Original Manuscript

Leukemia (2005) 19, 806–813. doi:10.1038/sj.leu.2403684 Published online 3 March 2005

Fusion Proteins

Cloning and functional characterization of MEF2D/DAZAP1 and DAZAP1/MEF2D fusion proteins created by a variant t(1;19)(q23;p13.3) in acute lymphoblastic leukemia

V Prima1, L Gore2,3, A Caires2, T Boomer2, M Yoshinari4, M Imaizumi5, M Varella-Garcia3,6 and S P Hunger1

  1. 1Department of Pediatrics, University of Florida College of Medicine and the University of Florida Shands Cancer Center, Gainesville, FL
  2. 2Department of Pediatrics, Denver, CO, USA
  3. 3University of Colorado Cancer Center, Denver, CO, USA
  4. 4Pediatric Oncology, Tohoku University Hospital, Sendai, Miyagi, Japan
  5. 5Department of Hematology and Oncology, Miyagi Children's Hospital, Sendai, Miyagi, Japan
  6. 6Department of Medicine, Denver, CO, USA

Correspondence: Dr SP Hunger, Pediatric Hematology-Oncology, University of Florida College of Medicine, PO Box 100296, Gainesville, FL 32610-0296, USA. Fax: +1 352 392 8725; E-mail: hungesp@peds.ufl.edu

Received 22 October 2004; Accepted 28 December 2004; Published online 3 March 2005.

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Abstract

We analyzed the TS-2 acute lymphoblastic leukemia (ALL) cell line that contains a t(1;19)(q23;p13.3) but lacks E2A-PBX1 fusion typically present in leukemias with this translocation. We found that the t(1;19) in TS-2 fuses the 19p13 gene DAZAP1 (Deleted in Azoospermia-Associated Protein 1) to the 1q23 gene MEF2D (Myocyte Enhancer Factor 2D), leading to expression of reciprocal in-frame DAZAP1/MEF2D and MEF2D/DAZAP1 transcripts. MEF2D is a member of the MEF2 family of DNA binding proteins that activate transcription of genes involved in control of muscle cell differentiation, and signaling pathways that mediate response to mitogenic signals and survival of neurons and T-lymphocytes. DAZAP1 is a novel RNA binding protein expressed most abundantly in the testis. We demonstrate that MEF2D/DAZAP1 binds avidly and specifically to DNA in a manner indistinguishable from that of native MEF2D and is a substantially more potent transcriptional activator than MEF2D. We also show that DAZAP1/MEF2D is a sequence-specific RNA-binding protein. MEF2D has been identified as a candidate oncogene in murine retroviral insertional mutagenesis studies. Our data implicate MEF2D in human cancer and suggest that MEF2D/DAZAP1 and/or DAZAP1/MEF2D contribute to leukemogenesis by altering signaling pathways normally regulated by wild-type MEF2D and DAZAP1.

Keywords:

leukemogenesis, oncogene, chromosomal translocation

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Introduction

Chromosome translocations play a critical role in leukemogenesis.1 The 19p13.3 gene E2A (also termed TCF3) is the target of several recurrent genomic rearrangements in acute lymphoblastic leukemia (ALL).2 The t(1;19)(q23;p13.3) is the second most common translocation in ALL.3, 4 In 90–95% of patients with a t(1;19), the molecular consequence is creation of an E2A-PBX1 fusion gene on the der(19) that encodes chimeric E2A-PBX1 proteins with transforming properties.5, 6, 7, 8, 9, 10 The remaining 5–10% of patients have translocations that appear identical by conventional cytogenetics, but do not involve E2A or PBX1.11 Yoshinari et al12 described the TS-2 cell line that was established from a 3-year-old girl with ALL and contains a t(1;19)(q23;p13.3), but lacks E2A or PBX1 abnormalities.

