postulated that E2A-HLF blocks a very early step within an evolutionarily conserved apoptotic pathway in pro-B lymphocytes (26).

To better understand the role of this fusion oncoprotein in the genesis of acute leukemia, we assessed the contributions of its various structural motifs to the antiapoptotic effect seen in murine pro-B cells. This strategy seemed necessary in view of research implicating E2A and related E proteins in early B-cell development. The E proteins form a class of helix-loop-helix (HLH) proteins that include the E2A gene products E12 and E47, as well as E2-2, HEB, and daughterless, a Drosophila protein (41). The E proteins have a wide tissue distribution and can bind to DNA either as homodimers or as heterodimers with other types of lineage-restricted bHLH proteins, such as MASH1 (mammalian achaete-scute homolog 1), ADD1, Scleraxis (Scl1), and Tal1/SCL (12, 18-20, 51, 54, 58). They also can interact with the Id family of proteins, which lack functional DNA-binding domains and are able to oppose the action of E proteins by sequestering them into nonfunctional complexes (7). Of particular relevance to the present study, two E2A gene products, E12 and E47, are essential for the establishment of normal B-cell differentiation (4, 5, 61).

The results presented here indicate that neither DNA binding nor protein dimerization through the bZIP domain of HLF is essential for the prolongation of cell survival after growth factor deprivation. Instead, apoptosis was uniformly inhibited by mutant proteins that contained either or both of the E2A transactivating domains in the context of a disabled HLF DNA-binding region. This finding emphasizes the importance of the transactivation domains of E2A and suggests that protein-protein interactions mediated by these domains allow the chimeric factor to affect the expression of genes involved in the apoptotic program.

	MATERIALS AND METHODS

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Construction of eukaryotic expression vectors. Expression plasmids containing wild-type and mutated E2A-HLF, E2A, and HLF cDNAs were constructed with the pMT-CB6+ eukaryotic expression vector (a gift from F. Rauscher III, Wistar Institute, Philadelphia, Pa.), which contains the inserted cDNA under control of a sheep metallothionein promoter, as well as the neomycin resistance gene driven by the simian virus 40 early promoter. Several of the mutant E2A-HLF expression constructs used in this study have been described in earlier publications (29, 60). Deletion mutants of E2A-HLF were prepared by PCR and standard cloning methods. E2A-HLF proteins carrying amino acid substitutions were designated by the relevant amino acid numbers, with single-letter codes used to designate the substitutions. DNA fragments generated by PCR were sequenced to eliminate the possibility that mutations had been introduced by the amplification procedure.

Cell culture and cell survival assay. FL5.12 pro-B lymphocytes (38) were cultured in RPMI 1640 medium containing 10% fetal calf serum and 10% WEHI-3B-conditioned medium (as a source of interleukin-3 [IL-3]). Transfectants were generated by electroporation of 2 × 10⁷ cells and 80 µg of DNA with a gene pulser (Bio-Rad, Hercules, Calif.) set at 300 V and 960 mF. The cells were then cultured in 24-well dishes and selected in the presence of the neomycin analog G418 (0.6 mg/ml) for 2 weeks. The induction of protein expression with zinc in G418-resistant cells was confirmed by immunoblot analysis, and three to six independent pools of cells expressing the expected protein at comparable levels were selected for further experimentation. For cell survival assays, protein expression was induced by treating cells with 100 µM ZnSO₄ for 16 h prior to growth factor deprivation. IL-3 was removed by repeated centrifugation in fresh medium, and the cells were adjusted to 5 × 10⁵ per ml on day 0 and cultured without IL-3. Viable cell counts were determined by trypan blue dye exclusion.

