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Originally published In Press as doi:10.1074/jbc.M408654200 on October 26, 2004

J. Biol. Chem., Vol. 279, Issue 53, 55362-55371, December 31, 2004
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E2A-PBX1 Interacts Directly with the KIX Domain of CBP/p300 in the Induction of Proliferation in Primary Hematopoietic Cells*{boxs}

Richard Bayly{ddagger}, Luan Chuen{ddagger}§, Richard A. Currie{ddagger}§, Brandy D. Hyndman{ddagger}, Richard Casselman{ddagger}, Gerd A. Blobel¶, and David P. LeBrun{ddagger}||

From the {ddagger}Queen's University Cancer Research Institute, Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, K7L 3N6, Canada and the Division of Hematology, The Children's Hospital, Philadelphia, Pennsylvania 19104

Received for publication, July 29, 2004 , and in revised form, October 26, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The E2A gene encodes DNA-binding transcription factors, called E12 and E47, involved in cell specification and maturation. E2A is also involved in a chromosomal translocation that leads to the expression of an oncogenic transcription factor called E2A-PBX1 in cases of acute leukemia. In the work described here, we elucidate the interaction between E2A-PBX1 and transcriptional co-activators. We confirm that the E2A portion can interact with CBP and PCAF and map required elements on E2A and CBP. On CBP, the interaction involves the KIX domain, a well characterized domain that mediates interactions with several other oncogenic transcription factors. On E2A, the interaction with CBP requires conserved {alpha}-helical domains that reside within activation domains 1 and 2 (AD1 and AD2, respectively). Using purified, recombinant proteins, we show that the E2A-CBP interaction is direct. Notwithstanding the previously demonstrated ability of AD1 and AD2 to function independently, some of our findings suggest functional cooperativity between these two domains. Finally, we show that the CBP/p300-interactive helical domains of E2A are important in the induction of proliferation in cultured primary bone marrow cells retrovirally transduced with E2A-PBX1. Our findings suggest that some aspects of E2A-PBX1 oncogenesis involve a direct interaction with the KIX domain of CBP/p300.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transcription factors encoded by the gene E2A are crucial for normal development and implicated in acute leukemia. E2A-null mice manifest a complete block of B-lymphoid development at the pro-B cell stage, prior to immunoglobulin gene rearrangement, and a propensity to develop T-lymphoblastic lymphoma (1–3). Despite these particular defects in the lymphoid compartment, the E2A gene products, called E12 and E47 (or "E2A proteins" for simplicity) are expressed widely in tissues and appear to function more generally in cell type specification and maturation (4, 5).

The E2A proteins belong to the basic helix-loop-helix (bHLH)1 class of transcription factors (6). The bHLH domain, located toward the C terminus in E2A proteins, mediates dimerization and DNA binding (see Fig. 1). Dimerization is required for DNA binding by bHLH proteins. E2A proteins bind DNA at sites, called "E-boxes," which contain the simple consensus sequence CANNTG. The widely expressed E2A proteins can heterodimerize with bHLH proteins whose expression is restricted to particular cell types to regulate target gene transcription in a tissue-specific manner.



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  		FIG. 1.
Domain organization of E2A, PBX1, and E2A-PBX1 proteins. The numbers indicate the first and last amino acid positions of activation domains 1 and 2, and the most C-terminal E2A position (483) before the fusion point with PBX1. AD1, activation domain 1; AD2, activation domain 2; bHLH, basic helix-loop-helix domain; HD, homeodomain. The sequence alignment shows the N termini of metazoan E2A orthologs and the CBP-interactive domain of c-Myb. Identical residues in the E2A orthologs are given a black background; similar residues have a gray background. The asterisks indicate amino acids conserved between human E2A proteins and c-Myb.

 
E2A proteins are transcriptional activators. Two activation domains have been mapped, largely using transient co-transfection of reporter plasmids with portions of E2A fused to heterologous DNA-binding domains (7–9). Activation domain 1 (AD1) encompasses 99 amino acids at the extreme N terminus of the proteins, whereas activation domain 2 (AD2) is located more centrally (see Fig. 1). These domains are considered to function independently through distinct mechanisms. For example, whereas AD1 can trans-activate reporter genes in various cell types, AD2 is differentially active in cells of pancreatic {beta}-cell origin (5).

Transcriptional activation domains mediate contacts between DNA-bound transcription factors and other proteins involved in gene transcription (10). They can recruit large, multiprotein assemblies called transcriptional co-activator complexes. These often include proteins with intrinsic acetyltransferase activity. Acetyltransferases catalyze the acetylation of histones and other chromatin-associated proteins, including transcription factors. The mechanism by which acetylation of these proteins facilitates gene transcription is poorly understood and likely complex (11).

Among the best characterized transcriptional co-activator proteins are CREB binding protein (CBP) and its close paralog p300. CBP and p300 are large, ubiquitously expressed nuclear proteins (12). They were originally identified separately and are encoded on separate genes, but their polypeptide sequences are highly conserved; they are often referred to collectively as CBP/p300. Although lacking a DNA-binding domain, CBP/p300 can be recruited through protein-protein interactions to the promoters of target genes regulated by DNA-bound transcription factors. These include both oncogenic proteins, including c-Myb and c-Jun, and tumor suppressors such as p53 and pRb. The manner of involvement of CBP/p300 in oncogenesis appears complex and context-specific. PCAF, another well characterized and potent acetyltransferase, is an ortholog of the prototypic yeast acetyltransferase protein GCN5 (13). PCAF exists as a stable complex with other proteins (the "PCAF complex") and, like CBP/p300, can function as a transcriptional co-activator through transcription factor-dependent recruitment to target gene promoters. Furthermore, PCAF can associate directly with CBP/p300 and this probably contributes to its co-recruitment with CBP/p300 to some promoters. Functional and biochemical evidence indicates that E2A proteins can interact with CBP/p300. For example, p300, CBP, and PCAF can be co-immunoprecipitated with E2A proteins from crude nuclear extracts and co-elute with E2A proteins on fractionation of nuclear extracts by size exclusion chromatography (14).

