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Originally published In Press as doi:10.1074/jbc.M309074200 on February 17, 2004

J. Biol. Chem., Vol. 279, Issue 19, 19512-19522, May 7, 2004
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Isoforms of the Ets Transcription Factor NERF/ELF-2 Physically Interact with AML1 and Mediate Opposing Effects on AML1-mediated Transcription of the B Cell-specific blk Gene*

Je-Yoel Cho{ddagger}§, Yasmin Akbarali{ddagger}, Luiz F. Zerbini{ddagger}, Xuesong Gu{ddagger}, Jay Boltax{ddagger}, Yihong Wang{ddagger}, Peter Oettgen{ddagger}, Dong-Er Zhang¶, and Towia A. Libermann{ddagger}||

From the {ddagger}BIDMC Genomics Center and the New England Baptist Bone and Joint Institute, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, Massachusetts 02115 and the Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037

Received for publication, August 15, 2003 , and in revised form, January 20, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously isolated different isoforms of a new Ets transcription factor family member, NERF/ELF-2, NERF-2, NERF-1a, and NERF-1b. In contrast to the inhibitory isoforms NERF-1a and NERF-1b, NERF-2 acts as a transactivator of the B cell-specific blk promoter. We now report that NERF-2 and NERF-1 physically interact with AML1 (RUNX1), a frequent target for chromosomal translocations in leukemia. NERF-2 bound to AML1 via an interaction site located in a basic region upstream of the Ets domain. This is in contrast to most other Ets factors such as Ets-1 that bind to AML1 via the Ets domain, suggesting that different Ets factors utilize different domains for interaction with AML1. The interaction between AML1 and NERF-2 led to cooperative transactivation of the blk promoter, whereas the interaction between AML1 and NERF-1a led to repression of AML1-mediated transactivation. To delineate the differences in function of the different NERF isoforms, we determined that the transactivation domain of NERF-2 is encoded by the N-terminal 100 amino acids, which have been replaced in NERF-1a by a 19-amino acid transcriptionally inactive sequence. Furthermore, acidic domains A and B, which are conserved in NERF-2 and the related proteins ELF-1 and MEF/ELF-4, but not in NERF-1a, are largely responsible for NERF-2-mediated transactivation. Because translocation of the Ets factor Tel to AML1 is a frequent event in childhood pre-B leukemia, understanding the interaction of Ets factors with AML1 in the context of a B cell-specific promoter might help to determine the function of Ets factors and AML1 in leukemia.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Immune system development is regulated by the combined action of cytokines, cell-cell interactions, and a distinct set of transcription factors that modulate and coordinate developmental stage- and lineage-specific gene expression. Because expression of a specific set of genes is distinct for each cell lineage and each developmental stage, analysis of transcription factors involved in the regulation of these lineage-specific genes is one approach to understand the molecular mechanisms underlying differentiation. Analysis of regulatory regions of B cell-specific genes has revealed the presence of DNA motifs that are repeatedly found in variations and different combinations in most B cell-specific genes. Most B cell-specific genes contain binding sites for different Ets factors such as Pu.1, Ets-1, ELF-1,1 NERF (ELF-2), and Ets-related gene and factors such as Oct-2, Ikaros, E2A, Rel/NF-{kappa}B, BSAP, lymphoid enhancer-binding factor-1, N-Myc, and early B cell factor (1). Cooperativity between these different factors leads to selective stage- and cell-specific expression of a particular gene. B cell specificity of a transcription factor such as the ubiquitously expressed E2A does not always coincide with its exclusive expression in B cells, but rather depends on formation of B cell-specific protein·protein complexes due to the combination of a particular set of factors expressed in B cells.

The Ets transcription factor family plays a key role in cellular differentiation, proliferation, and development; apoptosis; and immune responses, including the growth, survival, and activation of hematopoietic cells (2). More than 30 Ets family homologs have been cloned (3), and these homologs function as transcription factors under physiological conditions and transform cells when aberrantly expressed. All Ets factors share a highly conserved 80–90-amino acid DNA-binding domain, the Ets domain (4–6). This domain is sufficient to interact specifically with DNA; and due to the conserved DNA-binding domain, binding sites for Ets factors are similar, with a (A/G)GA(A/T) core binding motif and slight differences in flanking nucleotides for different Ets factors. Outside the DNA-binding domain, very little homology is common to all members of the Ets family. Ets-related proteins can be grouped into subclasses based on additional homologous domains unique for particular members of the Ets family (4–6) such as NERF/ELF-2, ELF-1, and MEF/ELF-4, which contain several homologous regions outside the Ets domain not found in other Ets factors. Protein-protein interactions are critical for the function of Ets-related proteins and occur with transcription factors of various other families. Thus, ERP, SAP-1, and ELK-1 (Ets-related protein, SRF accessory protein, and Ets-like) form a ternary complex with the serum response factor, whereas GA-binding protein-{alpha} interacts with GA-binding protein-{beta} (2, 6). Additional regulation of Ets factors involves phosphorylation by kinases activated via different signal transduction pathways (6, 7).

In an effort to search for novel members of the Ets family that might be relevant for B cell gene regulation, we previously identified and characterized cDNA clones encoding three alternative splice products of a novel member of the Ets gene family, NERF/ELF-2, NERF-1a, NERF-1b, and NERF-2, which differ in their N termini (8). NERF is most closely related to ELF-1 and MEF/ELF-4. We have demonstrated that both NERF and the related protein ELF-1 are involved in regulating a set of genes in B cells and myeloid cells and are highly expressed in B cells and myeloid cells (8, 9). We also showed that NERF-2 is expressed in endothelial cells and transactivates the regulatory regions of the tie2 gene (10). Interestingly, NERF-2 expression is also increased in endothelial cells in response to hypoxia and to angiopoietin-1, indicating functions for NERF in the immune system and vasculature (11).