EMSA. Binding reactions by electrophoretic mobility shift assay (EMSA) were performed with a ³²P-end-labeled DNA oligonucleotide probe (2 × 10⁴ cpm) containing the underlined HLF consensus binding site sequence (HLF-CS probe; 5'-GCTACATATTACGTAACAAGCGTT-3') in 10 µl of binding buffer (12% glycerol, 12 mM HEPES [pH 7.9], 4 mM Tris [pH 7.9], 133 mM KCl, 1.5 µg of sheared calf thymus DNA, 300 mg of bovine serum albumin per ml). Nuclear proteins were extracted from transfected FL5.12 cells by standard procedures as previously described (28). A 1,500-fold molar excess of the unlabeled M4 oligonucleotide, which contains a 4-bp mismatch (boldfaced) with the HLF-CS probe (5'-GCTACATAACACGTGTCAAGCGTT-3'), was added to the reaction mixture containing nuclear extracts from FL5.12 cells to block nonspecific binding. The entire mixture was incubated at 30°C for 15 min. Nondenaturing polyacrylamide gels containing 4% acrylamide and 2.5% glycerol were prerun at 4°C in a high-ionic-strength Tris-glycine buffer for 30 min, loaded with the samples containing protein-DNA complexes, run at 35 mA for approximately 90 min, dried under a vacuum, and analyzed by autoradiography.

Immunoblot analysis. Cells were solubilized in Nonidet P-40 lysis buffer (150 mM NaCl, 1.0% Nonidet P-40, 50 mM Tris [pH 8.0]), and total cellular proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After wet electrotransfer onto nitrocellulose membranes, immunoblotting was performed with anti-E2A or anti-HLF(C) rabbit serum (28). The blots were then stained with primary antibody followed by horseradish peroxidase-conjugated anti-rabbit Ig secondary antibodies and subjected to autoradiography by enhanced chemiluminescence (Amersham Life Science, Inc., Arlington Heights, Ill.).

Immunofluorescence studies. FL5.12 cells expressing each construct were cultured with 100 µM ZnSO₄ for 16 h in the presence of growth factor and then fixed with 3.7% paraformaldehyde in phosphate-buffered saline, treated with acetone, and stained with IgG-purified anti-E2A (1:500 dilution) or HLF(C) (1:3,000 dilution) rabbit serum followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG. Cell nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole).

	RESULTS

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E2A-HLF prolongs the survival of IL-3-dependent murine pro-B cells in the absence of growth factor. We conditionally expressed the E2A-HLF protein by using a zinc-regulated eukaryotic expression vector (pMT-CB6+) in FL5.12 cells, an IL-3-dependent line of murine pro-B lymphocytes. Each independent pool of cells isolated after G418 selection expressed E2A-HLF proteins in the presence of 100 µM ZnSO₄, at levels that were approximately 20- to 30-fold higher than background levels in medium lacking the metal (Fig. 1a). When IL-3 was removed from the medium, cells from the E2A-HLF-expressing pools survived for longer than 2 weeks when grown in the presence of zinc, whereas in medium lacking the metal, they rapidly underwent apoptosis, as did control cells transfected with an empty vector, regardless of the zinc concentration (Fig. 1b).

View larger version (31K): [in this window] [in a new window]   FIG. 1.   Effects of E2A-HLF protein expression in FL5.12 cells. (A) Immunoblot analysis with an HLF(C) antiserum of five independent E2A-HLF-positive, G418-resistant transfected pools of FL5.12 cells and an empty vector-transfected cell pool, all cultured in the presence (even-numbered lanes) or absence (odd-numbered lanes) of 100 µM ZnSO4. (B) Growth of FL5.12 cells induced to express the E2A-HLF protein, together with growth of control cells transfected with an empty vector. Cells growing exponentially in IL-3-supplemented medium for 16 h in the presence or absence of zinc were adjusted to 5 × 105 cells per ml on day 0 and cultured without IL-3 for 15 days. The growth factor was reintroduced to the cultures on day 15 (arrow). The shaded region indicates the ranges of cell counts for E2A-HLF-transfected pools in the absence of zinc and control cells in the presence or absence of the metal. Each symbol represents a discrete pool of E2A-HLF-transfected, G418-resistant cells; open circles, pool 104; open triangles, pool 112; open squares, pool 116; closed circles, pool 120; and open diamonds, pool 122. (C) Cell cycle phase distribution of a representative pool of transfected cells (pool 116; open squares in panel B) expressing E2A-HLF in the presence (+) or absence () of zinc, as determined from DNA histograms obtained by propidium iodide staining and flow cytometry.