The E2A locus on chromosome 19 is involved in at least two chromosomal translocations associated with acute lymphoblastic leukemia (ALL) (15). Translocation 1;19 creates the E2A-PBX1 fusion gene, which expresses chimeric, oncogenic proteins called E2A-PBX1a and E2A-PBX1b, in roughly 5% of cases of ALL (15). In these oncoproteins, the N-terminal two-thirds of E2A proteins, which includes AD1 and AD2, is fused to most of PBX1, including its homeodomain (Fig. 1). Considering this structural configuration, it has been proposed that E2A-PBX1 promotes oncogenic cell transformation through the aberrant transcriptional regulation of target genes that are normally regulated by PBX1-containing transcriptional regulatory complexes. Consistent with this hypothesis, reporter plasmids bearing consensus PBX1 binding sites are trans-activated by co-transfected E2A-PBX1, but not wild-type PBX1 proteins (16–18). Furthermore, engineered deletions that impair trans-activation by E2A-PBX1, such as those that include AD1, also impair oncogenicity (19–21). Therefore, it appears likely that, as with transcriptional regulation by wild-type E2A proteins, neoplastic transformation by E2A-PBX1 relies on specific interactions of polypeptide elements in AD1 or AD2 with one or more transcriptional co-regulators.

In the work described here, we elucidate functional interactions between the portion of E2A included in E2A-PBX1 (i.e. amino acids 1–483) and transcriptional co-activators. Our findings indicate that E2A can interact directly with the KIX domain of CBP. This interaction involves conserved helical domains within AD1 and AD2 and is associated with accelerated cell proliferation in retrovirally transduced primary hematopoietic progenitors.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—Plasmids conferring bacterial expression of glutathione S-transferase (GST) fusion proteins were constructed using PCR amplification. Plasmids containing the E2A-PBX1a and the murine CBP cDNAs were used as templates for the PCR. The PCR products were restriction-digested with BamHI and EcoRI (the appropriate restriction sites were included in the PCR primers) and ligated into the pGEX-2T bacterial expression plasmid in-frame with the GST open reading frame. All constructs made using PCR were sequenced to rule out the possibility of PCR-induced errors. Plasmids conferring mammalian expression of GAL4- or VP16-E2A fusion proteins were assembled by excising the various cDNAs from pGEX-2T and, using the BamHI and EcoRI restriction sites, ligating them into the pCVM1 mammalian expression plasmid in-frame with a portion of cDNA encoding the N-terminal 147 amino acids of GAL4 (i.e. the DNA-binding domain) or two VP16 activation domains arranged in tandem. The VP16 insert was generated by PCR from the plasmid "RSV-2X-VP16-Myb," kindly provided by Dr. Paul Brindle. The reporter plasmid p5X GAL4 LUC was a generous gift from Dr. Cornelius Murre.

Expression and Purification of Recombinant Proteins—The expression plasmids based on pGEX-2T were transformed into competent Escherichia coli (strain BL21-CodonPlus(DE3)-RP, Stratagene, La Jolla, CA) by electroporation. Recombinant protein production was induced in 500 ml of exponentially growing cells by the addition of isopropyl 1-thio-{beta}-D-galactopyranoside to 0.1 mM, and the cells were lysed 2.5 h later by sonication in 25 ml of phosphate-buffed saline (PBS) containing protease inhibitors. Triton X-100 was added to 1%, and the lysate was rocked gently at 4 °C for 30 min. Particulate material was pelleted by centrifugation, the supernatant was transferred to a fresh tube, and 250 µl of pre-swollen glutathione-Sepharose 4B (Amersham Biosciences) beads were added. The bead suspension was incubated overnight at 4 °C with gentle rocking and transferred to a chromatography column (Econo Column, Bio-Rad, Mississauga, Ontario). The beads were washed with 40 bed volumes of 1% Triton X-100 in PBS and then with 20 bed volumes of PBS. Recombinant GST fusion proteins were eluted in 9 bed volumes of elution buffer (20 mM reduced glutathione, 75 mM Tris, pH 8.8, 100 mM NaCl) containing protease inhibitors. Eluate fractions containing protein were pooled and dialyzed overnight at 4 °C against PBS. The resulting solution was combined with an equal volume of 100% glycerol and stored in aliquots at –20 °C. The quantity and integrity of the GST fusion proteins was verified by Coomassie Brilliant Blue staining of SDS-PAGE gels with comparison with known quantities of bovine serum albumin.

