AML1 (CBFα2) Cooperates with B Cell-specific Activating Protein (BSAP/PAX5) in Activation of the B Cell-specific BLK Gene Promoter*

AML1 plays a critical role during hematopoiesis and chromosomal translocations involving AML1 are commonly associated with different forms of leukemia, including pre-B acute lymphoblastic leukemia. To understand the function of AML1 during B cell differentiation, we analyzed regulatory regions of B cell-specific genes for potential AML1-binding sites and have identified a putative AML1-binding site in the promoter of the B cell-specific tyrosine kinase gene, blk. Gel mobility shift assays and transient transfection assays demonstrate that AML1 binds specifically to this site in the blk promoter and this binding site is important for blk promoter activity. Furthermore, in vitro binding analysis revealed that the AML1 runt DNA-binding domain physically interacts with the paired DNA-binding domain of BSAP, a B cell-specific transcription factor. BSAP has been shown previously to be important for B cell-specific regulation of the blkgene. Physical interaction of AML1 with BSAP correlates with functional cooperativity in transfection studies where AML1 and BSAP synergistically activate blk promoter transcription by more than 50-fold. These results demonstrate physical and functional interactions between AML1 and BSAP and suggest that AML1 is an important factor for regulating a critical B cell-specific gene,blk.

AML1 plays a critical role during hematopoiesis and chromosomal translocations involving AML1 are commonly associated with different forms of leukemia, including pre-B acute lymphoblastic leukemia. To understand the function of AML1 during B cell differentiation, we analyzed regulatory regions of B cell-specific genes for potential AML1-binding sites and have identified a putative AML1-binding site in the promoter of the B cell-specific tyrosine kinase gene, blk. Gel mobility shift assays and transient transfection assays demonstrate that AML1 binds specifically to this site in the blk promoter and this binding site is important for blk promoter activity. Furthermore, in vitro binding analysis revealed that the AML1 runt DNA-binding domain physically interacts with the paired DNA-binding domain of BSAP, a B cell-specific transcription factor. BSAP has been shown previously to be important for B cell-specific regulation of the blk gene. Physical interaction of AML1 with BSAP correlates with functional cooperativity in transfection studies where AML1 and BSAP synergistically activate blk promoter transcription by more than 50-fold. These results demonstrate physical and functional interactions between AML1 and BSAP and suggest that AML1 is an important factor for regulating a critical B cell-specific gene, blk.
AML1 is a member of the PEBP2/CBF family of transcription factors (1,2). These factors consist of heterodimers between the DNA binding ␣ subunit and the ␤ subunit, CBF␤, which does not bind DNA directly but enhances the binding of the ␣ subunit (3). Multiple ␣ subunit genes, including CBF␣1 (AML3), AML1 (CBF␣2), and CBF␣3 (AML2), as well as alternatively spliced isoforms of the ␣ and ␤ subunits have been detected (4,5). All of the CBF␣ proteins have a DNA-binding domain (the runt domain), which is similar to the Drosophila pair-rule gene, runt (6). AML1 and other PEBP2/CBF␣ proteins are transcription factors whose recognition sequence is required for tissue specific expression of several hematopoietic genes including M-CSF receptor, GM-CSF, IL-3, T cell receptors, immunoglobulin heavy chain, defensin NP-3, and myeloperoxidase (7)(8)(9)(10)(11)(12)(13)(14)(15). AML1 knockout mice indicate, furthermore, that AML1 is indeed a critical regulator of early hematopoiesis (16,17). The AML1 gene is frequently associated with chromosomal translocations in different forms of leukemia, including t(8;21), t (3; 21), in addition to t(12;21) (1, 18 -20). The ␤ subunit of PEBP2/ CBF is also involved in a chromosomal inversion, inv (16), associated with FAB M4eo AML (21). Therefore, each of the two chains of the PEBP2/CBF heterodimer is directly implicated in the pathogenesis of leukemia. T(12;21) is a common chromosomal abnormality in childhood pre-B acute lymphoblastic leukemia (20,22). This translocation generates two chimeric genes, TEL/AML1 and AML1/TEL. Only the TEL/AML1 chimeric gene product is consistently detected in cells with t(12; 21) (23). The TEL/AML1 chimeric gene expresses a fusion protein that contains the 333 NH 2 -terminal amino acids of the TEL protein encoding the Pointed dimerization domain but lacking the Ets DNA-binding domain, and almost the entire AML1 protein, including the AML1 DNA-binding domain. Therefore, the TEL/AML1 fusion protein can interact with AML1 DNA-binding sites and possibly interfere with AML1 function during B cell differentiation. This indicates that AML1 may play an important role in controlling gene expression during normal B cell differentiation.