AML1 (also known as RUNX1 (runt box-1), CBF{alpha}2, and polyoma enhancer-binding protein 2{alpha}2) is a transcription factor critical for definitive hematopoiesis (12, 13). The AML1 recognition sequence is required for tissue-specific expression of several hematopoietic genes, including the macrophage colony-stimulating factor, granulocyte/macrophage colony-stimulating factor, interleukin-3, and T cell receptors; the immunoglobulin µ-heavy chain; defensin NP-3; and myeloperoxidase (14–22). The AML1 gene is the most frequent target for chromosomal translocations in human leukemias. It is rearranged in distinct chromosomal translocations associated with AML (t(8,21), t(12,21), t(16,21), t(19,21)) (23–26), acute lymphatic leukemia (t(12, 21)) (27), and myelodysplastic syndrome (t(3,21)) (28, 29). AML1 (CBF{alpha}2) forms a heterodimer with CBF{beta}. CBF{beta} does not bind DNA directly, but enhances the binding of AML1 (30). Multiple {alpha}-subunit genes, including CBF{alpha}1(AML3), CBF{alpha}2(AML1), and CBF{alpha}3(AML2), as well as alternatively spliced isoforms of the {alpha}- and {beta}-subunits have been detected (31, 32). All of the CBF{alpha} proteins have a DNA-binding domain (the runt domain), which is similar to the Drosophila pair-rule gene runt (33). To understand the function and role of AML1 in leukemia, it is important to study the molecular mechanism of AML1-mediated regulation of gene expression.

Ets-related binding sites are evident in most B cell-specific genes. Hematopoietic genes containing high affinity NERF/ELF-1-binding sites include, among others, IgH and terminal deoxynucleotidyltransferase (34, 35), mb-1 (membrane-bound immunoglobulin IgM-{alpha}) and B29 (36), BSAP (37), lck (38), blk (B lymphoid kinase) (39), and lyn (40, 41). Blk is a B cell-specific tyrosine kinase that is expressed in pre-B and mature B cells, but not in plasma cells; this is similar to the expression of mb-1 (membrane-bound immunoglobulin IgM-{alpha}) and B29 (42). Blk is associated with the antigen receptor and is involved in signal transduction (39). The blk promoter contains a previously uncharacterized NERF/ELF-1-binding site adjacent to a BSAP and AML1 site. Not much is known about regulation of blk gene expression, except that the B cell-specific transcription factor BSAP plays an important role and that the transcription factor NF-{kappa}B/p50 interacts with the blk gene during B cell activation (41). We furthermore demonstrated that AML1 binds to the blk promoter and cooperatively transactivates the blk promoter in the presence of BSAP (43). AML1 has been shown previously to interact with a variety of Ets factors, including the related protein MEF, indicating that a possible interaction between AML1 and NERF may play a role in blk gene regulation (44).

We now report that NERF-2 and ELF-1 directly interact with the runt homology domain of AML1 through a basic region upstream of the Ets domain and cooperate with AML1 in activating blk promoter transcription. We also demonstrate that the NERF-1a isoform lacks the NERF-2 transactivation domain and represses AML1-mediated transactivation of the blk promoter.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Construction—Full-length proteins and different fragments of NERF-2, NERF-1a, and ELF-1 were cloned into the pGEX-5X-3 vector to make GST fusion proteins in Escherichia coli strain BL21 (Novagen). The expression vectors of full-length AML1, AML1-(1–208), and AML1-(87–208) are all derived from AML1b and were prepared as reported previously (45). lyn and blk promoter constructs were also prepared as reported previously (8). For expression of NERF-2 in mammalian cells, NERF-2 was cloned into the NotI site of the pCi vector, which has a cytomegalovirus promoter and enhancer.

For Gal4 expression constructs, NERF-2 and NERF-1 fragments were cloned in the reading frame into the BamHI site of the Gal4-(1–147) expression vector pSG424. The plasmids Gal4-NERF-2-(1–203), Gal4-NERF-2-(1–164), Gal4-NERF-2-(1–141), Gal4-NERF-2-(1–108), Gal4-NERF-2-(1–103), Gal4-NERF-1a-(1–155), and Gal4-NERF-1b-(1–155) were cloned into the Gal4 vector by deleting C termini using restriction enzymes BglII, EaeI, NcoI, EcoNI, NdeI, BglII, and BglII. Gal4-NERF-2-(1–103) MutA, MutB, MutC, MutD, and MutA+B were generated by site-directed mutagenesis by replacing glutamic acid with alanine.

The 5'-FLAG vector was prepared by inserting FLAG sequence-encoding oligonucleotides into the NheI and KpnI sites of the pcDNA3.1 plasmid. The inserted FLAG sequence was ATGGACTACAAAGACGATGACGACAAG. The 3'-Myc vector was prepared by inserting Myc epitope sequence-encoding oligonucleotides into the XhoI and ApaI sites of the pcDNA3.1 plasmid. The inserted Myc epitope sequence was GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG. NERF-2, MEF, PDEF, and ESE1 DNAs were PCR-amplified using Hi-Fidelity Taq polymerase (Invitrogen) and inserted into the BamHI and XhoI sites of the 5'-FLAG-pcDNA3.1 vector. PCR-amplified AML1 was cloned into the BamHI and XhoI sites of the 3'-Myc-pcDNA3.1 plasmid to construct AML1–3'-Myc-pcDNA3.1.