Pull-downs, Immunoprecipitations, and Immunoblotting—Nuclear extracts were prepared from RCH ACV cells using the Dignam and Roaeder method (22). For pull-down experiments, 10 µg of GST fusion protein was combined with 20 µl of pre-swollen glutathione-Sepharose beads in PBS containing 1% Triton X-100 to a final volume of 300 µl and rocked at 4 °C for 2 h. The beads were then pelleted, washed in HEGN buffer (20 mM Hepes-KOH, pH 7.9, 0.1 mM EDTA, 10% glycerol, and 0.1% Nonidet P-40) containing protease inhibitors, and nonspecific protein binding was blocked by incubation in HEGN buffer containing 1 mg/ml bovine serum albumin and 0.1 M KCl. The beads were pelleted, resuspended in 350 µl of nuclear extract (containing roughly 3 mg/ml protein in HEGN buffer), and incubated overnight at 4 °C with gentle rocking. The beads were pelleted and washed five times in HEGN buffer containing 0.2 M NaCl. They were then resuspended, and the bound proteins were eluted in reduced glutathione. The eluate was combined with SDS-containing electrophoresis sample buffer (New England Biolabs, Mississauga, Ontario) in preparation for SDS-PAGE and immunoblotting. To immunoprecipitate purified GST fusion proteins, roughly 0.032 pmol of each protein was combined in 300 µl of HEGN buffer containing 0.1 M KCl and 4.4 mg/ml bovine serum albumin. A 100-fold molar excess of GST was added to block protein-protein interactions mediated by the GST portion of the recombinant proteins. After incubation for 2 h at 4 °C with gentle rocking, 10 µg of pre-swollen, pre-blocked protein G-Sepharose beads (Amersham Biosciences) were added and the solution rocked for a further 2 h. Then, 1 µg of antibody was added, and the solution was rocked overnight. The beads were pelleted, washed three times in NNT wash buffer (100 mM NaCl, 1% Nonidet P-40, and 50 mM Tris, pH 8), and suspended in 40 µl of electrophoresis sample buffer. For immunoblotting, proteins were resolved on SDS-PAGE and transferred electrophoretically to a nitrocellulose membrane (Bio-Rad). The membrane was blocked by incubation for at least 1 h in PBS containing 5% nonfat dried milk and 0.1% Tween 20 and then rocked overnight with the primary antibody in PBS containing 3% dried milk at 4 °C. The membrane was then washed and incubated with a secondary antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories) for 2 h. After washing, the secondary antibody was detected by chemiluminescence, according to the manufacturer's instructions (PerkinElmer Life Sciences). The following antibodies were used in this study: anti-CBP (CBP (C-1) monoclonal antibody and (A-22) polyclonal antiserum, Santa Cruz Biotechnology (Santa Cruz, CA), anti-E2A (yae monoclonal antibody, Santa Cruz Biotechnology), anti-PCAF (a polyclonal antiserum kindly provided by Dr. Xiang-Jiao Yang, McGill University, Montreal, Canada), and anti-GAL4 (polyclonal antiserum, Santa Cruz Biotechnology).

Transfections and Luciferase Assays—RCH ACV cells were propagated in RPMI medium supplemented with 10% fetal bovine serum (FBS). For transient transfections, 1.5 x 107 cells in 400 µl of complete medium containing 20 µg of DNA (including 8 µg of expression plasmid (based on pCMV1), 8 µg of reporter plasmid (p5xGAL4 LUC), and 4 µg of a plasmid conferring constitutive expression of {beta}-galactosidase) were placed in an electroporation cuvette (gap 0.4 cm) and subjected to an electrical pulse (260 V, 1050 microfarads) using a Gene Pulser II apparatus (Bio-Rad). Immediately thereafter, the cells were diluted into 5 ml of complete medium in 60-mm Petri dishes. Luciferase and {beta}-galactosidase assays were carried out 18–24 h later using chemiluminescence reagents (Tropix Inc., Bedford, MA) according to the manufacturer's instructions. Each data point represents at least four transfections. The luciferase values were normalized to the {beta}-galactosidase or Renilla luciferase values, and the mean and standard deviation were calculated. For the mammalian two-hybrid experiments, 104 HeLa cells were plated into each well of a 12-well plate in Dulbecco's modified Eagle's medium containing 10% FBS. The next day, the cells were transfected with mammalian expression and reporter plasmids (3.5 µg of pCMV-GAL4-CBP 586–673, 0.5 µg of pCMV-2xVP16-E2A, 2 µg of p5xGAL4 LUC, and 0.3 µg of PRL-CVM (a Renilla luciferase-expressing plasmid kindly provided by Dr. Chris Mueller)) using a liposome-based technique described previously (23). The cells were lysed 40 h later in Passive Lysis Buffer (Promega), and a portion of the lysate was analyzed using Dual-Luciferase Assay reagents (Promega) and an LB96V MicroLumat Plus luminometer (EG & G Berthold Ltd., Bundoora, Australia).

Retroviral Transductions and Immortalization Assays—Bone marrow was harvested on day 1 of the experiment from the femurs and tibia of five CD-1 mice at 12–14 weeks of age and placed in prestimulation mix (IMDM medium containing 15% fetal bovine serum (FBS), 10 ng/ml IL-3, 10 ng/ml IL-6, and 75 ng/ml murine stem cell factor; all recombinant cytokines were purchased from Peprotech Inc., Rocky Hill, NJ). Retroviral packaging cells (293T) were co-transfected with MSCVneo (retroviral backbone) and MCV-ecopac (ecotropic packaging) plasmids by the calcium phosphate method in 100-mm Petri dishes. On day 2, the bone marrow cells were washed in IMDM containing 2% fetal calf serum and re-suspended in prestimulation mix. The packaging cells were exposed to 15 Gy of ionizing radiation and refed. The next day, the bone marrow cells were washed and split into the Petri dishes containing the packaging cells in fresh prestimulation mix containing Polybrene (8 µg/ml). On day 4, the bone marrow cells were washed again and re-plated on the packaging cells in fresh prestimulation mix with Polybrene. The next day, the bone marrow cells were washed and transferred to fresh, non-coated Petri dishes in fresh medium. On day 6, the cells were washed, re-suspended in IMDM containing 10% FBS, 10 ng/ml granulocyte macrophage-colony stimulating factor, and 100 µM 2-mercaptoethanol, and monitored for growth (this is designated as "day 1" in the growth curves). The serial replating assay was performed essentially as described previously (24). Briefly, 104 retrovirally transduced cells were plated on day 1 in IMDM supplemented with 15% FBS, 10 ng/ml IL-3, 10 ng/ml IL-6, 10 ng/ml granulocyte macrophage-colony stimulating factor, 200 µg/ml transferrin (Sigma), and 1% methylcellulose (Stem Cell Technologies, Vancouver, Canada). Colonies containing at least 50 cells were counted after 6 or 7 days. The cells were then re-suspended, washed twice in 2x IMDM, counted, and 104 cells were re-plated in methylcellulose-containing medium for the next round in the experiment. The medium contained G418 (1.2 mg/ml) in the first plating only. Cell cycle analysis by flow cytometry was carried out as described previously (25).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Oncogenic Portion of E2A Proteins Interacts with CBP— Motivated primarily by the consideration that interaction with transcriptional co-activators could play a role in leukemia induction, we set out to characterize the interaction between E2A-PBX1 and CBP/p300. To map CBP-interactive elements, we carried out pull-down experiments using portions of E2A-PBX1b expressed and purified as fusion proteins with glutathione S-transferase. These were bound in equivalent quantities to glutathione-Sepharose beads and then mixed with nuclear extract prepared from the cell line RCH ACV. These cells were established from a case of ALL and are associated with t(1;19) and expression of endogenous E2A-PBX1. Proteins retained on the beads after washing were analyzed by SDS-PAGE followed by immunoblotting with an anti-CBP monoclonal antibody.