To study the function of AML1 in B-cell differentiation, we analyzed regulatory regions of B cell-specific genes for the presence of AML1-binding sites. We observed a consensus AML1-binding site in the promoter of the blk gene. BLK, a Src family member, encodes a B cell-specific, 55-kDa protein tyrosine kinase p55 Blk (24). BLK is associated with the B cell antigen receptor and is involved in signal transduction events (25). The B cell antigen receptor complex is formed by membrane IgM noncovalently associated with heterodimers of B29 and mb-1 (26,27). Antigen cross-linking to the B cell antigen receptor leads to a rapid and transient increase in tyrosine kinase activity resulting in activation of several signal transduction pathways, including the mitogen-activated protein kinase pathway. The antigen receptor does not itself contain an intrinsic tyrosine kinase domain; therefore, tyrosine kinase activation is due to signal transduction mediated through interaction of two B cell antigen receptor-associated proteins, Ig␣ (mb-1) and Ig␤ (B29) with the cytoplasmic Src-like kinases, BLK, LYN, and FYN (28). This rapid and transient activation of tyrosine kinases triggers a cascade of downstream events, leading to changes in gene expression and either cell proliferation and differentiation or apoptosis. BLK is exclusively expressed in B cells at the pro-B, pre-B, and mature B cell stages, but not in plasma cells or non-B cells (29). The function of BLK in B cell antigen receptor signaling is not clear, although several studies have implicated the BLK kinase in antigen receptor cross-linking mediated growth arrest and apoptosis (30,31). However, transgenic mice expressing a constitutively activated BLK mutant in the B cell lineage develop B lymphoid tumors (32). Therefore, control of blk gene expression could be directly related to B cell development and neoplasia. B cell specificity of blk gene expression is primarily regulated at the transcriptional level and a 320-bp 1 promoter region of the murine blk promoter appears to contain most of the B cell-specific regulatory elements (29,33). Relatively little is known about the transcriptional regulation of blk gene expression. It has been reported that B cell-specific activator protein (BSAP/Pax5) binds to the blk promoter, and recently BSAP as well as NF-B were shown to transactivate the blk promoter (33,34). BSAP is a critical transcription factor for B cell development with a similar expression pattern as BLK (35,36). BSAP belongs to the paired box (Pax) gene family, which plays an important role during the development of the central nervous system and during B cell development (35)(36)(37). BSAP has been shown to act as both a positive and negative regulator of gene expression controlling the expression of several B cell-specific genes including CD19, blk, and various immunoglobulin genes (33, 38 -43). We now report that AML1 binds to a functionally important region of the blk promoter, directly interacts with the paired box DNA-binding domain of BSAP protein through its runt homology domain, and cooperates with BSAP in activating blk promoter transcription.

EXPERIMENTAL PROCEDURES
Electrophoretic Mobility Shift Assay (EMSA)-The probe for the EMSA was a double-stranded oligonucleotide with bp Ϫ47 to Ϫ28 of the blk upstream region (Fig. 1). A 32 P-labeled probe was prepared by T4 kinase phosphorylation in the presence of [␥-32 P]ATP (NEN Life Science Products Inc.). Nuclear protein for the EMSA was prepared from Ba/F3 cells according to the method published by Schreiber et al. (44). Approximately 0.5 ng of probe was incubated with 5 g of nuclear protein or 1 l of in vitro translated protein in 20 l of binding buffer containing 10 mM HEPES, pH 7.9, 30 mM KCl, 5 mM MgCl 2 , 1 mM dithiothreitol, 1 mM EDTA, 0.4 mM phenylmethylsulfonyl fluoride, 12% glycerol at 4°C for 20 min (45). All reactions contain 1 g of poly(dI-dC). Reactions were electrophoresed at 10 V/cm on a 6% polyacrylamide gel (bisacrylamide/acrylamide ratio ϭ 1:29) in 0.5 ϫ TBE (45 mM Tris borate, 1 mM EDTA) at 4°C. For supershift experiments, 1 l of polyclonal rabbit antiserum raised against a 17-amino acid NH 2 -terminal peptide from AML1 was added to the binding reaction mixture 10 min prior to addition of the probe (46).