Wild-type E2A (E12 and E47) and HLF proteins do not promote survival after growth factor deprivation. HLF is normally expressed in liver, kidney, and brain but not in B-lymphoid cells, whereas E2A is expressed in a variety of cell types, including pro-B lymphoblasts (24, 27). To determine whether wild-type E2A or HLF possesses intrinsic antiapoptotic properties in the absence of translocation-induced recombination in pro-B lymphocytes, we tested the effects of enforced expression of HLF and two different splice forms of E2A (E12 and E47) (42) on the survival of FL5.12 cells. Human E12, E47, and HLF cDNAs were introduced separately into the cells under control of the zinc-regulated metallothionein promoter. Immunoblots of lysates of the transfected cells confirmed regulated expression of the desired proteins (Fig. 2A).

View larger version (34K): [in this window] [in a new window]   FIG. 2.   Antiapoptotic activity of wild-type E2A (E12 and E47) and HLF proteins in FL5.12 cells. (A) Immunoblot analysis with an HLF(C) antiserum (lanes 1 to 6) or E2A antiserum (lanes 7 to 14) of proteins in transfected FL5.12 pools cultured in the presence (even-numbered lanes) or absence (odd-numbered lanes) of zinc. (B) EMSA of DNA-protein complexes formed with an HLF-CS probe in nuclear extracts of transfected FL5.12 clones in the presence of zinc. (C) Comparison of the antiapoptotic activities of normal E2A and HLF proteins with that of the E2A-HLF protein. The numbers of living cells in pools expressing the designated protein are represented by bars in the absence (upper) or presence (lower) of zinc after 4 days of culture without IL-3. The values are the means of results from at least three independently analyzed transfected cell pools; standard deviations are given at the ends of the bars. TAD, transactivation domain; PAR, proline and acidic amino acid-rich region; NLS, nuclear localization signal.

Antiapoptotic activity of non-DNA-binding mutants of E2A-HLF. We constructed a set of E2A-HLF mutants to test the requirement for DNA binding in the antiapoptotic activity of the intact fusion protein. We first tested a basic region mutant (BX), containing substitutions of six critical basic amino acids in the DNA-binding portion of the HLF bZIP domain. Even though the mutant's level of expression was comparable to that of intact E2A-HLF in FL5.12 cells (Fig. 3A, lanes 1 and 2), it did not form complexes with DNA, as judged by EMSA with the HLF-CS probe (Fig. 3B, lane 3), indicating the expected loss of DNA-binding activity. Surprisingly, this mutant efficiently promoted cell survival in the absence of IL-3 (Fig. 3C), suggesting that the antiapoptotic property of E2A-HLF does not depend on interaction with DNA through the basic region of HLF.

View larger version (56K): [in this window] [in a new window]   FIG. 3.   Antiapoptotic activities of E2A-HLF and HLF proteins with deletions or amino acid substitutions in the bZIP domain. (A) Immunoblot analysis with an HLF(C) antiserum (lanes 1 to 4) or E2A antiserum (lanes 5 to 12) of proteins in transfected FL5.12 pools cultured in the presence (even-numbered lanes) or absence (odd-numbered lanes) of zinc. (B) EMSA of DNA-protein complexes formed with an HLF-CS probe in nuclear extracts of transfected FL5.12 clones in the presence of zinc. (C) Comparison of the antiapoptotic activity of the protein expressed from each construct with that of the E2A-HLF protein. Diagrams of the E2A-HLF mutant proteins analyzed are shown on the left. Bars indicate the numbers of living cells in each pool that expressed the designated proteins in the absence (upper) or presence (lower) of zinc after 4 days of culture without IL-3. The values are the means of at least three independent pools; standard deviations are given at the ends of the bars. Notation is as for Fig. 2C.