The results indicate that the oncogenic portion of E2A (i.e. E2A 1–483), which is invariant between E12 and E47, but not full-length PBX1a, is capable of interacting with CBP under the conditions of this experiment (Fig. 2B). Therefore, additional GST fusion proteins were designed to map CBP-interactive elements within the E2A portion of E2A-PBX1. E2A 1–273, which encompasses AD1 but excludes AD2, also retained considerable CBP, although substantially less than intact E2A 1–483; 10-fold dilution of the eluate from the E2A 1–483 pull-down indicated that E2A 1–273 retained roughly 20% of the CBP retained by the larger polypeptide (Fig. 2C). Deletion of the N-terminal 28 amino acids from E2A 1–273 (i.e. 29–273) completely abrogated the CBP interaction. Use of E2A 1–99 (i.e. isolated AD1) reduced the CBP interaction relative to E2A 1–273 but did not abrogate it. Therefore, the N-terminal 28 amino acids of AD1 are required for the CBP interaction in the context of E2A 1–273. E2A segment 274–483 (containing AD2 but not AD1) retained CBP but at barely detectable levels. E2A 1–483 retains considerably more CBP than is retained by the sum of the CBP retained by E2A 1–273 and E2A 274–483, suggesting a cooperative interaction between elements contained within these two protein segments.



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  		FIG. 2.
Mapping CBP-interactive domains on E2A-PBX1. A, portions of E2A expressed as GST fusion proteins in this study. B, pull-down/immunoblot assays. GST fusion proteins were bound to glutathione-Sepharose beads. These were mixed with nuclear extract from the t(1;19)-associated cell line RCH ACV, and proteins retained on the beads after washing were analyzed by immunoblotting with anti-CBP. An equal portion of the beads was run on a separate, Coomassie-stained gel to ensure equivalent loading of the ligands. In this experiment, twice as many of the PBX1a-bound beads were used in the pull-down, relative to GST and GST-E2A, to compensate for the relative under-loading of the beads with PBX1a protein evident in the Coomassie stain. C, mapping CBP-interactive elements within E2A. The lane labeled "1–483 (1:10)" contains 0.1x the quantity of eluate loaded into the adjacent "1–483" lane. Note that E2A 1–273 retains perhaps 0.2-fold the CBP retained by E2A 1–483. A barely visible band is present in the lane labeled "274–483". The Coomassie-stained gel indicates equivalent loading. D, pull-down assays with GST-E2A 1–483 proteins followed by immunoblotting with anti-CBP or anti-PCAF. The relatively faint CBP band visible in the {Delta}16–23 lane on overexposure of the blot was reproducible in several experiments. The PCAF band is indicated by an asterisk at the left of the image. Most of the extraneous bands visible in the anti-PCAF blot are due to cross-reactivity of the anti-serum with (presumably the GST portion of) the GST fusion proteins.

 
Interaction with CBP and PCAF Requires Conserved Helical Domains within AD1 and AD2 of E2A Proteins—Amino acids 11–28 of E2A are phylogenetically conserved and predicted to adopt a {alpha}-helical conformation (26). This helical domain interacts with the SAGA transcriptional co-activator complex in yeast and is required in the transcriptional function of AD1. Our results suggested that the {alpha}-helical domain of AD1 would also be important for CBP recruitment in the context of E2A 1–483. Deletion of most of this domain ({Delta}16–23) in the context of E2A 1–483 drastically reduced, but did not abrogate, CBP recruitment in the pull-down assay (Fig. 2D). Therefore, we suspected that elements within AD2 might be compensating to some degree for the loss of the AD1 helical domain from E2A 1–483. Although no sequence conservation exists between the two E2A activation domains, AD2, like AD1, contains a short, conserved domain (residues 397–406) that is predicted to form a {alpha}-helix and required in the transcriptional function of AD2 (8). Deletion of the AD2 helical domain ({Delta}397–405) reduced CBP recruitment slightly relative to wild-type E2A 1–483, whereas simultaneous deletion of both helical domains ({Delta}16–23/{Delta}397–405) completely abrogated the interaction of E2A 1–483 with CBP (Fig. 2D). Re-probing the same blot with a polyclonal antiserum against PCAF indicated recruitment of this protein by E2A 1–483, {Delta}16–23, and {Delta}397–405 in quantities that paralleled those of CBP. Although the quality of this blot is relatively poor, the result was reproducible in at least three experiments. Thus, in these experiments, helical domains within both AD1 and AD2 contribute to the recruitment of CBP and PCAF from lymphoid nuclear extract, although the AD1 helical domain makes a considerably greater contribution.