Plasmid Construction-A murine blk promoter (bp Ϫ191 to ϩ136) DNA fragment was subcloned into the KpnI and XhoI sites of the pXP2 luciferase reporter gene construct to form pBlk-luc (47). pBlk-luc-(mAML1), which contains a mutation at the AML1 binding was generated using QuickChange Mutagenesis Kit (Stratagene). The AML1 site was changed from TGTGGT to TGCACT. AML1 and CBF␤ expression constructs, pCMV5-AML1 and pCMV5-CBF␤, were received from Dr. Scott Hiebert (46). BSAP cDNA was received from Dr. Steve Desiderio (33) and inserted into the NotI site of the pCI expression vector. The GST-BSAP full-length fusion protein was generated by inserting a blunt-ended NotI fragment of the BSAP cDNA into the SmaI site of pGEX-5X-3. GST-BSAP 1-312 and GST-BSAP 1-159 were generated by deleting a SmaI-XhoI fragment or a SacII-XhoI fragment from the carboxyl terminus of GST-BSAP, respectively. GST-BSAP 234 -391 was generated by inserting a PvuII-NotI fragment of BSAP into BamHI (blunt ended) and NotI sites of pGES-5X-3. Full-length AML1, AML1 1-208, and AML1 87-208 were as reported previously (48).
Cell Culture and Transfection-The human B cell line BJA-B, promonocytic cell line THP-1, and murine B cell line Ba/F3 were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine. THP-1 cell culture medium contained 2 ϫ 10 5 M 2-mercaptoethanol. Ba/F3 cell culture medium also contained 10% conditioned WEHI-3B medium as a source for interleukin-3. Maintenance and transfection of CV-1 cells were as described previously (48). BJA-B and Ba/F3 cells were transfected by electroporation in RPMI 1640 medium at 960 microfarads and 220 or 270 V, respectively. Cells were harvested 5 h after transfection. Rous sarcoma virus-human growth hormone expression construct was co-transfected with luciferase reporter constructs for normalizing transfection efficiency (45).
In Vitro Translation-Protein in vitro translation was performed with TnT T7-coupled reticulocyte lysate system according to the manufacturer's protocol (Promega). The TnT lysate contains approximately 150 g/l endogenous protein. Each in vitro translation reaction uses 25 l of TnT lysate in a 50 l of reaction.
GST Pull-down Experiments-The GST-pull down experiments were as described previously (49).

AML1 Interacts with a Sequence 3Ј of the BSAP Site of the
Murine blk Promoter-The TEL/AML1 fusion protein is associated with pre-B acute lymphoblastic leukemia. Since the TEL/AML1 protein has lost the Ets DNA-binding domain of TEL, but still contains the intact AML1 DNA-binding domain, this fusion protein could possibly generate leukemia by interfering with the function of normal AML1 or other PEBP2/CBF family members in the activation of critical B cell developmental genes. Therefore, we searched the regulatory regions of several B cell-specific genes for putative AML1-binding sites and found an AML1 binding consensus sequence "TGTGGT" in the B cell-specific blk promoter at bp Ϫ39 to Ϫ34, just downstream of the BSAP-binding site (Fig. 1). In addition to the putative AML1 site we also detected putative binding sites for several other transcription factors including three putative Ets-binding sites and a helix-loop-helix protein-binding site (Fig. 1). To test whether AML1 can interact with the blk promoter, oligonucleotides containing the AML1 consensus sequence from the blk promoter were used in EMSA experiments with nuclear extracts from the B cell line, Ba/F3, or with in vitro translated AML1. As shown in Fig. 2A, Ba/F3 nuclear extract formed a specific protein-DNA complex with the blk promoter bp Ϫ47 to Ϫ28 oligonucleotide. This protein-DNA complex was specifically competed by unlabeled self-oligonucleotide or an oligonucleotide encoding the Moloney murine leukemia virus enhancer PEBP2/CBF-binding site (50), but not by an unlabeled non-PEBP2/CBF binding oligonucleotide. These data suggested that this complex contains a PEBP2/CBF related protein. To further characterize this protein, antiserum raised against the NH 2 -terminal region of one of the PEBP2/ CBF family members, AML1 (46), was used in the EMSA analysis. Upon the addition of the anti-AML1 antiserum, the specific protein-DNA complex was drastically reduced and a supershifted band was detected indicating that the protein in Ba/F3 cell nuclear extracts interacting with the blk promoter AML1 site is either identical with AML1 or closely related. Additional EMSA analysis demonstrated that in vitro translated AML1 formed a complex with the same radiolabeled blk promoter probe (Fig. 2B). These results clearly demonstrate that PEBP2/CBF transcription factors, such as AML1, can interact specifically with the blk promoter AML1 site.