E2A structural requirements for inhibition of apoptosis in the presence and absence of an intact HLF bZIP domain. To determine the portion of the E2A amino-terminal region that was responsible for the antiapoptotic effects observed in Fig. 3C, we generated a series of mutants with deletions or substitutions of amino acids in E2A functional domains (Fig. 4C), including the AD1 and AD2 transactivation domains and adjacent E2A sequences. The resulting constructs were fused either with an intact HLF bZIP domain or with one in which multiple amino acid substitutions were introduced within the basic region to prevent specific DNA binding. When expressed in the presence of zinc (Fig. 4A), each mutant protein's DNA-binding properties by EMSA analysis depended on whether the HLF bZIP domain was intact or contained basic region mutations (Fig. 4B). A clear pattern of FL5.12 cell survival was observed for cells that expressed fusion proteins with disabled HLF DNA-binding domains (Fig. 4C), in that virtually all of these transfectants were rescued from apoptosis after IL-3 deprivation, whether functional activity was retained by the AD1 or AD2 domain of E2A and whether inactivation of these regions was achieved by deletion of the entire sequence or by point mutation of specific amino acids known to have critical functional roles (37, 48). Only two of the non-DNA-binding mutants failed to promote survival (Fig. 4C). One (1-142,277-412) lacked both the AD1 and AD2 regions, while the other (19L-R,22F-R/403V-R,404L-R) incorporated point mutations that inactivate the transactivating potential of both the AD1 and AD2 domains in a single protein.

View larger version (32K): [in this window] [in a new window]   FIG. 4.   Antiapoptotic activities mutations in E2A and E2A-HLF proteins with intact or disabled (BX) HLF DNA-binding domains. (A) Immunoblot analysis with an HLF(C) antiserum of proteins in transfected FL5.12 cell pools cultured in the presence (even-numbered lanes) or absence (odd-numbered lanes) of zinc. (B) EMSA of DNA-protein complexes formed with an HLF-CS probe in nuclear extracts of transfected FL5.12 clones in the presence of zinc. (C) Comparison of the antiapoptotic activities of E2A-HLF proteins with mutations in E2A and intact (left) or disabled (right) HLF DNA-binding domains. Diagrams of the mutant proteins are shown on the left. Bars indicate the numbers of living cells in each pool of transfected cells that expressed the designated proteins in the absence (upper) or presence (lower) of zinc after 4 days of culture without IL-3. The values are means from at least three independent pools; standard deviations are given at the ends of the bars.

Nuclear localization of intact and altered E2A-HLF polypeptides. Nuclear localization of transcription factors is essential for their function in gene regulation (13). To examine the possibility that deletion or mutation of E2A and/or HLF sequences affected the subcellular localization (hence the antiapoptotic properties) of our mutants, we studied FL5.12 cells by immunofluorescence, using antisera specific for E2A and HLF epitopes. As expected, intact E2A-HLF (Fig. 5B and G), normal E2A (E12 [Fig. 5H] and E47 [Fig. 5I]), and normal HLF (Fig. 5C) proteins were all found in the nucleus. Each of the E2A-HLF mutants prepared in this study was also targeted to the nucleus, as shown for the representative mutants E2A-HLF/BX (Fig. 5B), 1-142 (Fig. 5E), and 484-574 (Fig. 5J), whereas cells transduced with an empty vector did not show evidence of staining with these antibodies (Fig. 5A and F). These findings indicate that all polypeptides used in this study (whether intact or modified) functioned as nuclear proteins, as one would predict from their nuclear localization signal in the amino-terminal segment of E2A (amino acids 170 to 175).