We found that the 1;19 translocation in TS-2 fuses the 1q23 gene MEF2D (Myocyte Enhancer Factor 2D) to DAZAP1 (Deleted in Azoospermia-Associated Protein 1) located at chromosome 19p13.3. MEF2D is a member of MEF2 family of transcription factors that bind specifically to the MEF2 element present in the regulatory regions of many genes active in cells of muscle or neural lineage. MEF2D dimers regulate expression of genes involved in muscle-specific and/or growth factor-related transcription.13, 14, 15 Murine retroviral insertional mutagenesis studies have identified MEF2D as a candidate oncogene involved in the pathogenesis of leukemia.16, 17 DAZAP1 is an RNA-binding protein that may be essential for spermatogenesis.18 It is expressed primarily in testis, and to a lower level, in thymus. DAZAP1 shows dynamic subcellular distribution during spermatocyte development, consistent with its presumed role in mRNA transport or processing.18, 19 In this report, we show that in-frame MEF2D/DAZAP1 and DAZAP1/MEF2D fusion transcripts are expressed in TS-2 and define the DNA-, RNA-binding, and transcriptional regulatory properties of the resultant chimeric proteins. Our studies identify MEF2D and DAZAP1 fusion proteins as components of novel pathways that contribute to human leukemogenesis.

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Materials and methods

ALL cell lines

ALL cell lines were grown in RPMI-1640 and CV-1 cells were grown in DMEM supplemented with 10% fetal calf serum at 37°C in a humidified atmosphere containing 5% CO2. Cell lines utilized included TS-2, RCH-ACV (kindly provided by Dr Ram Seshadri) that has a t(1;19)(q23;p13) and E2A-PBX1 fusion, and the B-precursor ALL cell line REH.12, 20

Fluoresence in situ hybridization (FISH)

Single and multiplex FISH was performed as described previously using cosmids provided by Anne Olsen and the human chromosome 19 mapping group at Lawrence Livermore National Labs.21, 22

Molecular analyses

Nucleic acids were extracted from cell lines and lambda phage using commercial reagents (QIAGEN, Valencia, CA, USA; Invitrogen Life Technologies, Carlsbad, CA, USA; Promega, Madison, WI, USA). Southern blot, Western blot and PCR analyses were performed essentially as described previously.23, 24, 25, 26, 27

A TS-2 genomic library was constructed using the Lambda DASH II kit (Stratagene, San Diego, CA, USA). Approximately one million recombinant phage were screened with a DAZAP1 cDNA probe kindly provided by Dr Pauline Yen (Torrance, CA, USA); positive clones were purified to homogeneity and the phage inserts were characterized by nucleotide sequence analysis as described previously.26

cDNA cloning, expression and reporter constructs

First-strand cDNA synthesis was performed on total RNA using a mix of oligo(dT) primers and random hexamers with SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). To clone full-length cDNAs, first-strand cDNA was used as PCR template with the primer sets DAZAP1 (f/r), MEF2D (f/r) (oligonucleotide sequences are listed in Supplementary Table 1), and Expand High Fidelity PCR System (Roche Molecular Systems, Alameda, CA, USA). The reverse primers added FLAG tags (underlined) to the 3'-ends of cDNAs. PCR products were cloned into pCR2.1-TOPO and then subcloned into pBlue-TOPO (T7 promoter) and pRc/RSV vectors (Invitrogen). Reporter plasmid p4XMEF2-FLuc containing four MEF2 sites upstream of the c-fos minimal promoter and luciferase gene was a kind gift from Dr Ron Prywes (Columbia University). The plasmid pCMVbetagal contains the beta-galactosidase gene under control of a CMV promoter.

In vitro translation and immunoblot analysis

In vitro transcriptions and translations (IVT) were performed using the Promega TNT Quick Coupled Transcription/Translation System (1 mug/50mul reaction). Following separation on 12% SDS-PAA gels, protein expression was determined by Western blot analysis using the M2 anti-FLAG primary antibody (Sigma, St Louis, MO, USA) and goat-anti-mouse Ig-HRP secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The chemiluminescent signal was detected using the ECL Plus detection kit (Amersham, Arlington Heights, IL, USA).

Electrophoretic mobility shift assay (EMSA)

EMSA was performed at 20°C with IVT proteins. The double-stranded MEF2 consensus binding oligonucleotide, 5'-GATCGCTCTAAAAATAACCCTGTCG-3' (Santa Cruz Biotechnology), was end-labeled with italic gamma-32P-ATP using T4 polynucleotide kinase (Promega). The DNA binding reaction mixtures in 20 mul contained 1 ng of 32P-labeled probe, 1 mug/mul of poly(dI-dC) (Sigma), 10 mM Tris-HCl, pH 7.5; 1 mM DTT; 1 mM EDTA; 50 mM NaCl; 5% glycerol and 5 mul IVT reaction. For competition analyses, 50-fold molar excess of unlabeled consensus or mutant (5'-GATCGCTGTAAACATAACCCTGTCG-3') MEF2 double-stranded oligonucleotide was added to the binding assay. Samples were separated in a 0.5 times TBE 5% PAA gel at 20 mA constant current, 4°C, and gels were dried and autoradiographed.