AML1 Induces blk Promoter Transcription-To determine whether the AML1-binding site is functionally relevant for blk promoter activity, the AML1 site was mutated from TGTGGT to TGCACT in the blk promoter luciferase construct to generate pBlk(mAML1)-luc. As shown in Fig. 3A, transient transfection into the B cell line BJA-B demonstrated that the blk gene upstream region from bp Ϫ191 to ϩ136 has strong promoter activity as previously shown (33). A 25-fold increase in luciferase expression was observed in comparison to the promoterless luciferase construct pXP2. Mutation of the AML1-binding site in the blk promoter reduced the promoter activity to 67% when compared with the wild type promoter. Furthermore, the same mutation also reduced the promoter activity to a similar level in another B cell line, Ba/F3. This indicates that the AML1-binding site in the blk promoter is a functionally relevant site, although not absolutely essential. To test whether AML1 can indeed transactivate the blk promoter, co-transfection experiments were performed in CV-1 cells with expression vectors for AML1 and its heterodimer partner CBF␤. As shown in Fig. 3B, exogenous AML1 together with CBF␤ expression increases transcription of the wild type blk promoter-luciferase construct by 7-fold. Mutation of the AML1-binding site reduced transactivation of the blk promoter by AML1/CBF␤ to less than 2-fold. These data together with the results from the DNAprotein interaction studies demonstrate that AML1 or a related

FIG. 3. AML1 is an important transcription factor for blk promoter activity.
A, mutation of the AML1-binding site significantly reduces blk promoter activity. The promoterless pXP2 luciferase construct, the wild-type blk promoter-luciferase construct (pBLK-luc), and the AML1 site mutated construct (pBLK(mAML1)-luc) were transiently transfected into B cell lines BJA-B (f) and Ba/F3 (u). B, AML1 stimulates transcription from the blk promoter. CV-1 cells were transfected by the Ca 3 (PO4) 2 precipitation method with 3.5 g of the wild type blk promoter-luciferase construct (pBLK-luc) or the AML1-binding site mutated blk promoter construct (pBLK(mAML1)-luc). These constructs were co-transfected with or without 0.5 g each of AML1 and its heterodimer partner CBF␤ expression vector pCMV-AML1B and pCMV-CBF␤. The averages and standard deviations were generated from three separate experiments. Luciferase activities were normalized for transfection efficiency with the co-transfected growth hormone plasmid Rous sarcoma virus-human growth hormone. The error bars indicate the mean Ϯ S.D. family member plays a significant role in B cell-specific regulation of the blk promoter.