View larger version (108K): [in this window] [in a new window]   FIG. 5.   Subcellular localization of E2A-HLF, HLF, E2A, and representative mutant proteins. FL5.12 cells expressing each construct were immunostained with either IgG-purified anti-HLF(C) serum (A to E) or the anti-E2A serum (F to J). Simultaneous staining with DAPI was performed to permit visualization of cell nuclei (A' to J'). The FL5.12 cells studied either contained the empty vector (A, A', F, and F') or expressed E2A-HLF (B, B', G, and G'), HLF (C and C'), E2A-HLF/BX (D and D'), 1-142 (E and E'), E2A (E12) (H and H'), E2A (E47) (I and I'), or 484-574 (J and J').

	DISCUSSION

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In earlier studies, E2A-HLF blocked apoptosis in murine pro-B lymphocytes (26), but the structural motifs that were essential for this activity remained unclear. Here we demonstrate that in mutants bearing a disabled HLF basic region, the presence of either transactivation region in the E2A amino-terminal segment (AD1 or AD2) is sufficient to rescue pro-B lymphocytes from apoptosis triggered by growth factor deprivation. The requirement for these domains in the antiapoptotic activity of polypeptides containing the amino-terminal sequences of E2A was apparent from experiments in which specific point mutations that abolished the transactivating capacity of AD1 and AD2 (37, 48) also abolished the ability of the mutants to block apoptosis (Fig. 4C). Neither wild-type E47 nor wild-type E12 protein was capable of mediating survival when overexpressed in FL5.12 cells, indicating that amino-terminal E2A sequences have antiapoptotic activity only when they are expressed outside the context of their normal linkage to the bHLH domain. Thus, E2A-HLF appears able to prevent apoptosis through a mechanism that depends critically on the AD1 and AD2 domains, even in the absence of sequence-specific DNA binding mediated by the HLF bZIP domain.

Exactly how the AD1 and AD2 motifs contribute to the antiapoptotic effects of E2A-HLF is unclear, although two mechanisms seem plausible. We favor a model in which these transactivation domains are guided to the promoters of downstream genes through protein-protein interactions mediated by sequences within the amino terminus of E2A, allowing the chimera to function as a transcriptional coactivator. Precedents for functional activity of mutant transcription factors lacking DNA-binding domains can be found in the Fushi tarazu (Ftz) Drosophila homeodomain protein and the glucocorticoid and estrogen receptors (1, 2, 17). Most pertinent to the present study are results of mutational analyses showing that DNA binding is not required for transformation mediated by the chimeric transcription factor E2A-PBX1 (9, 30, 40). In each of the above cases, amino acids adjacent to the DNA-binding domain appeared to mediate protein-protein interactions that allowed the mutant transcription factor to function as a coactivator or corepressor of gene expression. We suspect that a similar mechanism enables E2A to inhibit apoptosis when its HLF partner lacks a functional bZIP domain.

Alternatively, the effects we observed may involve competition by the AD1 and AD2 domains of E2A for transcriptional cofactors or adaptors that are critically involved in the cell death programs of early B cells. Examples of the biologic activity of E2A transactivator regions include overexpression of a GAL4-AD1 chimera, leading to a slow-growth phenotype in yeast (37). Thus, the AD1 sequences can produce functionally significant phenotypic changes in the absence of bHLH-mediated DNA binding. In addition, the E2A amino-terminal region caused cell cycle arrest when overexpressed in NIH 3T3 fibroblasts (47), suggesting that in these cells it can interact with and neutralize transcriptional cofactors required for the expression of proteins involved in the regulation of cell proliferation. Further study of downstream effectors of E2A-HLF antiapoptotic activity is needed to distinguish between these two possibilities.