Cell transfections and reporter gene assays

CV-1 cells were cultured in six-well plates and transfected using Lipofectamine 2000 (Invitrogen). In total, 2 mug of the test expression plasmids (empty pRc/RSV, pRc/RSV-DAZAP1-FLAG; pRc/RSV-MEF2D-FLAG; pRc/RSV-DAZAP1/MEF2D-FLAG; pRc/RSV-MEF2D/DAZAP1-FLAG) was cotransfected with 1.5 mug of p4xMEF2Fluc and 0.5 mug of pCMVbetagal. Luciferase and beta-galactosidase expressions were determined using commercial reagents (Promega). Luciferase values are expressed as meanplusminuss.e.m. of fold induction seen compared to luciferase values obtained with the empty expression vector and are the averages of at least three separate experiments with duplicate transfections normalized to beta-galactosidase values.

In vitro RNA-binding assay

Binding of IVT 35S-labeled proteins to RNA homopolymers immobilized on agarose bead (Sigma) was carried out in a total of 0.5 ml of binding buffer (10 mM Tris-HCl, pH 7.4; 2.5 mM MgCl2; 0.5% Triton X-100; 0.1 M NaCl) for 10 min with rotation at 4°C. Equal amounts of 35S-labeled proteins were incubated with equal volume of agarose-linked RNA homopolymers as described.18 To study binding-specificity, 50-fold molar excess of soluble RNA homopolymers were included as competitors for protein binding. The beads were briefly pelleted in a microcentrifuge and washed five times with binding buffer prior to resuspension in 70 mul of SDS-PAGE loading buffer. Bound protein was eluted from the nucleic acid by boiling 5 min in gel loading buffer, resolved on 12% SDS-PAA gel, and visualized by autoradiography.

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Results

Localization of the chromosome 19 genomic breakpoint in TS-2

To localize the TS-2 translocation breakpoint with respect to E2A, we performed metaphase FISH with TS-2 and RCH-ACV cell lines using cosmids containing genomic sequences from 19p13 (Supplementary Figure 1). These studies first established that the TS-2 chromosome 19 breakpoint is located within the 1500 kilobases (kb) of DNA telomeric to E2A (Supplementary Table 2). This is shown in Figure 1a and b using the two-color split signal E2A FISH assay that we described previously.22 Additional FISH studies narrowed the interval of interest to an approximately 400 kb region between E2A and POL2RE (Supplementary Table 2). Detailed FISH studies using a panel of 12 cosmids that spanned the region between E2A and POL2RE revealed that the breakpoint occurred in the region contained in cosmid 21277, which was split in TS-2 and hybridized to both the der(1) and the der(19) (Supplementary Table 2 and Figure 1c). To better visualize the chromosome 19 breakpoint in TS-2, we developed a more robust two-color split signal FISH assay. A pool of four cosmids located immediately centromeric to cosmid 21277 and telomeric to cosmid 27377 serves as one probe, while the other probe is composed of a pool of four cosmids located immediately telomeric to 21277. Split signals in metaphase (Figure 1d) and interphase (data not shown) FISH confirmed localization of the TS-2 chromosome 19 breakpoint to the region homologous to cosmid 21277.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Localization of TS-2 chromosome 19 breakpoint via FISH. (a) Two-color E2A split signal FISH was performed using a pool of four cosmids located immediately centromeric (green) and telomeric (red) to E2A. FISH with RCH-ACV shows a breakpoint within E2A with a fused signal on the normal 19 and split signals on the der(19) (green) and der(1) (red). (b) Two-color E2A split signal FISH with TS-2 shows fused signals on the normal and der(19) homologs, indicating that the chromosome 19 breakpoint occurs telomeric to both probes. (c) Cosmid 21277 hybridizes to the normal 19, der(1) and der(19) chromosomes in TS-2 metaphases. (d) Two-color split signal FISH using pools of cosmids located immediately centromeric (green) and telomeric (red) to cosmid 21277 was performed with TS-2 metaphases. One fused red/green and split red and green signals confirm that the chromosome 19 breakpoint occurs within cosmid 21277. For the schematic depiction of FISH experiments see Supplementary Figure 1.