E2A Interacts Directly with the KIX Domain of CBP—CBP is a large protein with multiple protein-protein interaction domains. To map E2A-interactive elements on CBP, GST fusion proteins were generated with contiguous or overlapping segments of murine CBP that collectively span the entire protein. These were used in pull-down experiments with RCH ACV nuclear extract; proteins retained on the beads were immunoblotted with an anti-E2A monoclonal antibody. Only one segment of CBP, extending from residue 461 to 915, retained E2A proteins beyond background level (Fig. 3B). This region contains the KIX domain (residues 586–666, also called the "CREB-binding domain"), a well characterized protein-protein interaction domain known to mediate interactions with multiple transcriptional activation domains, including the prototypic interaction with CREB (Fig. 3A) (27, 28).



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  		FIG. 3.
Mapping an E2A-interactive domain on CBP. A, portions of mouse CBP expressed as GST fusion proteins for pull-down experiments. B, GST-CBP pull-down assays followed by anti-E2A immunoblotting. E2A-PBX1b runs faster in the 461–915 pull-down lanes due to co-migration of the (invisible) GST fusion protein.

 
We carried out additional pull-down assays to confirm interaction of E2A with the KIX domain of CBP. Deletion of the crucial {alpha}3 helix (residues 646–661) within the KIX domain in the context of the relatively large segment of CBP (residues 461–915) capable of interacting with E2A completely abrogated the interaction (Fig. 3B). Furthermore, a small segment of CBP (amino acids 586–673) incorporating the KIX domain with only seven additional, C-terminal amino acids was fully capable of interacting with E2A proteins. In some experiments, CBP 586–673 showed an apparent preference for interacting with wild-type E2A proteins relative to the fusion proteins (i.e. compare the intensities of the bands in the left and right panels of Fig. 3B). Under the conditions of these experiments, the KIX domain of CBP is required and sufficient to mediate the interaction with E2A proteins.

CBP can interact with numerous proteins besides PCAF and E2A, raising the question as to whether the interaction with E2A is direct. Therefore, we attempted to reconstitute the interaction between E2A and CBP using purified, recombinant proteins. GST-E2A 1–483 was mixed in solution with GST-CBP 1–720 (a segment that includes the KIX domain), and immunoprecipitation was carried out using a polyclonal anti-serum directed against the N terminus of CBP. Subsequent immunoblotting of the precipitated proteins with an anti-E2A monoclonal antibody indicated co-precipitation of GST-E2A 1–483 (Fig. 4B). Under the conditions of these experiments, the {alpha}-helical domains within AD1 and AD2 of E2A appeared redundant for the CBP interaction, because deletion of either one had no observable effect upon co-immunoprecipitation of CBP. However, simultaneous deletion of both of these regions completely abrogated the CBP interaction. Co-immunoprecipitation of the E2A protein was not observed with GST-CBP 1–460, which lacks the KIX domain, or with an antiserum against the hemagglutinin epitope, which served as a negative control. Coomassie staining of a duplicate gel indicated equivalent loading of the various GST fusion proteins (Fig. 4C). These results indicate that E2A can interact directly with CBP without the participation of additional proteins. Furthermore, they suggest that both of the {alpha}-helical domains contained within AD1 and AD2 make direct contact with the KIX domain or elements in its immediate vicinity (i.e. within amino acid positions 461–720 of CBP).



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  		FIG. 4.
Co-immunoprecipitation of CBP and E2A using purified, recombinant proteins. A, portions of CBP expressed as GST fusion proteins in this experiment. B, protein complexes were immunoprecipitated with an anti-CBP antiserum (or anti-HA as a negative control) and then immunoblotted with anti-E2A. All of the indicated portions E2A and CBP were used as GST fusion proteins. C, Coomassie-stained gel prepared from an equal portion of the protein mixes used in the co-immunoprecipitation experiment showing equivalent loading of the various GST fusion proteins. GST-CBP 1–460 is difficult to see as it co-migrates with GST-E2A 1–483.

 
These results using purified proteins differ somewhat from those involving pull-down assays from crude nuclear extract. In the pull-down assays, the AD2 helical domain shows much less activity than the AD1 domain in recruiting CBP (Fig. 2), whereas in the context of purified proteins the two helical domains appear equivalent and redundant (Fig. 4). This apparent discordance may be due to our use of relatively high, presumably non-limiting concentrations of pure proteins in the co-immunoprecipitation experiment, or the absence in this experiment of additional proteins or post-translational modifications that may have modulated the interaction in the context of pull-down assays from crude nuclear extract.