Deletion mutants of NERF-2 (NERF-2(del108–180)) were produced by removing the NERF-2 DNA sequences between two EcoNI sites at amino acids 108 and 180 in the pCi-NERF-2 vector. Briefly, the plasmid was digested with EcoNI, and isolated bands were religated with linkers 5'-TTGAGGGATTCAAGAAGTCCTGA-3' and 5'-CTCAGGACTTCTTGAAT CCCTCA-3', which were previously annealed to fuse the N-terminal 108 amino acids of NERF-2 in-frame with the C terminus of NERF-2 starting at amino acid 180. For NERF-2(del108–180)-FLAG, the pCi-NERF-2(del108–180) vector was used as a template for PCR amplification with primers 5'-CGCGGATCCATGACATCAGCAGTGGTTGAC-3' and 5'-CGCGTCGACTTTCTCACATGTCACTAGTCC-3', which contain BamHI and SalI restriction enzyme sites. The PCR-amplified mutant NERF-2 DNA was inserted in-frame into the 5'-FLAG-pcDNA3.1 vector.

Cell Culture and Transfection—CV-1 and human embryonic kidney 293T cells were grown in Dulbecco's modified Eagle's medium (BioWhittaker, Inc.) containing 10% fetal bovine serum and penicillin/streptomycin. Cotransfection of 3 x 105 CV-1 cells was carried out with 2 µgof reporter gene construct DNA and 3 µg of expression vector DNA using 12.5 µl of LipofectAMINE (Invitrogen) as described previously (45). The cells were harvested 16 h after transfection and assayed for luciferase activity as described previously (43). Transfection for each construct was performed independently in duplicates or triplicates and repeated three to four times with two different plasmid preparations with similar results.

In Vitro Translation—In vitro protein translation was performed using the TNT T7-coupled reticulocyte lysate system (Promega) according to the manufacturer's protocol. The TNT lysate contains ~150 µg/µl endogenous protein. Each in vitro translation reaction uses 25 µlofTNT lysate/50-µl reaction.

GST Pull-down Experiments—The GST pull-down experiments were performed as described previously (46) with some modification. The integrity of the bacterially expressed GST fusion proteins was examined by SDS-PAGE, followed by Coomassie Blue staining. Approximately equal amounts of the fusion proteins were used for each reaction. Briefly, the GST fusion proteins were expressed in E. coli strain BL21; expression was confirmed and quantified by SDS-PAGE; and GST fusion proteins were immobilized on glutathione-agarose beads for pull-down assays as described (46). Recombinant 35S-labeled AML1 and 35S-labeled rhombotin-2 were produced by in vitro transcription/translation (TNT coupled reticulocyte lysate system kit) from pCi-AML1 and pCi-rhombotin-2 plasmid templates. Recombinant proteins were incubated with GST fusion proteins at 4 °C for 1 h in 20 mM Tris (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.25% bovine serum albumin, 0.5% Nonidet P-40, and 0.1 mM dithiothreitol. The beads were then washed three times, and associated proteins were resolved by SDS-PAGE and visualized by autoradiography.

Co-immunoprecipitation and Western Blotting—Human embryonic kidney 293T cells grown on 100-mm dishes were cotransfected overnight with 6 µg of FLAG-tagged Ets constructs and 6 µg of Myc-tagged AML1 expression vector or the parental vector using LipofectAMINE Plus (Invitrogen). The cells were then changed to fresh growth medium for 24–36 h and collected in lysis buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris (pH 8.0), 0.5% Igepal (Nonidet P-40 substitute), 0.5% of Triton X-100, 10% glycerol, and 1:30 diluted protease inhibitor mixture (Roche Applied Science)). The insoluble cell debris was removed by centrifugation at 14,000 x g for 20 min at 4 °C. The supernatants were transferred to a new tube. Total cell lysates were diluted at a 4:6 ratio with immunoprecipitation dilution buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 1:30 diluted protease inhibitor mixture). Then, 20 µl of anti-FLAG monoclonal antibody-conjugated agarose beads (M2-agarose, Sigma) were added to the cell lysates. Immunoprecipitations were carried out overnight at 4 °C with a slow rotating motion. The immunoprecipitated complex was washed five times with 25 mM Tris, 2.7 mM KCl, and 137 mM NaCl (pH 7.4). After the final washing, the bound proteins were eluted in nonreducing SDS sample buffer (63 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 0.005% bromphenol blue) or with 3x FLAG peptide (150 ng/µl final concentration; Sigma). The samples were boiled for 3 min and loaded onto 10% Tris/glycine gel (Bio-Rad). After electrophoresis, gels were transferred to a polyvinylidene difluoride membrane for 1 h. The membranes were blocked overnight in 5% dry milk in 25 mM Tris, 2.7 mM KCl, 137 mM NaCl (pH 7.4), and 0.1% Tween 20 at 4 °C. The transferred membrane was incubated with horseradish peroxidase-conjugated anti-Myc polyclonal antibody (1:1250 dilution; Santa Cruz Biotechnology) for 1.5 h at room temperature. The signal was detected by ECL detection reagents (Amersham Biosciences) on x-ray film.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
NERF-2 Physically Interacts with AML1—Our previous studies established that the promoter region of the B cell-specific blk gene contains Ets- and AML1-binding sites in close proximity to each other (43). Because AML1 has been shown to interact and cooperate with other Ets transcription factors, including Ets-1 and MEF, we explored the possibility that NERF-2, which activates the blk promoter, can cooperate with AML1 in regulating blk gene expression (8). We first tested whether NERF-2 directly interacts with AML1 using a GST pull-down assay in which E. coli cells expressing GST fusion proteins immobilized on glutathione-agarose beads were incubated with in vitro translated 35S-labeled proteins. As shown in Fig. 1, in vitro translated full-length AML1 could be specifically retained on agarose beads containing the GST-NERF-2 fusion protein, but not on glutathione beads containing only GST. In contrast to AML1, rhombotin-2 did not bind to NERF-2 in the GST pull-down assay. This result shows that NERF-2 can specifically interact with AML1.