Full figure and legend (305K)

DAZAP1 is fused to MEF2D by the t(1;19) in TS-2

A Genbank search for open reading frames within cosmid 21277 identified exons of DAZAP1, which encodes a protein with novel RNA-binding properties that is expressed most abundantly in the testis and had been mapped previously to 19p13.3.18 Southern blots containing TS-2 and control DNA were probed with a DAZAP1 cDNA probe. Rearranged bands were seen only in TS-2, establishing that the t(1;19) interrupts the 19p13.3 gene DAZAP1 in TS-2, with the breakpoint region in approximately the middle of the gene (Figure 2a).

Figure 2.
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The t(1;19) in TS-2 fuses DAZAP1 to MEF2D. (a) Southern blots of TS-2 and control samples were hybridized with a labeled DAZAP1 cDNA probe. Rearranged bands present in TS-2 establish that the t(1;19) in TS-2 interrupts DAZAP1. TS-2, RCH-ACV, REH: ALL cell lines; NL1, NL2: normal DNA. Arrows marked G or R indicate germline or rearranged DNA fragments. (b) Schematic depiction of the cloned lambda fragment containing TS-2 der(19) genomic DNA. Arrows denote primers used for sequencing (Supplementary Table 1); black boxes represent DAZAP1 exons. Nucleotide sequences of the fusion sites of chimeric chromosomes are shown in Supplementary Figure 2. (c) RT-PCR amplification of DAZAP1, MEF2D, DAZAP1/MEF2D and MEF2D/DAZAP1 transcripts shows that wild-type DAZAP1 and MEF2D transcripts are expressed in both TS-2 and REH, while only TS-2 expresses the fusion DAZAP1/MEF2D and MEF2D/DAZAP1 transcripts. For nucleotide sequences of the fusion transcripts see Supplementary Figure 3.

Full figure and legend (90K)

We constructed a TS-2 genomic library, screened it with a DAZAP1 cDNA probe, and identified a lambda phage clone that contained a rearranged DNA fragment of approximately 13 kb. Nucleotide sequence analysis showed that this lambda clone contained a portion of DAZAP1 fused to a region of chromosome 1 that contained MEF2D, which encodes one of the four members of the MEF2 family (Figure 2b).15 We then amplified TS-2 DNA with breakpoint-flanking primers and sequenced the genomic fusion sites from both the der(1) (Genbank accession AY681493) and der(19) (accession AY681494) (Supplementary Figure 2). The genomic breakpoints occur in introns of MEF2D and DAZAP1. There is a five base pair (bp) insertion at the site of genomic fusion on the der(19) that is not derived from either germline chromosome 1 or 19. Homologous breakpoints occur on the der(1) chromosome with a deletion of 97 bp and a 21 bp GC-rich insertion.

Wild type and chimeric DAZAP1 and MEF2D transcripts are expressed in TS-2

To study expression of DAZAP1, MEF2D and the predicted fusion transcripts, we performed RT-PCR on TS-2 and REH, a control B-precursor ALL cell line. Both TS-2 and REH expressed DAZAP1 and MEF2D transcripts, but MEF2D/DAZAP1 and DAZAP1/MEF2D were expressed only in TS-2 (Figure 2c). Sequence analysis of PCR products showed that MEF2D/DAZAP1 and DAZAP1/MEF2D fusion transcripts were in-frame (Supplementary Figure 3).

Structure and synthesis of wild type and chimeric DAZAP1 and MEF2D proteins

Native DAZAP1 (NP_061832) includes two identified RNA recognition motifs (RRM) specified by amino acids 1–87 and 105–190 (Figure 3a). MEF2D (NP_005911) has conserved N-terminal MADS-box (MCM1, Agamous, Deficiens and Serum response factor28) and MEF2 domains involved in protein dimerization, DNA binding and protein–protein interactions, and two transcriptional activation domains (TAD), one which is poorly localized carboxy-terminal to the MEF2 domain, and one within the C-terminus.29 DAZAP1/MEF2D fusion cDNAs (accession AY678451) are predicted to encode a chimeric protein that contains all of the first DAZAP1 RRM and a truncated portion of the second RRM joined to the carboxy-terminal portion of MEF2D that includes the second TAD. Reciprocal MEF2D/DAZAP1 fusion transcripts (accession AY675556) are predicted to encode a chimera that includes the MEF2D MADS-box, MEF2 domain, and the first TAD joined to the carboxy terminus of DAZAP1 including a truncated portion of RRM 2.