AML1 Physically Interacts with BSAP-As shown in Fig. 1, the AML1-binding site is relatively close to the transcription initiation site of the blk gene. Six base pairs upstream of the AML1 site is a region that has been identified as a BSAPbinding site (33,34). BSAP is a B cell-specific activating protein, which is a critical transcription factor for B cell-specific gene expression and B cell development. Since AML1 has been shown to interact and cooperate with other transcription factors, and the AML1 site is adjacent to the BSAP-binding site, we analyzed whether AML1 can cooperate with BSAP in regulating blk gene expression. First, we studied their physical interaction using GST-pull down assays, in which Escherichia coli expressing GST fusion proteins immobilized on glutathione-agarose beads were incubated with in vitro translated 35 Slabeled proteins. As shown in Fig. 4A, in vitro translated fulllength AML1 can be specifically retained on agarose beads containing the fusion protein made from BSAP (GST-BSAP), but not on glutathione beads containing only GST. This result indicates that AML1 can physically interact with BSAP. To analyze this interaction in more detail, DNA constructs containing only the NH 2 -terminal portion of AML1 (a.a. 1-208) or the AML1 runt homology domain (a.a. 87-208) were in vitro translated in the presence of [ 35 S]methionine. As shown in Fig.   4, B and C, both peptides can specifically interact with GST-BSAP fusion protein, but not GST protein alone. These results indicate that the runt homology domain of AML1 is responsible for the interaction with BSAP. To determine which domain of BSAP is responsible for its interaction with AML1, GST-fusion proteins containing COOH-terminal deletion mutants of BSAP were used in the binding reactions. Removal of the carboxyl terminus up to a.a. 312 did not have any effect on AML1 interaction (Fig. 4E, lane 3). The BSAP NH 2 -terminal portion (a.a. 1-159) containing the paired DNA-binding domain retained the interaction with AML1 as well (Fig. 4E, lane 4). In contrast, GST-fusion protein containing only the BSAP COOHterminal portion (a.a. 234 -391) did not show any interaction with AML1. These results demonstrate that AML1 can directly interact with BSAP and this interaction is through the runt homology domain of AML1 and the paired domain of BSAP (6,42,51).
AML1 Cooperates with BSAP in Transactivation of the blk Promoter-Both AML1-binding site mutation analysis and transactivation analysis indicate that AML1 is an important transcription factor for blk promoter activity (Fig. 3). BSAP has been shown to be a critical regulator of blk promoter activity (33,34) and data in Fig. 4 have demonstrated the physical interaction between these two transcription factors. To determine whether physical interaction between AML1 and BSAP is associated with functional cooperativity in activating blk gene expression, we performed co-transactivation experiments in monkey kidney CV-1 cells as shown in Fig. 5. In the presence of only the parental expression vector (control), luciferase expression directed by the blk promoter is ϳ2-fold over the promoterless construct. When BSAP was used alone in the transactivation experiments, there was an 8-fold induction of blk promoter activity. AML1 together with its heterodimer partner CBF␤ activated the blk promoter 12-fold. When all three transcription factors, BSAP, AML1, and CBF␤ were used in the same transactivation experiment, blk promoter activity was induced by 51-fold. These results demonstrate a strong transcriptional synergy of blk promoter activity in the presence of all three factors, indicating that physical interaction between AML1 and BSAP leads to functional cooperativity in the context of the blk promoter. DISCUSSION AML1 is a transcription factor identified by studying t(8;21) associated acute myeloid leukemia (1,(52)(53)(54). The function of AML1 in hematopoiesis has been demonstrated by analyzing its role in regulating the expression of critical hematopoietic genes and by studying AML1 null mice (55,56). AML1 knockout mice die during embryogenesis with the block of definitive hematopoiesis (16,17). These studies indicate that AML1 plays an important role during early hematopoietic progenitor cell formation. AML1 binds to the regulatory elements of genes specifically expressed in different lineages of hematopoietic cells or of genes important for lineage development. Recently, AML1 has been found to associate with a chromosomal translocation, t(12;21), commonly associated with B-cell lineage differentiation, indicating an important role for AML1 during B cell development (20,22). Nevertheless, only one potential B cell-specific target gene for AML1, the immunoglobulin heavy chain gene enhancer (57), has been identified to date. Therefore, we decided to search for other potential target genes for AML1 in B cells. We identified a putative AML1-binding site in the promoter region of the B cell-specific blk gene and tested the potential role of AML1 in B-cell specific blk gene regulation. We provide strong evidence here that AML1 directly interacts with a functionally important region of the blk promoter and transactivates the promoter. Furthermore, we demonstrate that AML1 physically interacts with another critical B-cell transcription factor BSAP and synergizes with BSAP in regu-lating blk promoter activity. There are different alternatively spliced forms of AML1, which have been named as AML1 (AML1a), AML1A (AML1b), and AML1B (AML1c). In this paper, we used "AML1" to include different forms of the AML1 protein. AML1B was used in the transactivation and in vitro transcription reactions.