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  		FIG. 1.
The NERF-2 protein binds to AML1 in vitro. GST pull-down experiments showed that GST-NERF-2 protein from bacteria interacted with in vitro translated (IVT) 35S-labeled AML1 proteins. The AML1 protein was pulled down with GST-NERF-2 fusion proteins, but not with GST alone in vitro. RBTN2, rhombotin-2.

 
A Basic Region Upstream of the Ets Domain of NERF-2 Interacts with AML1—To map the region of NERF-2 responsible for interaction with AML1, 35S-labeled AML1 was incubated with a series of GST-NERF-2 deletion mutant proteins (Fig. 2). As shown in Fig. 2B and summarized in Fig. 4, mutants containing N-terminal amino acids 1–203 (fourth lane) still interacted with AML1 proteins as efficiently as full-length NERF-2 (amino acids 1–581) (third lane). However, C-terminal mutants containing amino acids 204–581 (fifth lane) lost interaction activity with AML1.



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  		FIG. 2.
A basic domain of NERF-2 physically interacts with AML1. A, domain structures of NERF-2 and the fragments used for the GST pull-down experiments shown in B. B, GST pull-down experiments demonstrating AML1 interaction with various fragments of NERF-2 and NERF-1a. These data show that as few as amino acids 121–203 of NERF-2, which are just N-terminal to the Ets domain, can bind efficiently to AML1 proteins. IVT, in vitro translated 35S-labeled AML1 protein; RBTN2, rhombotin-2.

 



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  		FIG. 4.
Interaction of NERF-2 and AML1 synergistically activates the blk promoter. A, blk promoter-luciferase assays were performed with NERF-1a, NERF-2, or AML1 (+CBF{beta}) or with NERF-2 + AML1 (+CBF{beta}) or NERF-1a + AML1 (+CBF{beta}) expression constructs in CV-1 cells. NERF-2 + AML1 (+CBF{beta}) coexpression resulted in greater luciferase activity compared with the combined increase of NERF-2-alone and AML1-alone activities, demonstrating that physical interaction of NERF-2 and AML1 has a cooperative function in activating the blk promoter. However, NERF-1a interaction with AML1 showed a suppressive function with AML1-induced blk promoter activity. RLU, relative luciferase units. B, two clones of the NERF-2(del108–180) deletion mutant fused to the FLAG peptide in the FLAG vector (Mut #1 and Mut #17) were generated as described under "Materials and Methods." These mutant DNAs were cotransfected with AML1-Myc into 293T cells, and their proper expression was tested by Western blot analysis using anti-Myc and anti-FLAG antibodies for AML1 and NERF-2 mutants, respectively. As a control, wild-type FLAG-NERF-2 was also transfected. C, FLAG-tagged wild-type NERF-2 and NERF-2(del108–180) mutant constructs were transiently cotransfected with Myc-tagged AML1 into 293T cells. Cell lysates were co-immunoprecipitated (IP) using anti-FLAG monoclonal antibody M2. Western blotting (WB) was performed using anti-Myc polyclonal antibody to detect the AML1 protein. AML1 was detected only when NERF-2-FLAG and AML1-Myc were cotransfected into the cells, but not with NERF-2(del108–180) deletion mutants with AML1, vector only, or NERF-2 or AML1 plus vector. D, NERF-2(del108–180) deletion mutants were cotransfected with or without AML1 (+CBF{beta}) along with the blk promoter construct into CV-1 cells. The blk promoter activities were measured and are presented as -fold increase relative to the control without AML1. The -fold cooperation was calculated by dividing the activation of the promoter in the presence of both factors by the expected additive result after background subtraction. The mutants showed only an additive effect for AML1 and NERF-2 transactivation, whereas wild-type NERF-2 showed strong transcriptional cooperativity. E, the C terminus of AML1 is required for cooperativity with NERF-2. Transient transfections were carried out with NERF-2, CBF{beta}, and either AML1 or mutants of AML1 to identify the region of AML1 required for functional interaction with NERF-2. AML1-(1–289), AML1-(1–351), and AML1-(1–381) are C-terminal truncations of AML1; the numbers represent the amino acids that are encoded. The results represent the means ± S.E. The -fold cooperation was calculated by dividing the activation of the promoter in the presence of both factors by the expected additive result after background subtraction.

 
The N terminus of NERF-2 contains three domains (A, B, and C) that are highly conserved in three members of the Ets subfamily, NERF, ELF-1, and MEF. No specific function has been attributed to any of these domains until now. To examine whether any of these N-terminal homology domains (A, B, or C) is involved in the physical interaction with AML1, we generated NERF-2-(1–203{Delta}A), NERF-2-(1–203{Delta}B), NERF-2-(1–203{Delta}C), and NERF-2-(1–203{Delta}A/B) mutants by replacing acidic amino acids with alanine and performed GST pull-down assays with AML1. All of the deletion mutants tested still showed physical interactions, indicating that none of those regions are important for the interaction with AML1. Furthermore, GST fusion proteins with NERF-2 deletion mutants containing residues 1–103 (Fig. 2B, fifteenth lane), 1–141 (sixth lane), and 1–164 (eleventh lane) did not bind AML1 either, indicating that the domain of NERF-2 interaction with AML1 is concentrated between residues 165 and 203. Indeed, NERF-2 mutants containing residues 105–203 (sixteenth lane) and 121–203 (seventeenth lane) still retained full interaction capacity with AML1. Similarly, the NERF-2 mutant containing residues 111–180 (fourteenth lane) maintained full interaction activity, whereas that containing residues 165–203 (twelfth lane) exhibited somewhat reduced binding activity. These results demonstrate that the minimal domain for NERF-2 interaction with AML1 is located between amino acids 165 and 180, although region 111–165 appears also to contribute to the interaction with AML1 possibly by stabilizing the principal contact regions. To further confirm the importance of the basic region of NERF-2 in interaction with AML1, we also generated a GST fusion protein containing the N terminus of NERF-1a from amino acids 1 to 143 (eighteenth lane), which still contains the basic domain of NERF-2, but lacks the N-terminal part of NERF-2. GST pull-down assay showed that the N terminus of NERF-1a interacted with AML1 as well, further supporting that the basic region of NERF is involved in binding to AML1.