Figure 3.
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Structural features, expression and RNA-binding properties of wild type and chimeric MEF2D and DAZAP1 proteins. (a) Predicted functional domains of DAZAP1, MEF2D, DAZAP1/MEF2D and MEF2D/DAZAP1 proteins. Arrows indicate predicted protein breakpoints. (RRM – RNA recognition motif; MADS – DNA binding and protein dimerization domain; MEF2 – cofactor interactions domain; TAD – transcriptional activation domain). (b) Transient transfection of expression constructs with proteins detected via anti-FLAG Western blot. (c) IVT of proteins with detection via anti-FLAG Western blot. (d) IVT with 35S-labeled proteins detected via autoradiography. Lane 1: DAZAP1; lane 2: MEF2D; lane 3: DAZAP1/MEF2D; lane 4: MEF2D/DAZAP1. (e) In vitro binding to RNA homopolymers immobilized on agarose beads. IVT 35S-labeled proteins were incubated with agarose beads containing bound RNA homopolymers. Beads were collected via centrifugation and washed to remove nonspecific binding. Bound proteins were then visualized via gel electrophoresis and autoradiography. (f) To determine specificity of RNA-binding, experiments were performed in the presence of 50-fold molar excess of the soluble RNA homopolymer competitors. The first lane for each protein represents 1/3 of the IVT amount used in binding experiments.

Full figure and legend (165K)

We assembled full-length wild type and fusion MEF2D and DAZAP1 cDNAs with added C-terminal FLAG epitope tags for IVT and expression in mammalian cells. The proteins were detected following polyacrylamide gel electrophoresis (PAGE) either by anti-FLAG immunoblot or by autoradiography (Figure 3b–d). For IVT DAZAP1/MEF2D (Figure 3c and d), only the highest band on PAGE corresponds to the predicted full-size product observed following transient transfections (Figure 3b). This may reflect the lesser fidelity of translation in vitro allowing alternative downstream start codons. The smaller DAZAP1/MEF2D IVT fragments have the C-terminal FLAG tag but not the complete N-terminal RNA-binding domains (see Figure 3e and f).

In vitro RNA binding of DAZAP1, MEF2D and their derivatives

DAZAP1 displays salt-dependent RNA-binding properties; Tsui reported that IVT DAZAP1 could bind to RNA homopolymers immobilized on agarose beads, with preferential binding to poly(U) and poly(G), less avid binding to poly(A), and no binding to poly(C).18 We performed analogous experiments and found that IVT DAZAP1 bound strongly to poly(U) and poly(G) at 0.1 M NaCl, whereas DAZAP1/MEF2D bound to the same homopolymers to a lesser degree (Figure 3e). Neither protein bound to poly(C) agarose (data not shown). The decreased binding of DAZAP1/MEF2D as compared to native DAZAP1 may be due to the fact that DAZAP1/MEF2D fusion proteins expressed in TS-2 include an intact first RRM but only approximately one-half of the second RRM (Figure 3a). Unexpectedly, we found that both MEF2D and MEF2D/DAZAP1 bound strongly to poly(G), but not to poly(U) or poly(C) (Figure 3e and data not shown). To determine if the observed RNA-binding was specific; we performed RNA binding experiments in the presence of soluble RNA homopolymers to act as competitors to the agarose-linked RNA homopolymers. The presence of 50-fold molar excess of soluble poly(U) but not poly(C) abolished both DAZAP1 and DAZAP1/MEF2D binding to poly(U)-agarose, indicating that this binding was specific (Figure 3f). In contrast, MEF2D RNA binding was nonspecific, because both soluble poly(G) and poly(U) failed to affect MEF2D binding to poly(G)-agarose beads (Figure 3f). We observed a similar pattern of nonspecific RNA binding by MEF2D/DAZAP1 and the hepatic leukemia factor protein24, 30 (data not shown), consistent with weak RNA-binding properties of DNA-binding motifs found in many transcription factors.31

MEF2/DAZAP1 retains DNA-binding properties of wild-type MEF2D

We compared DNA-binding properties of wild type and chimeric MEF2D and DAZAP1 IVT proteins via EMSA using a radiolabeled MEF2 consensus binding site oligonucleotide. Both IVT MEF2D and MEF2D/DAZAP1 proteins bound avidly to the consensus MEF2 site, while there was no binding by DAZAP1 or DAZAP1/MEF2D (Figure 4). The observed binding was specific, because preincubation of MEF2D and MEF2D/DAZAP1 with 50-fold molar excess of the unlabeled consensus oligonucleotide markedly reduced binding to the labeled oligonucleotide, while there was no effect of pre-incubation with 50-fold molar excess of an unlabeled mutant oligonucleotide.