Correlation of CBP Interaction with Transcriptional Activity of E2A Proteins—In view of the potential relevance of these findings to transcriptional regulation by E2A proteins and E2A-PBX1-associated leukemia, we wished to correlate our pull-down results with transcriptional function in B-lineage, ALL-derived cells. Therefore, we constructed plasmids that conferred expression in mammalian cells of portions of E2A fused to a heterologous DNA-binding domain derived from GAL4. These were then co-transfected into RCH ACV cells with a reporter plasmid that confers luciferase expression regulated by tandem GAL4 binding sites. As expected, transfection of GAL4-E2A 1–483 was associated with relatively abundant luciferase activity (Fig. 5A). GAL4-E2A 1–273, which contains AD1, trans-activated the reporter minimally; although luciferase activity was consistently above background, it was only 1.8% of that associated with E2A 1–483. E2A 274–483, which contains AD2 but is still substantially less active than E2A 1–483, was considerably more active than E2A 1–273 (14% versus 1.8%). In contrast to earlier reports, these findings suggest that AD2 can be transcriptionally active in lymphoid cells and appears, in this context, to be more active than AD1 (9). Furthermore, in demonstrating that the covalent linkage of polypeptide elements contained within segments 1–273 and 274–483 of E2A produces a greater than additive effect on both co-activator recruitment (Fig. 2B) and transcriptional activation (Fig. 5A), the results suggest functional cooperativity between elements within these portions of the protein.



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  		FIG. 5.
Contributions of E2A domains in transcriptional activation. A, trans-activation by GAL4-E2A fusion proteins. RCH ACV cells were co-transfected with portions of E2A fused to the DNA-binding domain of GAL4 along with a reporter plasmid that confers luciferase expression regulated by tandem GAL4 binding sites. Luciferase values were normalized to {beta}-galactosidase activity conferred by a constitutively expressing, co-transfected plasmid. Each data point represents the mean of four transfections. Numerical values and standard deviations (in parentheses) are listed to the right of the graph. B, expression of GAL4-E2A fusion proteins. The anti-GAL4 immunoblot used lysates from HeLa cells transiently transfected with the indicated GAL4 fusion proteins along with the {beta}-galactosidase plasmid; loading of the lanes was normalized according to the {beta}-galactosidase activity in the lysates. C, nuclear localization of GAL4-E2A fusion proteins. The in situ immunofluorescence studies used transiently transfected HeLa cells grown on coverslips and immunostained with the same anti-GAL4 antiserum used in the immunoblot.

 
Targeted deletion of the helical domain from AD1 ({Delta}16–23) in the context of E2A 1–483 reduced relative luciferase activity to 68% of that seen with wild-type E2A 1–483, whereas deletion of the AD2 helical domain ({Delta}397–405) had a more drastic effect, reducing activity to 14% (Fig. 5A). Simultaneous deletion of both domains resulted in an apparent reduction of transcriptional activity to 11% of that observed with full-length E2A 1–483. Expression levels of the various GAL4 fusion proteins were comparable, as verified by immunoblotting the cell lysates used in the reporter gene assays with an anti-GAL4 antiserum (Fig. 5B). Furthermore, immunofluorescence microscopy using the same antiserum indicated nuclear localization of each of the GAL4-E2A fusion proteins (Fig. 5C).

We established a mammalian two-hybrid assay to evaluate the E2A-KIX interaction in the context of intact cells. Co-transfection of HeLa cells with 2X-VP16-E2A 1–483 (i.e. E2A 1–483 fused to two, N-terminal VP16 transcriptional activation domains) and GAL4-KIX (CBP residues 586–673) resulted in potent activation of the GAL4 reporter in a manner that was dependent on elements within both E2A and KIX (Fig. 6). Deletion of the AD1 helical domain ({Delta}16–23) reduced luciferase activity to 31% of that produced by E2A 1–483. Deletion of the AD2 helical domain ({Delta}397–405) had a somewhat greater effect, reducing luciferase activity to 16%. This reduction is comparable to the effect of the same deletion on autonomous transactivation by E2A (14%, Fig. 5). These findings indicate that the interaction between E2A and the KIX domain of CBP can occur within the intact nuclei of epithelial (HeLa) cells and are consistent with involvement of the helical domains of AD1 and AD2.



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  		FIG. 6.
Mammalian two-hybrid assay. HeLa cells were transiently co-transfected with portions of E2A fused to two VP16 activation domains, the KIX domain of CBP fused to the DNA binding domain of GAL4 (GAL4-CBP 586–673), and a GAL4-responsive reporter plasmid. Luciferase values were normalized to Renilla luciferase activity conferred by a co-transfected plasmid. "2xVP16" indicates a negative control in which the VP16-E2A fusion protein was replaced by the VP16 activation domains alone; "1–483 + GAL4-KIX {Delta}646–662" indicates a negative control in which GAL4-KIX is replaced by the GAL4 domain fused to an E2A binding-defective deletion mutant of the KIX domain.

 
Interaction between E2A-PBX1b and CBP Correlates with Proliferation in Primary Hematopoietic Cells—Retroviral transduction of primary mouse bone marrow with E2A-PBX1 induces the outgrowth in tissue culture of immortal, highly proliferative, cytokine-dependent cells with immunophenotypic features of primitive myeloid progenitors (29). We used this assay to investigate possible involvement of the CBP/KIX interaction in oncogenesis by E2A-PBX1. The deletions targeted to the AD1 or AD2 helical domains were incorporated into E2A-PBX1b and cloned into the MSCVneo retroviral backbone vector. Anti-E2A immunoblotting of cell lysates from retrovirally transduced, drug-selected NIH 3T3 cells showed equivalent expression and nuclear localization of the recombinant E2A-PBX1b proteins (Fig. 7). Retrovirally transduced mouse bone marrow cells were plated in semisolid, methylcellulose-containing medium, and the resultant colonies were counted after 6 or 7 days. The cells were then temporarily re-suspended in liquid medium, counted, and re-plated in semisolid medium for the next round in the experiment.



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  		FIG. 7.
Expression of E2A-PBX1b and deletion mutants in retrovirally transduced, drug selected NIH 3T3 fibroblasts. Immunoblotting (A) and immunofluorescence (B) microscopy with anti-E2A.