NERF-2 Binds to AML1 in Vivo—To confirm that NERF-2 can bind to AML1 in vivo, we performed a co-immunoprecipitation experiment. For this purpose, we generated expression vectors for a fusion protein of NERF-2 containing the FLAG tag at the N terminus and for a fusion protein of AML1 containing the Myc tag at the C terminus. These constructs were either individually transfected or cotransfected into 293T cells. Total cell lysates were immunoprecipitated using anti-FLAG antibody-conjugated agarose beads, followed by Western blot analysis with anti-Myc antibody. Vector alone, NERF-2-FLAG, or AML1-Myc did not give any signal. However, AML1 was detected clearly when NERF-2-FLAG and AML1-Myc were cotransfected into the cells (Fig. 3A). These data most vividly demonstrate that the AML1 protein binds to the NERF-2 protein in vivo.



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  		FIG. 3.
NERF-2 interacts with AML1 in vivo. A, the FLAG-tagged NERF-2 construct was transiently cotransfected with Myc-tagged AML1 into 293T cells. Cell lysates were co-immunoprecipitated (IP) using anti-FLAG monoclonal antibody M2. Western blotting was performed using anti-Myc polyclonal antibody to detect the AML1 protein. AML1 was detected only when NERF-2-FLAG and AML1-Myc were cotransfected into the cells, not with vector only or NERF-2 or AML1 plus vector. B, other Ets factors were also cloned into the FLAG vector and cotransfected with the AML1 construct. These co-immunoprecipitation/Western blot (WB) experiments showed that AML1 interacted with the NERF-2 and MEF proteins, but not with the PDEF and ESE1 proteins.

 
AML1 Interacts with Other Selected Members of the Ets Family in Vivo—To test whether AML1 interacts with other Ets family members in vivo, the AML1-Myc construct was cotransfected with NERF-2-FLAG, MEF-FLAG, PDEF-FLAG, or ESE1-FLAG into 293T cells (Fig. 3B). The expression of each Ets-FLAG construct was then tested by SDS-PAGE and Western blotting using anti-FLAG antibody. Immunoprecipitation with anti-FLAG antibody followed by Western blot analysis with anti-AML1 antibody revealed that AML1 bound to NERF-2 and MEF, as expected. However, no binding of AML1 to PDEF or ESE1 was detected, demonstrating that AML1 interacts with a selected subset of the Ets family.

NERF-2 Cooperates with AML1 in Transactivation of the blk Promoter, but NERF-1a Represses AML1-mediated Transactivation—To evaluate whether AML1 interaction with NERF leads to cooperativity in the context of the blk promoter, we performed cotransfection experiments. NERF-2 and AML1 together with its non-DNA-binding heterodimer partner CBF{beta}, either alone or in combination, were cotransfected along with the blk promoter-luciferase construct into CV-1 cells, and luciferase assays were performed 16 h later. NERF-2 activated the blk promoter by 3.6-fold and AML1c (which is longest form of the AML1 splice variants, with 480 amino acids) by 12-fold. However, the combination of NERF-2 and AML1c led to a synergistic increase in blk promoter activity of 42-fold (Fig. 4A), which is significantly more than would be expected due to an additive effect. This experiment clearly demonstrates that NERF-2 cooperatively enhances AML1-mediated transactivation of the blk promoter. In contrast to the transactivator NERF-2, the NERF-1a isoform by itself did not significantly transactivate the blk promoter and, in combination with AML1, drastically inhibited AML1-mediated blk promoter transactivation, suggesting that NERF-1a might work as a transcriptional repressor.

The domain of NERF-2 that retained maximum interaction with AML1 contains residues 111–180, including a basic domain conserved in the three Ets family members NERF-2, ELF-1, and MEF. To evaluate whether cooperative stimulation of the blk promoter requires physical interaction between NERF-2 and AML1, we generated NERF-2 deletion mutants (NERF-2(del108–180)) that lack the AML1 interaction domain between amino acids 108 and 180, including the basic domain. We derived two different clones from these NERF-2(del108–180) mutants fused to the N-terminal FLAG peptide in the FLAG vector and first confirmed their proper expression after transfection into 293 cells and the size of the proteins by Western blot analysis (Fig. 4B). Fig. 4B demonstrates that both mutant NERF-2 proteins were expressed at similar levels compared with wild-type NERF-2 and with the expected molecular masses. By co-immunoprecipitation and Western blot analysis, we then tested whether these NERF-2 deletion mutants had lost their ability to physically interact with AML1 in vivo. As shown in Fig. 4C, both NERF-2(del108–180) mutants were unable to physically interact with AML1, whereas wild-type NERF-2 efficiently interacted with AML1, confirming that deletion of amino acids 108–180 eliminates the AML1 interaction domain.