AML1 is a member of the CBF protein family. All three ␣ subunits of the CBF family contain the highly conserved runt homology domain, which encodes the DNA-binding domain. Therefore, all three ␣ subunits recognize the same "TGTGGT" DNA sequence. Previous studies by others have identified different members of the CBF␣ protein in B cell lines and a B cell-enriched tissue spleen, using antibodies specifically against three different CBF␣ subunits. Their results show that AML1 and AML2 are expressed at similar levels in B lineage cells; AML3 is not detectable (58). The AML1 antiserum that we used in Fig. 2 is raised against the AML1 NH 2 -terminal 17 amino acids. The NH 2 -terminal region is conserved between all three CBF␣ proteins. Since both AML1 and AML2 are expressed at similar levels in B cells and both AML1 and AML2 have a similar DNA binding activity (58), it is possible that both of them are in the supershifted protein-DNA complex as shown in Fig. 2.
It has been reported previously that CBF␤ enhances AML1 binding to DNA (3) and CBF␤-AML1 forms a slower mobility shifted band with DNA than AML1-DNA complex (46). As shown in Fig. 2B, in vitro translated AML1 binds to DNA and addition of CBF␤ enhances the binding of AML1 with the DNA. However, we did not detect an obvious slower mobility complex. This is probably due to the lack of a particular modification in the in vitro translation system that is required for forming a stable complex of a heterodimer CBF protein with DNA, or the specific experimental conditions do not favor the formation of the complex during gel electrophoresis.
Mutation of the AML1 site in the blk promoter significantly reduced promoter activity. However, this mutation did not abolish the promoter activity, indicating that AML1 is an important factor for the promoter activity. Other transcription factors that bind to the blk promoter could also contribute to the promoter activity. As shown in Fig. 1, besides the AML1binding site, there are several other potential transcription factor-binding sites including a binding site for the B cellspecific BSAP transcription factor just upstream of the AML1 site. BSAP has been reported previously to play a crucial role in the regulation of the blk promoter (33,34). Therefore, we analyzed whether AML1 and BSAP could interact with each other and whether there is any synergy between these two factors in promoter activation. The results from GST-pull down assays demonstrated a strong interaction between these two factors, and the interaction is between the AML1 runt homology domain and the BSAP NH 2 -terminal paired domain. The original function of the runt homology domain includes direct binding to DNA and formation of heterodimers with CBF␤. There have been reports about AML1 physically interacting with other transcription factors through the runt homology domain, including Ets-1, PU.1, and CAAT/enhancer-binding protein family members (14,48,49). Similarly, the paired domain of BSAP has been shown to be involved in protein-protein interactions with members of the Ets family (59). The interaction between AML1 and BSAP represents a new class of interaction between AML1 and other transcription factors. Interestingly, it is the DNA-binding domain that is involved in the interaction of both transcription factors. It will be interesting to further study the specific amino acids involved in the different interactions. The interaction and synergy between Ets-1 and AML1 is crucial for the enhancer function of several T cell receptor genes (14, 60); FIG. 5. AML1 and BSAP synergistically activate the blk promoter. CV-1 cells were transfected by the Ca 3 (PO 4 ) 2 precipitation method with 3.5 g of blk promoter-luciferase construct (pBLK-luc) in the presence or absence of 0.5 g of the AML1, CBF␤, and BSAP expression constructs pCMV-AML1B, pCMV-CBF␤, or pCI-BSAP, respectively, as indicated. The averages and standard deviations were generated from three separate experiments. Luciferase activities were normalized for transfection efficiency with the co-transfected growth hormone plasmid Rous sarcoma virus-human growth hormone. The error bars indicate the mean Ϯ S.D. Fold increases in the promoter activity are relative to that in the absence of any additional transcription factors. furthermore, the interactions and synergies between AML1 and PU.1 (49), Myb (61), and CAAT/enhancer-binding protein (48) have been demonstrated to serve critical functions during activation of myeloid specific gene expression. Both AML1 and BSAP are critical regulators of hematopoiesis (16,17,62). In addition, both genes have been directly implicated in B cell malignancies (20,63). The interaction and synergy between AML1 and BSAP could play a significant role in B cell-specific gene expression and disruption of this interaction due to chromosomal translocations or other chromosomal abnormalities could play a critical role in B cell transformation. Further experiments using other B cell-specific gene targets will be extremely interesting.