 
After the first round of growth, conducted in the presence of G418, a greater number of drug-resistant colonies was produced by cells infected with the empty murine stem cell virus vector than with any of the E2A-PBX1-expressing constructs (Fig. 8A). However, these cells showed a marked reduction in colony-forming ability as of the fourth round of replating. This contrasted with the behavior of cells transduced with E2A-PBX1b. In these cells, clonagenicity increased markedly after the first plating and was maintained at a high level through fully six rounds. Therefore, as demonstrated previously, retrovirally transduced E2A-PBX1 is capable of extending the in vitro self-renewal capacity of primary hematopoietic progenitors (30). Deletion of the AD1 helical domain ({Delta}11–28), alone or in combination with the AD2 helical domain ({Delta}16–23/397–405) resulted in no consistent, substantial reductions in clonagenicity relative to the full-length oncoprotein. Therefore, this experiment failed to clearly demonstrate a role for CBP in the prolongation of hematopoietic progenitor self-renewal associated with enforced expression of E2A-PBX1.



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  		FIG. 8.
Immortalization of primary hematopoietic progenitors by E2A-PBX1b. A, serial replating assay using retrovirally infected bone marrow cells. Bone marrow was harvested from mice and transduced with retroviruses conferring expression of the various recombinant proteins. Cells were plated in semisolid medium containing methylcellulose and cytokines (IL-3, IL-6, and granulocyte macrophage-colony stimulating factor). Every 7 days, the colonies were counted and re-suspended in liquid medium, and 104 cells were re-plated for the next round in the experiment. The asterisk indicates a missing data point. B, retrovirally transduced bone marrow cells were propagated in liquid medium containing granulocyte macrophage-colony stimulating factor and non-adherent cells that excluded trypan blue were counted every 7 days.

 
However, we observed that the AD1 deletions appeared to correlate with production of smaller colonies by retrovirally transduced progenitors, suggesting reduced rates of proliferation among clonagenic cells and their progeny (not shown). Cultivation of the retrovirally transduced cells in liquid medium without drug selection, carried out in parallel with the replating assay, demonstrated a reduced rate of cell accumulation associated with deletion of the AD1 ({Delta}11–28), but not the AD2 ({Delta}397–405), helical domain (Fig. 8B). Deletion of the AD2 helical domain had no measurable effect in the context of an intact AD1 helical domain, whereas combined deletion involving both helical domains ({Delta}16–23/397–405) further reduced cell proliferation relative to deletion of the AD1 domain alone. Cell numbers in the {Delta}16–23/397–405 flask showed a downward trend when the experiment was terminated. The replating and growth curve experiments were carried out twice with similar results (see Supplementary Material). Differential cell proliferation was confirmed by flow cytometric cell cycle analysis performed on day 25 of the experiment. In cells transduced with full-length E2A-PBX1b or the AD2-deleted protein, 29 and 35% of cells, respectively, were in S-phase (Fig. 9). In contrast, the S-phase fraction was reduced to 13% in association with the AD1 deletion and 9% in association with combined deletions in both AD1 and AD2. There were commensurate increases in the G1 fraction. Consistent with earlier studies, limited immunophenotyping of the immortalized cell populations (i.e. those transduced with full-length E2A-PBX1b or the {Delta}11–28 or {Delta}397–405 deletion mutants) by flow cytometry showed expression of CD11b, supporting myeloid differentiation (data not shown). These results in primary hematopoietic cells suggest a particular role for the AD1 helical domain and, by inference, the CBP/p300 interaction in mediating the induction of cellular proliferation by E2A-PBX1.



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  		FIG. 9.
Cell cycle progression in hematopoietic progenitors. Note the lower proportion of cells in S-phase associated with deletions involving the AD1 helical domain ({Delta}11–28 or {Delta}16–23/397–405).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The interaction between E2A proteins and CBP/p300 was evident in earlier studies. Co-transfected p300 enhanced E47-dependent trans-activation mediated by tandem E-box elements in reporter plasmids in NIH 3T3 fibroblasts (31). In an electrophoretic mobility shift assay using nuclear extract from (lymphoma-derived) BJAB cells, an anti-p300 antibody supershifted a low mobility complex formed by E47 homodimers on an E-box-containing DNA probe. Co-transfected p300 potentiated trans-activation by the E2A activation domains expressed as heterologous fusion proteins with the GAL4 DNA-binding domain in (pancreatic {beta}-cell-derived) HIT cells (32). Bacterially expressed GST-E12 could interact with in vitro translated p300 in a pull-down assay (33). In addition, p300, CBP, and PCAF were co-immunoprecipitated with E2A proteins from lymphoid nuclear extracts; the same co-activators co-eluted with E2A proteins on fractionation of nuclear extracts by size exclusion chromatography (14). Our current work extends these earlier results by demonstrating that the E2A-CBP interaction is direct, mapping interactive elements at higher resolution and in the context of E2A-PBX1, demonstrating involvement of the KIX domain of CBP, evaluating the functional roles of the AD1 and AD2 helical domains in the context of large portions of E2A or E2A-PBX1, and demonstrating a correlation between the CBP/p300 interaction and induction of proliferation by E2A-PBX1 in primary hematopoietic progenitors.

In view of the earlier suggestion of preferential activity of AD1 in most cell lineages, we were surprised to observe greater transcriptional activity related to AD2 in ALL-derived RCH ACV cells (9). Significantly, we obtained similar results in HeLa cells, in which Aronheim et al. (9) observed far greater activity of AD1 relative to AD2 (data not shown). This apparent discrepancy may be related to differences in the E2A segments used; Aronheim et al. used GAL4 fusion proteins containing amino acids 3–153 (AD1) or 369–485 (AD2) of E2A, whereas we used 1–273 or 274–483, respectively. Therefore, the relatively low level of trans-activation by AD2 observed by Aronheim and colleagues may have been due to exclusion of key polypeptide elements from their GAL4-AD2 fusion protein.