Figure 4.
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DNA binding properties of MEF2D, DAZAP1 and chimeric proteins. EMSA was performed with the indicated IVT unlabeled proteins and a 32P-labeled MEF2 consensus oligonucleotide. Where indicated 50-fold molar excesses of either unlabeled consensus MEF2 oligonucleotide or unlabeled mutant oligonucleotide were included as competitors.

Full figure and legend (83K)

MEF2D/DAZAP1 is a more potent transcriptional activator than wild-type MEF2D

To characterize the transcriptional regulatory properties of MEF2D/DAZAP1, we transiently transfected CV-1 cells with expression constructs and p4xMEF2FLuc, which has four consensus MEF2 binding sites located 5' to a luciferase reporter gene. MEF2D/DAZAP1 was a substantially more potent transcriptional activator than wild-type MEF2D (Figure 5). DAZAP1 or DAZAP1/MEF2D did not activate transcription of p4xMEF2FLuc.

Figure 5.
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MEF2D/DAZAP1 is a more potent transcriptional activator than MEF2D. Expression constructs and reporter genes were transiently transfected into CV-1 cells. The relative luciferase levels were normalized to the levels of beta-galactosidase expression. The average fold inductionsplusminuss.e. of the means of three separate experiments are indicated.

Full figure and legend (41K)

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Discussion

We report that the variant t(1;19)(q23;p13) present in TS-2 fuses MEF2D to DAZAP1, show that TS-2 expresses MEF2D/DAZAP1 and DAZAP1/MEF2D chimeric transcripts, and characterize the in vitro RNA-, DNA-binding, and transcriptional regulatory properties of the encoded fusions proteins.

There are four vertebrate (MEF2A-D) and one Drosophila (D-MEF2) MEF2 genes that encode proteins originally characterized as muscle-specific transcription factors that bind as homodimers and heterodimers to an A/T-rich DNA sequence present in target gene promoters and regulate transcription of genes critical for myogenic differentiation (reviewed by McKinsey).15 MEF2 proteins have highly similar amino-terminal regions that contain conserved elements including the MADS box with an adjacent MEF2 domain involved in dimerization and DNA binding, and more divergent carboxy-terminal domains. Members of this protein family interact with a variety of other proteins involved in transcriptional regulation including histone deacetylases (HDAC), p300 and Cabin1/Cain.32, 33, 34, 35 MEF2 proteins are essential for cardiac morphogenesis in mice (mef2c) and terminal differentiation of all muscle lineages during Drosophila embryogenesis.36, 37, 38 MEF2 proteins also have critical functions in other cell types. They are transcriptional effectors of mitogenic signaling pathways initiated by mitogen-activated protein kinases (MAPKs) including p38 and ERK5 (extracellular signal-related kinase 5)/Big MAPK-1, and also play critical roles in calcium-regulated signaling pathways that control survival of neurons and T-cells.15, 33, 39 The effects of MEF2 proteins on survival can be either pro- or antiapoptotic.39, 40, 41 While MEF2 proteins have not previously been implicated in human cancer, MEF2D can induce expression of c-jun, a known transforming oncogene, and has recently been identified in murine retroviral mutagenesis studies as a candidate oncogene involved in the pathogenesis of lymphoid malignancies.16, 17, 42

In contrast to MEF2D, relatively little is known concerning DAZAP1, a protein originally isolated by virtue of its interaction with DAZ in a yeast two-hybrid assay.18 DAZAP1 is an RNA-binding protein expressed primarily in testis that contains two conserved RRMs and a proline-rich C-terminal domain.18, 19 There are dynamic changes in temporal and spatial expression of DAZAP1 during spermatogenesis in the mouse, with suggestions that expression may be associated with active transcription.19 DAZAP1 mRNA is also expressed in thymus,18 and we show in this report that the transcript is detected via RT-PCR in human B-precursor ALL cell lines.