To evaluate whether these mutants are able to cooperate with AML1 in transactivation of the blk promoter, CV-1 cells were transiently cotransfected with wild-type or mutant NERF-2 and AML1 together with CBF{beta}, either alone or in combination (Fig. 4D). Deletion of amino acids 108–180 resulted in complete loss of cooperativity with AML1 compared with the activity induced by wild-type NERF-2 (Fig. 4D). Transactivation of the blk promoter by these mutants alone was also slightly reduced, which may be the result of reduced transactivation capacity due to either lack of interaction with endogenous AML1 or an effect on the basal transactivation capacity of NERF-2, even though neither the transactivation domain (see below) nor the DNA-binding domain was changed. These data clearly demonstrate that disruption of NERF-2 interaction with AML1 results in the loss of transcriptional cooperativity of NERF-2 with AML1 and that the AML1 interaction domain is critical for cooperativity.

To determine whether the transactivation domain of AML1 is essential for cooperativity with NERF-2, AML1 mutants truncated at the C terminus were transfected in the absence or presence of NERF-2 into CV-1 cells (Fig. 4E). Although termination of AML1 at amino acids 381 and 351 did not affect cooperativity with NERF-2 or transactivation by AML1 alone, termination at amino acid 289 drastically reduced cooperativity and correlated with loss of transactivation capability of AML1 itself. These results reveal that the C-terminal transactivation domain of AML1 is necessary for synergy with NERF-2.

NERF-2, but Not NERF-1a, Contains a Transactivation Domain Encoded by the N-terminal 100 Amino Acids—Although NERF-2 and NERF-1a were able to interact with AML1, only NERF-2 acted as a transcriptional activator and cooperated with AML1. This result suggests that the AML1 interaction domain is distinct from the NERF-2 transactivation domain. To define the transactivation domain of NERF-2 in more detail, we generated C- and N-terminal deletions of NERF-2 as shown in Fig. 5A. Cotransfection experiments were performed with expression vectors encoding full-length NERF-2 and deletion mutants and the lyn promoter Ets site-luciferase construct, another B cell target for NERF that is highly inducible by NERF-2. Deletion of the C terminus of NERF-2 (NERF-2-(1–381) or NERF-2-(1–510)) decreased transactivation slightly, whereas deletion of the N-terminal 103 amino acids (NERF-2-(104–581)) completely abolished NERF-2 transactivation capacity (Fig. 5A). NERF-1a, which lacks the N terminus of NERF-2 and instead has a distinct N terminus, did not transactivate the lyn promoter Ets site either and actually slightly decreased promoter activity compared with the parental pCI expression vector. These data provide strong evidence that the main NERF-2 transactivation domain is located at the N terminus that is absent in NERF-1a. These data also demonstrate that the transactivation domain is distinct from the AML1 interaction domain.



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  		FIG. 5.
Transcriptional activation of the lyn promoter Ets site. A, CV-1 cells were cotransfected with the indicated NERF-1a, wild-type (WT) NERF-2, or NERF-2 deletion mutant expression constructs and luciferase constructs containing two copies of the lyn promoter Ets site. When the first 103 amino acids of NERF-2 were deleted, the transactivation activity dropped to a basal level similar to that with pCi vector transfection. RLU, relative luciferase units. B, lyn promoter luciferase assay was carried out with 5–6 amino acid deletions in NERF-2 for MutA, MutB, and MutC or with the mutation of 2 glutamic acid residues to alanine for MutD, as shown in Fig. 7B. MutA+B showed the most reduction in lyn promoter activity, suggesting that domains A and B are largely responsible for the transactivation activity induced by NERF-2.

 
To confirm that the N-terminal deletion of NERF-2 does not affect protein expression, protein stability, or binding to the lyn promoter, we performed EMSAs using whole cell extracts of CV-1 cells transfected with either pCI-NERF-2 (wild-type) or pCI-NERF-2(del1–103) and oligonucleotides of the lyn promoter NERF-binding site. Deletion of the N-terminal 103 amino acids of NERF-2 did not affect protein expression levels or binding to the lyn promoter as demonstrated by the EMSA (Fig. 6), suggesting that the loss of transactivation activity of N-terminally deleted NERF-2 is not due to either decreased protein amounts of mutant NERF-2 or the loss of DNA binding activity of mutant NERF-2, but rather to the loss of the transactivation domain of NERF-2.



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  		FIG. 6.
EMSA showing DNA binding of full-length NERF-2 or NERF-2 mutants to the blk and lyn promoter Ets sites. DNA binding analysis was performed using extracts from cells transfected with full-length and mutant NERF-2 expression vectors in an EMSA with oligonucleotides encompassing the lyn promoter Ets site. The results show that the NERF-2 mutants used for the experiments in Fig. 5(A and B) did not affect or had little effect on the DNA binding activity for the blk or lyn promoter. WT, wild-type.

 
The Conserved N-terminal Domains A and B Are Part of the NERF-2 Transactivation Domain—To analyze in more detail which domains of the N terminus of NERF-2 might be part of the transactivation domain, we mutated three domains (A, B, and C) in the N-terminal region of NERF-2 that are not present in NERF-1a, but that are conserved in NERF-2, ELF-1, and MEF (Fig. 7A). We deleted 5–6 amino acids each in domain A (MutA), domain B (MutB), and domain C (MutC) or mutated 2 glutamic acid residues in domain B (MutD) to alanine (Fig. 7, A and B). We hypothesized that evolutionary conservation of these acidic domains may indicate an important function for these domains. Deletions of domains A, B, and D reduced transactivation to ~50% compared with wild-type NERF-2, and a combined deletion of domains A and B (MutA+B) further reduced transactivation, but not to the level observed with the full-length N-terminal deletion (Fig. 5B). In contrast, the deletion of domain C (MutC) did not affect transactivation capacity (Fig. 5B). These results demonstrate that the conserved N-terminal domains A and B (but not C) are integral components of the NERF-2 transactivation domain, which is either absent or truncated in NERF-1a and NERF-1b, respectively. To confirm that the differences in transactivation capability of the NERF-2 deletion mutants are not due to reduced levels of expression in transfected cells or reduced DNA binding, we performed EMSAs using aliquots of the cell extracts from transfected CV-1 cells (Fig. 6). All NERF-2 deletion mutants formed complexes with the expected mobility and similar intensity, suggesting that decreases in transactivation are not due to a lack of or reduced expression or a lack of DNA binding. The fact that deletion of NERF-2 domains A and B together drastically impaired, but did not completely abolish, transactivation activity suggests that there is an additional region in the N terminus (amino acids 1–103) that contributes to the transactivation of NERF-2 (Fig. 5B).



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  		FIG. 7.
A, comparison of the amino acid sequence of NERF with those of ELF-1 and MEF. The five major homologous regions (domains A, B, C, D, and E (Ets domain)) are boxed. B, the sequence of the highly acidic transactivation domain and basic domain, which is N-terminal to the Ets domain. The sequence shown is amino acids 1–203 of NERF-2, with some of the mutations made for the luciferase experiment shown in Fig. 5 and for the Gal4 assay shown in Fig. 8.

 
To further confirm that the N terminus of NERF-2, but not NERF-1a, exhibits transactivation activity, we fused different domains of NERF-2 and NERF-1 to the Gal4 DNA-binding domain and tested their transactivation activities on the luciferase reporter containing three binding sites for Gal4 fused to a minimal promoter. We cotransfected each of these constructs into COS cells along with the Gal4-luciferase reporter (pSGE1bluc or pSGluc1b). As predicted by our deletion mutants, the N terminus of NERF-2 strongly transactivated the Gal4-luciferase reporter by up to 900-fold compared with the Gal4 DNA-binding domain alone (Fig. 8). This activation was as strong as that of the N terminus of the related protein ELF-1 and significantly stronger than that of the ELK transactivation domain. C-terminal deletions of the NERF-2 N terminus down to amino acid 103 (NERF-2-(1–103)) did not diminish transactivation capacity, indicating that the transactivation domain is indeed encoded by the N-terminal 103 amino acids, which contain domains A, B, and C. Consistent with the internal deletion mutants shown in Fig. 5B, Gal4 fusions containing the N-terminal 103 amino acids combined with deletions of domains A, B, and D showed significant loss of transactivation activity, whereas MutC still maintained full transactivation activity. MutA+B showed again a further decrease in transactivation. In concordance, the N terminus of NERF-1a or NERF-1b (amino acids 1–155), which lacks domains A and B, did not exhibit any transactivation activity (Fig. 8), further highlighting the importance of domains A and B for transactivation activity (8).



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  		FIG. 8.
Transactivation of N-terminal NERF-2 in Gal4 constructs. COS cells were cotransfected with the indicated Gal4-NERF-2 mutant fusion constructs and the Gal4-luciferase reporter construct (pSGE1bluc or pSGluc1b), which contains the minimal promoter and three Gal4 DNA-binding elements. As shown here, NERF-2-(1–103) showed high transactivation activity. Consistent with the promoter assay shown in Fig. 5B, MutA, MutB, MutD, and MutA+B showed significant loss of transactivation activity, whereas MutC still maintained transactivation activity.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Translocations of Ets factors and AML1 have been shown to be of critical importance in leukemias and various other types of cancer, including translocation of the Ets factor Tel/ETV6 to AML1 itself (27). Furthermore, several members of the Ets family have been demonstrated to enhance AML1-mediated transactivation of various genes via direct protein-protein interaction through the Ets DNA-binding domain and the AML1 runt domain (2, 44). This finding implies also that Ets factors should affect transactivation in leukemias mediated by AML1 translocation proteins such as Tel/AML1 and AML1/ETO (eight twenty one) due to these protein-protein interactions. We have previously demonstrated that AML1 directly interacts with the B cell-specific transcription factor BSAP/PAX-5 and that this interaction leads to synergistic enhancement of transactivation of the B cell-specific blk gene promoter (43). We also previously showed that the blk promoter is regulated by the Ets factors NERF-2 and ELF-1 expressed in B cells (8).

In this study, we examined the physical interaction of NERF-2 and NERF-1 with AML1 and the functional consequences of these interactions in the context of the B cell-specific blk gene promoter. We have demonstrated that the two NERF isoforms NERF-2 and NERF-1 directly interact with AML1. Using various GST-NERF-2 and GST-NERF-1 fusion proteins, we identified the basic domain of NERF-2 (amino acids 111–180) upstream of the Ets DNA-binding domain as the major domain for protein-protein interaction with AML1. Both NERF-2 and NERF-1a interacted with AML1, indicating that both isoforms could affect AML1 activity. Indeed, whereas NERF-2 enhanced AML1-mediated transactivation of the blk promoter, NERF-1a drastically repressed AML1-mediated transactivation. Part of the explanation for these opposing activities of NERF-2 and NERF-1a is the lack of a transactivation domain in NERF-1a and the location of a transactivation domain within the N-terminal 103 amino acids of NERF-2 as shown by deletion and mutation studies as well as heterologous Gal4 fusion proteins. The opposite effects of NERF-2 and NERF-1a on AML1 activity is highly interesting because both NERF-2 and NERF-1a isoforms are expressed in B cells and other cell types, although their relative ratio changes in different cell types and under different conditions. NERF-2 and NERF-1a are actually regulated by different promoters, suggesting that different physiological settings could determine the relative level of NERF-2 versus NERF-1a. Because NERF-2 is a positive regulator of transcription and NERF-1a acts as a transcriptional repressor, regulated changes in the ratio of NERF-2 to NERF-1a are expected to either enhance or repress expression of target genes. In this context, the interaction of both NERF isoforms with AML1 would imply that AML1-mediated transactivation could be highly dependent on the ratio of NERF-2 versus NERF-1a within leukemic cells. With regard to AML leukemic cells that contain AML1 translocations crucial for transformation, NERF isoforms may be able to enhance or reduce the transforming capacities of AML1 translocation proteins.

The AML1 interaction domain of NERF-2 was mapped to a basic domain upstream of the Ets domain, which differs from the domain for Ets-1 binding to AML1. AML1 binds to the Ets domain of Ets-1 and autoinhibitory domains (negative regulatory domain for DNA binding and exon VII domain) (47). We have also shown, as has previously been demonstrated in vitro (48), that MEF, a NERF-2-homologous protein, binds to AML1 in vivo. MEF has also been reported to interact with AML1 through a region N-terminal to the Ets domain, although this region was not further defined. Therefore, it is likely that the basic domain D, which is conserved in E74 Ets family members, is the region through which NERF-2, MEF, and ELF-1 interact with AML1. Thus, this AML1 interaction domain appears to represent a novel protein-protein interaction domain, suggesting that AML1 can bind to different members of the Ets family via different interaction domains.

Because various isoforms of NERF are expressed in B cells, NERF is likely to play a role in B cell function or differentiation. The NERF-AML1 and BSAP-AML1 interactions and synergistic activation of the blk promoter support the notion that NERF, AML1, and BSAP regulate blk gene expression. Because BSAP has been demonstrated to interact with the Ets domain of several Ets factors, we are now also in the process of evaluating whether NERF interacts with BSAP and forms a NERF·BSAP·AML1 complex that regulates blk gene expression. Blk is a B cell-specific tyrosine kinase of the Src family important for B cell activation after cross-linking of antigens via the B cell antigen receptor. In peripheral lymphoid tissues, cross-linking-initiated signaling activates B cells to enter the G1 phase of the cell cycle, which will direct B cells to respond to proliferative signals (49). Subsequently, proliferating B cells differentiate into antibody-producing plasma cells. Expression of constitutively active Blk(Y495F) in the B cell lineage induces malignant transformation of early lymphoid progenitors in mice, suggesting a role for Blk in the control of proliferation during B cell development (50). Our results show that physical interaction of NERF-2 with AML1 synergistically activates the blk promoter, whereas NERF-1a inhibits AML1-mediated transactivation. Previously, we demonstrated that all NERF isoforms bind with comparable affinity to the same Ets sites in a variety of B cell-specific genes, including blk, although only NERF-2, but not NERF-1a and NERF-1b, function as transcriptional activators of B cell-specific promoters (8). NERF-1a may act as a competitive inhibitor of endogenous NERF-2 or possibly other Ets factors by replacing NERF-2 on the blk promoter and thus inhibiting AML1 transactivation activity, which might be NERF-2-dependent. Alternatively, NERF-1a may be an active repressor that interacts with a corepressor and actively inhibits AML1-mediated transactivation. We have shown here that the transactivation domain of NERF-2 does not overlap with the basic AML1 protein interaction domain, but is located in the N-terminal 103 amino acids. This also explains why NERF-1a and NERF-1b, which differ at their N terminus from NERF-2, do no exhibit transactivation activity. Indeed, the N terminus of NERF-1a does not contain a transactivation domain, as shown by our Gal4 heterologous transactivation assay. Recently, a potent transactivation domain of MEF, a NERF-2-homologous protein, has been mapped to the N-terminal region encompassing amino acids 1–52 (51). There is significant sequence homology within the N-terminal 103 amino acids among NERF-2, ELF-1, and MEF, particularly in domains A, B, and C. These conserved domains contain many acidic amino acids, and our point mutations replacing acidic amino acids with alanine or deleting acidic amino acids provide evidence that acidic residues are involved in transactivation function. Acidic transactivation domains have been observed in many transcription factors, including other members of the Ets family, and appear to interact with several general transcription initiation factors (52–56).

The synergistic and repressor activities of NERF-2 and NERF-1a, respectively, in conjunction with AML1 provide support for the notion that different NERF isoforms and their regulation may modulate AML1 function both during normal B cell development and in leukemic cells with translocated AML1. Future studies will focus on determining the effect of NERF isoforms on AML1 translocation proteins in leukemic cells.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants PO1 CA72009 (to T. A. L. and D.-E. Z.) and RO1 CA76323 (to T. A. L.). 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. Back

§ Present address: Dept. of Oral Biochemistry, School of Dentistry, Kyungpook National University, Daegu, 700-422 Korea. Back

|| To whom correspondence should be addressed: Beth Israel Deaconess Medical Center, 4 Blackfan Circle, Boston, MA 02115. Tel.: 617-667-3393; Fax: 617-975-5299; E-mail: tliberma{at}bidmc.harvard.edu.

1 The abbreviations used are: ELF, E74-like factor; NERF, new Ets-related factor; BSAP, B cell lineage-specific activator protein; MEF, myeloid ELF-1-like factor; AML, acute myeloid leukemia; CPF, core binding factor; GST, glutathione S-transferase; PDEF, prostate-derived Ets factor; EMSA, electrophoretic mobility shift assay. Back



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