The KIX domain includes 81 amino acids, from position 586 to 666 of mouse CBP. Its three-dimensional structure in a complex with a synthetic peptide derived from the CREB activation domain has been determined by NMR spectroscopy (34). KIX is a compact, globular domain incorporating three mutually interacting {alpha}-helices. Side chains from amino acids that mostly reside in the third {alpha}-helix (called {alpha}3) form a shallow hydrophobic groove on the surface of the KIX domain that accommodates phosphorylated CREB. Besides CREB, the KIX domain mediates interactions with transcriptional activation domains in numerous transcription factors, including c-Myb, c-Jun, MLL, p53, and MyoD (35–39). The motif {Phi}XX{Phi}{Phi} (where {Phi} represents a neutral, non-polar amino acid), frequently LXXLL, is often present in KIX-interactive regions (40). Direct interactions between the KIX domain and the CREB and c-Myb activation domains are particularly well characterized (35, 37). Alignment of the KIX-interactive region of c-Myb with the N-terminal {alpha}-helical domain of E2A and its metazoan orthologs shows conservation of the LXXLL motif and additional amino acids (Fig. 1). Because the c-Myb activation domain interacts directly with the KIX domain of CBP, the sequence conservation with E2A corroborates our demonstration of a direct interaction between E2A and KIX.

The LXXLL motif is present within the CBP/p300-interactive domain of the p160 family of transcriptional co-activators that function in trans-activation by nuclear receptors (41). The motif is also philogenetically conserved in vertebrate bHLH proteins, where it occurs as a direct N-terminal extension of the "LDFS" domain that contributes to the interaction with the SAGA complex in yeast (42). Interestingly, while the LDFS domain is conserved in metazoans and yeast, the LXXLL extension appears to be restricted to the former. Because there is no yeast ortholog of CBP/p300, the correlation between the existence of CBP/p300 orthologs and the LXXLL motif in metazoan bHLH proteins is consistent with a functional interaction.

Our studies suggest that, like AD1, AD2 may also make direct contact with the KIX domain of CBP. The helical domain of AD2 shows no sequence conservation with that of AD1; no LXXLL domain is present in AD2, although a sequence conforming to the {Phi}XX{Phi}{Phi} motif (415-MHTLL) is present. Further studies are required to investigate possible cooperative binding of AD1 and AD2 to the KIX domain and map the molecular surfaces involved.

Kasper and colleagues (43) recently demonstrated a physiological requirement for the KIX domain in whole animals. Using homologous recombination, mutations were introduced into the germ line of mice so as to substitute key amino acids known to mediate interactions between KIX and either CREB or c-Myb. Whereas mice bearing homologous mutations of the CBP KIX domain (CBPKIX/KIX) were essentially normal, similar substitutions in p300 resulted in multilineage hematopoietic defects. Interestingly, several of these abnormalities, including B-lymphopenia and abnormal thymic development, were also evident in E2A–/– animals (1, 2). CBP and p300 are very highly conserved within the KIX domain, so the results we report here for CBP are probably applicable to p300 (34). In light of our results demonstrating involvement of the KIX domain in mediating the E2A-CBP/p300 interaction, the similarity between the KIX–/– and E2A–/– phenotypes provides circumstantial support for a requirement for the E2A-KIX interaction in normal hematopoiesis.

The relative contributions made by AD1 and AD2 to transcriptional induction seem discordant with their roles in CBP recruitment and cellular proliferation. Whereas the AD2 helical domain appears important for transcriptional induction and CBP recruitment by E2A in the context of intact cells, it appears dispensable, at least in the presence of intact AD1, in the induction of cell proliferation by E2A-PBX1b. Our results provide no explanation for this discrepancy, which could reflect differential participation by additional proteins or domains in transcriptional versus cell cycle regulation by E2A proteins.

Our primary objective in undertaking this work was the elucidation of protein-protein interactions that could contribute to leukemogenesis by E2A-PBX1. To this end, we have demonstrated that targeted deletions of the conserved helical domains within AD1 and AD2 impair both the direct interaction with CBP and the induction of proliferation in primary bone marrow cells expressing E2A-PBX1b. That these deletions do not appear to substantially affect the prolongation of ex vivo replating activity mediated by E2A-PBX1 suggests either that interactions with additional factors are involved, or that the engineered deletions within AD1 and AD2 do not fully abrogate the CBP/p300 interaction in the context of this experiment. Nonetheless, our results argue that the contribution of the E2A portion to oncogenesis by E2A-PBX1 may be understood at least in part in terms of its ability to mediate a direct interaction with the KIX domain of CBP/p300.


   FOOTNOTES
 
* This work was supported in part by research grants (to D. P. L.) from the Canadian Institutes of Health Research and the National Cancer Institute of Canada. 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

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Fig. S1. Back

§ Recipients of Leukemia Research Fund of Canada Studentships. Back

|| To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, Richardson Laboratory, Queen's University, Kingston, Ontario K7L 3N6, Canada. Tel.: 613-533-3209; Fax: 613-533-6830; E-mail: lebrun{at}cliff.path.queensu.ca.

1 The abbreviations used are: bHLH, basic helix-loop-helix; AD1, -2, activation domains 1 and 2; PCAF, p300/CREB-binding protein-associated factor; ALL, acute lymphoblastic leukemia; GST, glutathione S-transferase; PBS, phosphate-buffered saline; FBS, fetal bovine serum; IMDM, Iscove's modified Dulbecco's medium; IL-3, interleukin-3. Back



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