The activities of E2A-HLF in this study contrast sharply with those previously ascribed to E2A-PBX1, which is generated by the t(17;19) translocation in childhood pre-B-cell acute lymphoblastic leukemia. Unlike our experience with E2A-HLF, attempts to constitutively express E2A-PBX1 in lymphoid cell lines have been unsuccessful, and in conditional systems the chimeric protein induced (rather than blocked) apoptosis (52). Moreover, in transgenic mice, constitutive expression of E2A-PBX1 caused profound deficiencies of both T and B cells and rendered thymocytes susceptible to apoptosis (14). These activities resemble those of the Myc oncoprotein, which mediates both programmed cell death and malignant transformation in susceptible lymphoid progenitors (3, 16, 32, 46). The apoptotic activity of conditional E2A-PBX1 expression in hematopoietic precursors required both the PBX1 homeodomain and the 12 flanking amino acids that form the HOX cooperativity motif (52), which mediates interactions between PBX1 and the major HOX proteins (9, 10, 36, 44, 56). Importantly, a mutant lacking all PBX1-derived sequences and consisting solely of the E2A portion of the chimera was incapable of inducing apoptosis (52). How, then, does one account for the very different effects of the E2A-PBX1 and E2A-HLF chimeras on cell survival? Most likely, the cooperative DNA-binding activity of the PBX1 homeodomain and the HOX cooperativity motif positions the AD1 and AD2 domains near genes whose expression can upregulate a p53-independent apoptotic pathway (52).

We have proposed that E2A-HLF blocks apoptosis in pro-B lymphocytes by disrupting an evolutionarily conserved pathway analogous to the cell death program mediated by ces-2, the C. elegans ortholog of HLF (26, 39). Thus, the chimeric protein is thought to compete with a mammalian CES-2-like transcription factor for a common promoter binding site and to transactivate (rather than repress) a ces-1-like gene, preventing the death of pro-B cells that otherwise would be targeted for destruction. Our findings in the present study, in which the AD1 and AD2 domains of E2A were sufficient to suppress the activation of the cell death program in growth factor-dependent pro-B lymphocytes, precluded analysis of additional contributions mediated through the bZIP domain of HLF. Evidence that such activity could contribute to the overall oncogenic effect of E2A-HLF comes from studies of NFIL3/E4BP4, a growth factor-regulated bZIP protein that binds to the HLF consensus sequence in FL5.12 cells and blocks apoptosis when its expression is enforced in the absence of IL-3 (25). In addition, TEF, a closer relative of HLF and potent transactivator of gene expression in multiple cell lines (15, 23), efficiently mediated cell survival after IL-3 withdrawal in our experimental system (29a). Moreover, TEF mutants with alterations in the bZIP basic region similar to those of the BX mutants of E2A-HLF in the present study were unable to block apoptosis. Thus, because TEF lacks amino acid regions with sequence homology to the AD1 and AD2 domains of the E2A protein and interacts with DNA through a highly conserved bZIP region shared with HLF and CES-2, its ability to promote the survival of pro-B cells likely depends on inappropriate transactivation of downstream responder genes. Finally, in each case of t(17;19) pro-B leukemia studied to date, HLF sequences are fused in frame with amino-terminal E2A sequences. In some cases, this occurs through a direct in-frame joining of E2A exon 12 with HLF exon 4 (type II fusions [22]). In other cases, however, E2A exon 13 is joined with HLF exon 4, which are in different translational reading frames (type I fusions [21, 22, 24, 27]). In these leukemias, the reading frame is restored by a complex joining exon, which contains intronic sequences from both the E2A and HLF genes as well as N-region nucleotides inserted at the breakpoints of the fused chromosome.

Consistent preservation of the reading frame linking the AD1 and AD2 domains of E2A with the bZIP DNA-binding and protein dimerization domain of HLF in leukemogenic E2A-HLF fusion proteins indicates essential roles for both components in the oncogenic activity of the chimera. These results suggest that E2A-HLF possesses a dual capacity to block apoptosis, one depending on protein-protein interactions mediated by the amino terminus of E2A and the other depending on classical transcriptional regulation through sequence-specific DNA binding. Such versatility is in keeping with the enormous selection pressure to generate fusion proteins that drive leukemic transformation with maximum efficiency.