Since TS-2 expresses both MEF2D/DAZAP1 and DAZAP1/MEF2D fusion transcripts, there are several potential mechanisms by which the chimeras might contribute to leukemogenesis. We demonstrate that MEF2D/DAZAP1 is a substantially more potent transcriptional activator than wild-type MEF2D in transient transfection assays. MEF2D/DAZAP1 might directly activate transcription of genes critical for lymphocyte growth and/or survival such as interleukin-2, a known transcriptional target of MEF2D in T-cells.43 Alternatively, MEF2D/DAZAP1 could contribute to leukemogenesis via dysregulated activation of MAPK-mediated cell proliferation pathways, analogous to constitutive activation of a growth factor receptor. Since MEF2 proteins are subject to modification through complex interactions with various transcriptional co-activators and co-repressors, it is also possible that replacement of the C-terminus of MEF2D with that of DAZAP1 critically alters regulatory influences on MEF2D function.

It is also important to consider the possibility that DAZAP1/MEF2D is critical for leukemogenesis. DAZAP1/MEF2D proteins expressed in TS-2 retain all of the first and approximately one-half of the second RRM of DAZAP1, fused to a truncated C-terminus of MEF2D. Studies of other RNA-binding proteins show that each RRM can form a globular domain that, in at least some cases, can bind to RNA independently.44 This is consistent with our data demonstrating that DAZAP1/MEF2D retains sequence-specific RNA-binding properties. A number of other genes that encode proteins with RNA binding domains are involved in translocations in human cancer including EWS, TLS/CHOP, RBM15, and RSI2.45, 46, 47, 48 As is the case in TS-2, each of these RNA-binding proteins is fused to a transcription factor with DNA-binding properties, but there is no clear understanding whether, and if so how, their RNA binding properties contribute to transformation.

We have performed FISH with chromosome 19 cosmids on several primary hyperdiploid childhood ALL cases with a t(1;19)(q23;p13) but lacking E2A-PBX1 fusion, and did not find evidence that DAZAP1 was interrupted (AC and SPH, unpublished observations). However, samples were limited and of poor quality making these conclusions tentative. A t(1;19)(q23;p13) has also been reported occasionally in AML, T-cell ALL, and in some lymphomas.4, 49, 50, 51 These cases should be analyzed to determine the potential involvement of DAZAP1 and/or MEF2D, and their possible role(s) in the oncogenic properties of such malignancies.

While this manuscript was being prepared for submission, Yuki et al52 reported cloning of identical MEF2D/DAZAP1 and DAZAP1/MEF2D fusion transcripts from TS-2. Their independent studies showed that transiently transfected MEF2D, DAZAP1, MEF2D/DAZAP1 and DAZAP1/MEF2D are localized to the nucleus in HeLa cells.52 MEF2D-DAZAP1 was co-immunoprecipitated with wild-type MEF2D from HEK293 cells, suggesting that the wild type and chimeric MEF2D proteins could form heterodimers and/or associate with one another in a higher order protein complex in vivo.52 MEF2D/DAZAP1 co-localized with HDAC4 in HEK293 cells and could be co-immunoprecipitated.52 Their data also showed that wild-type MEF2D, MEF2D/DAZAP1 and DAZAP1/MEF2D, but not wild-type DAZAP1, could promote soft agar colony formation by HeLa cells.52 When these data are taken together with our data summarized above and the murine retroviral insertional mutagenesis studies,16, 17 it suggests that native MEF2D has latent transforming properties that can be unmasked via aberrant protein expression. MEF2D-DAZAP1 has similar DNA binding properties to MEF2D, but is a more potent transcriptional activator and also may associate more strongly with other proteins involved in transcriptional regulation (eg HDAC4). These alterations may confer more potent transforming properties to MEF2D/DAZAP1, which can be further augmented by co-expression with the reciprocal DAZAP1/MEF2D chimera. In future studies, it will be critical to directly test the transforming properties of native and chimeric MEF2D and DAZAP1 proteins in lymphoid cells, and determine the mechanisms by which they contribute to leukemogenesis.

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Acknowledgements

We thank Anne Olsen for supplying cosmids and BACs, Pauline Yen for the DAZAP1 cDNA, Ron Prywes for the p4xMEF2FLuc reporter construct, Amanda Rice and Chuhua Zhong for technical advice and assistance. This work was supported by grants from the Loewenstern Family Foundation and Monfort Family Foundation to SPH, NCI Cancer Center Core Grant CA 46934, and by the grants from the Leukemia Research Foundation and the Cancer League of Colorado to LG.

Supplementary Information

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu).