Original Paper

Oncogene (2003) 22, 699–709. doi:10.1038/sj.onc.1206124

The MN1 oncoprotein synergizes with coactivators RAC3 and p300 in RAR-RXR-mediated transcription

Karel H M van Wely1,4, Anco C Molijn1,4, Arjan Buijs2, Magda A Meester-Smoor1, Albert Jan Aarnoudse1, Anita Hellemons1, Pim den Besten1, Gerard C Grosveld3 and Ellen C Zwarthoff1

  1. 1Department of Pathology, Erasmus University, PO Box 1738, 3000 DR Rotterdam, The Netherlands
  2. 2Department of Hematology, University Hospital Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
  3. 3Department of Genetics, St. Jude's Children's Research Hospital, Memphis, TN 38101-0318, USA

Correspondence: Dr EC Zwarthoff, Department of Pathology, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail: zwarthoff@path.fgg.eur.nl

4The first two authors contributed equally to this paper

Received 6 June 2002; Revised 2 October 2002; Accepted 9 October 2002.

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Abstract

The t(12;22) creates an MN1–TEL fusion gene leading to acute myeloid leukemia. The fusion partner TEL (ETV6) is a member of the ETS family of transcription factors. The nature of the other fusion partner, MN1, has not been investigated in detail until now. We recently described that MN1 activates the transcription activity of the moloney sarcoma virus long terminal repeat, indicating that this protein itself may act as a transcription factor. We show here that MN1 comprises multiple transcription activating domains. A search for a bound DNA sequence revealed that MN1 has affinity for retinoic acid responsive elements. A DR5 retinoic acid responsive element was observed in the LTR. The combination of MN1 and ligand-activated retinoic acid receptor leads to a synergistic induction of expression directed by the LTR. Cotransfection of MN1 with RAC3 or p300, known coactivators of retinoic acid receptors, leads to a further synergistic induction of transcription. In addition, the effect of MN1 can be inhibited by the wild-type adenovirus ElA protein that inhibits p300 function, but not by an E1A mutant lacking the p300-binding site. GAL4-MN1-mediated transcription can be enhanced directly by RAC3 and p300. Taken together, our results indicate that MN1 is a transcription coactivator rather than a sequence-specific transcription factor, and that it may stimulate RAR/RXR-mediated transcription through interaction with p160 and p300.

Keywords:

translocation, leukemia, retinoic acid, hormone receptor, coactivator

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Introduction

The t(12;22)(p13;q11) leads to the formation of an MN1–TEL fusion gene in acute myeloid leukemia (Lekanne Deprez et al., 1995; Buijs et al., 2000). The TEL gene, also called ETV6, is a member of the ETS family of transcription factors, and is a partner in many different translocations leading to leukemia. Examples of its partner genes are AML1, ABL, PDGFbeta-R and EVI1. In these fusion proteins TEL contributes its N-terminal helix–loop–helix, or pointed domain, that can act as an oligomerization domain for the fusion partners (Golub et al., 1994,1996). In other translocations, TEL contributes its ETS-like DNA binding domain that is located at the C-terminus of the protein. In this case, the N-terminal part of the fusion is contributed by BTL, STL, PAX5, HLXB9 or MN1. PAX5 and HLXB9 encode transcription factors, leading to the hypothesis that the formation of a fusion protein results in altered specificity of both fusion partners (Beverloo et al., 2001; Cazzaniga et al., 2001). We have shown before that MN1–TEL acts as an altered transcription factor, since it could activate transcription from the moloney sarcoma virus long terminal repeat (MSV-LTR) and from a reporter construct carrying 5 TEL–responsive elements (Buijs et al., 2000). In addition, MN1–TEL was able to transform NIH3T3 cells as deduced from its ability to stimulate colony growth in soft agar. The MN1 protein contains two proline–glutamine stretches plus a glutamine stretch of 28 residues, encoded by a reiteration of CAG and CAA triplets. These features suggest that the MN1 protein itself may also have a function in transcription regulation. This notion was underscored by the finding that MN1 by itself localizes to the nucleus and was also able to activate the MSV-LTR (Buijs et al., 2000).

Recent experiments have addressed the properties of several leukemia-associated fusion proteins, among which are TEL–AML, PML–RARalpha and AML–ETO (Gelmetti et al., 1998; Shao et al., 2000; Shurtleff et al., 1995). These fusion proteins not only merely function as altered transcription factors, but behave as dominant negative factors, inhibiting the function of both partner proteins encoded by the unaltered alleles (Fenrick et al., 1999; Lin and Evans, 2000; Minucci et al., 2000). Thus, the balance between inhibition and induction of gene expression by master regulators is affected in leukemias that are caused by such fusion proteins. One of the key regulators of proliferation and differentiation of blood cell lineages is the retinoic acid receptor RARalpha. RARalpha is a member of the nuclear receptor family and works as a heterodimer with the retinoid X receptor (RXR). The RAR–RXR dimer binds to a 6 nucleotide long repeat separated by a string of five random base pairs, called a direct repeat 5 or DR5. The transcription of downstream genes is stimulated in the presence of the ligand all-trans retinoic acid (ATRA). Alternatively, the RAR–RXR heterodimers may bind to DR1 elements, a repeat separated by a single random base pair. In this case, transcription of downstream genes is repressed rather than induced (Zechel et al., 1994). Conversely, RXR–RXR homodimers bind DR1 elements and stimulate transcription when provided with their ligand 9-cis retinoic acid. In the presence of ATRA, members from the p160 family of transcription coactivators are recruited to RAR–RXR (Leo and Chen, 2000). These proteins bind to the ligand-binding domain of nuclear receptors and in turn recruit other factors, among which is the coactivator p300/CBP. P300/CBP can positively affect gene expression not only by forming a bridge between the sequence-specific transcription factors and the general transcription machinery, but also by acetylation of histones. The latter activity is thought to lead to a more open configuration of chromatin (Goodman and Smolik, 2000). In the absence of ligand, the DNA-bound RAR–RXR heterodimer downregulates expression by recruiting corepressors like NCoR and SMRT. These proteins also bind to the ligand-binding domain of the receptors, but they in turn recruit the histone deacetylase complex SIN3.

In the leukemias that are caused by altered transcription factors such as PML–RARalpha, the balance between inhibition and induction of gene expression is affected. The transcription downregulating effect of PML–RARalpha relative to RARalpha is because of the ability of the PML moiety in the fusion protein to bind the corepressors NCoR and SMRT more tightly than RARalpha can. Only in the presence of nonphysiological levels of ATRA, the corepressors can be released from PML–RARalpha and gene expression induced. Indeed, patients with acute promyelocytic leukemia caused by the PML–RARalpha translocation have been treated successfully with pharmacological concentrations of ATRA (Warrell et al., 1993). Downregulation of RAR responsive genes, for example by PML–RARalpha, results in a block in differentiation and an increase in the number of myelocytic precursor cells (Lin and Evans, 2000; Wang et al., 1998). Such changes in the balance between active transcription and inhibition of transcription through an altered recruitment of corepressors and activators are now thought to be the basis of the leukemogenic effects of most translocations in which transcription factors participate. It is therefore not unlikely that the MN1–TEL fusion plays a similar role.

To gain more insight into the function of this fusion protein in leukemia, we started by studying the biological role of the least well-understood partner, the MN1 protein. In the work described here, we show that MN1 can synergize with a sequence specific transcription factor, such as the retinoic acid receptor, and with the p160 and p300 coactivators in expression driven by the MSV-LTR.

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Results

MN1 comprises several transcription activating domains

To identify possible transcription activating domains (TADs), we constructed fusion proteins in which different regions of MN1 are coupled to the yeast GAL4 protein DNA binding domain (GAL4-DBD). Figure 1 shows an outline of the different fusion proteins used. The nuclear localization and expression of all constructs was verified using an antibody against the GAL4 moiety (results not shown). The GAL4-DBD-VP16 construct was used as a positive control. When the entire (AA 1-1319) or almost entire (AA 48-1319) MN1 protein was fused to the GAL4-DBD, a transactivating activity similar to the GAL4-DBD-VP16 fusion protein was measured. Deletion mapping of the MN1 protein revealed that the region between amino acids 48–139 harbors the major transactivating activity. A weaker but persistent transactivating effect was also observed with constructs containing amino acids 365–520 and 576–1319. Domains 48–139 and 365–520 are also active in the induction of transcription in yeast (results not shown). These results suggest that MN1 indeed has a function in regulation of transcription.

Figure 1.
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MN1 encompasses transcription activating domains. Different regions of MN1 were fused in frame to the DNA binding domain of the yeast GAL4 transcription factor and used for transient transfection of Hep3B cells together with a luciferase reporter harboring GAL4 responsive elements. Fusion constructs are depicted in the left panel and the corresponding activity is shown on the right. The nuclear localization of all constructs was verified using an antibody against the GAL4 moiety, and Western blots of lysates of transfected cells revealed that most proteins were expressed to a similar extent

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MN1 recognizes retinoic acid responsive elements

In order to identify a putative DNA binding site for MN1, selection and amplification of high-affinity binding sites from a pool of random oligonucleotides was performed (Blackwell and Weintraub, 1990). Oligonucleotides containing a 15 nucleotide random central region flanked by two constant regions were incubated with cellular extracts of HtTA–MN1 cells expressing the MN1 cDNA. After five rounds of selection, using the MN1-specific monoclonal antibody 2F2, PCR fragments (only obtained in the experiment with MN1 expressing cells) were cloned and 50 plasmid inserts were sequenced. The sequences revealed a consensus element comprising a CACCC sequence that was observed 30 times (to be published elsewhere) and a perfect DRl 9-cis retinoic acid responsive element (RARE) consensus sequence (AGGTCAAAGGTCA) was found 7 times. The remaining 13 clones contained apparently random sequences. Close inspection of the MSV-LTR, a natural promoter that is activated by MN1 (Buijs et al., 2000), revealed the presence of two putative DR5 ATRA responsive elements related to the DRl sequence. Probably no DR5 sequence was obtained from the oligonucleotide selection because of the length of this repeat (17 nucleotides), which is more than the random core available in the oligonucleotide mixture. Both DR1 (AGGTCAAAGGTCA) and DR5 (AGTTCAGATCAAGGTCA) sequences were used for electrophoretic mobility shift assays. Band shifts were observed for both sequences, suggesting that these elements are genuine transcription factor binding elements (Figure 2a). However, no differences were observed in the migration pattern when cell lysates from MN1-expressing or nonexpressing cells were used. Also, the addition of an anti-MN1 monoclonal antibody did not result in a supershift, suggesting that MN1 is not binding to DRl and DR5 sequences under the conditions of the mobility shift experiments (results not shown). Very likely, the conditions in this assay are unfavorable for the necessary interactions. As an alternative method to determine the binding of MN1 to these sequences, we incubated immobilized oligonucleotides with lysates of cells expressing HA-MN1 and detected bound MN1 with the antihemagglutinin-specific antibody 12CA5 (Figure 2b). Identical results were obtained when the 2F2 antibody was used for detection. Empty beads and beads loaded with a random oligonucleotide were used as a negative control. Some background binding to these beads was observed, but the immobilized DR1 or DR5 oligonucleotides bound significantly more MN1 protein. This shows that MN1 indeed is capable of specifically recognizing these sequences. Since the DR1 and DR5 sequences represent classical binding sites for RXR and RAR, we determined the binding of the latter to the immobilized oligonucleotides as a control. RARalpha, expressed endogenously in the cells used for lysate preparation, was found to bind to the immobilized DR1 or DR5 oligonucleotides regardless of MN1 expression (Figure 2c). These results show that MN1 recognizes DR1 and DR5 elements. However, the binding of MN1 to these elements most probably is mediated by other proteins.

Figure 2.
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(a) Band shift assay showing that DR1 (AGGTCAAAGGTCA) and DR5 (AGTTCAGATCAAGGTCA) consensus sequences bind proteins. Band shifts can be competed by cold oligonucleotide. The presence of MN1 does not alter the band shift pattern. (b and c) Immobilized DR1 and DR5 sequences bind RARalpha and hemagglutinin-tagged MN1 in 3T3 cell lysates. Only HA–MN1 is recognized by the antibody, as the eluates from the non-MN1–expressing 3T3 cells are negative

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MN1 synergizes with the retinoic acid receptor in transcription from the MSV-LTR

We observed before that MN1 stimulates transcription from the MSV-LTR (Buijs et al., 2000). To investigate whether retinoic acid receptors and MN1 work together on the MSV-LTR, the reporter construct was cotransfected to human Hep3B cells with an MN1 cDNA expression construct in the presence or absence of the RAR ligand ATRA. Note that MN1 is not expressed endogenously in the used cell lines (results not shown).

As can be expected from the presence of RAREs, the MSV-LTR can also be induced by ATRA, and not only by MN1. Moreover, the effects of both MN1 and ATRA on this promoter were synergistic rather than additive (Figure 3a). The control construct for the MSV-LTR is a deletion construct (MSV20), which encompasses only the TATA box of the promoter. The expression from this reporter is very low, and also the level of induction by MN1 is low when compared to that of the full-length LTR. To demonstrate the importance of the RAREs for the effect of MN1 on the LTR, we have used a number of deletion constructs (Figure 3b). Upon deletion of the first RARE, a reduction in the effect of MN1 was observed, although some activity remained. The construct containing only the second RARE and the basal promoter (-123) was activated even more than the somewhat larger constructs. Deletion of the second RARE (nucleotide -33 with respect to the transcription start site) results in a construct that is hardly activated by MN1, ATRA, or both. The effect of coexpression of MN1 on two constructs in which the major part of the LTR was deleted are shown in Figure 3c. In constructs MSV–DR5 and RAR–DR5, the 5' DR5 from the MSV-LTR and the canonical DR5 from the RARbeta gene promoter were inserted at nucleotide -51 of the LTR, thereby deleting almost the entire LTR. Both DR5 elements collaborate efficiently with MN1, whereas the control containing only the TATA-box is not induced. Similar constructs in which the DR5 elements were inserted in front of another basic promoter, such as the promoter of the Herpes Simplex virus TK gene, did not lead to induction by MN1 nor did they result in synergy between MN1 and RAR/RXR-induced expression (data not shown). These results suggest that collaboration between MN1 and retinoic acid receptors is specific for certain promoter sequences.

Figure 3.
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(a) Synergistic induction of the MSV-LTR by MN1 and ATRA. MSV1 and MSV20 reporters were cotransfected with increasing amount of an MN1 expression construct. MN1 enhances transcription directed by the MSV-LTR in a dosage-dependent manner. Note that the Hep3B cells used for the transfections express RAR and RXR endogenously. The relative activation by MN1 is indicated in folds. (b) Effect of ATRA and MN1 on MSV-LTR deletion constructs. Various deletions of the MSV1 reporter (indicated in basepairs from the transcription start site) were cotransfected with MN1 expression construct. A decrease in the activation by MN1 is observed upon deletion of the first and second RARE in the LTR (indicated with an asterisk). The activation by MN1 is indicated in folds. (c) MN1 and ATRA also collaborate on a truncated promoter in which the MSV 5' DR5 or the DR5 from the RARbeta gene were inserted 51 nucleotides upstream of the transcription start site, at the same time deleting the remainder of the LTR

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To investigate the effects of MN1 and ATRA on expression directed by the LTR in more detail, we transfected a fixed amount of the MN1 expression construct and varied the ATRA concentration in the experiment. A synergistic effect of MN1 on ATRA-induced expression is observed over the whole concentration range (Figure 4a). Likewise, increasing amounts of the MN1 expression construct synergize with a fixed concentration of ATRA (Figure 4b). The most important conclusion from these experiments is that MN1 can synergize efficiently with RAR–RXR in the context of the MSV-LTR.

Figure 4.
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MN1 synergizes with RAR in expression directed by the MSV-LTR. The MSV1 reporter was cotransfected with various amounts of MN1 expression construct in the presence of different concentrations of ATRA. (a) A fixed amount of MN1 synergizes with a range of concentrations of ATRA and (b) increasing amounts of MN1 synergize with a fixed concentration of ATRA. Transfections were performed as described for Figure 3

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MN1 may be recruited by p160 or p300

Upon binding of ligand, nuclear hormone receptors activate gene transcription by recruiting coactivators from the p160 family, such as SRC-1, GRIP1/TIF2 or pCIP/RAC3 (Leo and Chen, 2000). In turn, these coactivators can recruit secondary coactivators from a much wider variety of proteins including p300/CBP. A possible explanation for the effect of MN1 on transcription may be that the protein is recruited to RAR–RXR by the p160 family of coactivators or p300/CBP. To test this hypothesis, cotransfections with increasing amounts of RAC3 or p300 expression plasmids were performed in the absence or presence of MN1 and/or ATRA. The results indicate synergy between RAC3 and MN1 and between RAC3 and RAR–RXR (Figure 5a). A combination of MN1, ATRA and RAC3 leads to an even higher induction of transcription. In this experiment, RAC3 could be replaced by the other p160 family member TIF2, but not by SRC1 (results not shown). Nearly identical results were also obtained when RAC3 was replaced by p300 (Figure 5b), resulting in comparable induction levels. Cotransfection of the coactivator p/CAF had no or only a minor effect on ATRA-induced expression of the LTR, and no influence of this coactivator on MN1 was noted (results not shown).

Figure 5.
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Effect of MN1 on transcription is mediated by p160 and p300. Cotransfection of increasing amounts of an RAC3 (a) or p300 (b) expression construct shows that these coactivators synergizes with MN1 and that a combination of MN1, ATRA, and RAC3 or p300 is very efficient to obtain high expression levels. Transfections were performed as described for Figure 3

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Since p300 appears to have an important role in the effect of MN1 on the MSV-LTR, its inhibition is expected to impair MN1 as well. The multiple inhibitory effects of the adenovirus E1A oncoprotein on the coactivator functions of p300/CBP have been described extensively before, and include inhibition of histone acetyl transferase activity, of transactivating activity, and of assembly of coactivation complexes (Yang et al., 1996; Chakravarti et al., 1999; Hamamori et al., 1999; Xu et al., 2000). The product of the 12S wild-type ElA mRNA strongly inhibits activation of the MSV-LTR in a transfection experiment (Figure 6a). We also used an ElA mutant in which part of the N-terminal p300-binding domain is deleted. This mutant is no longer able to inhibit the activity of p300 but still inhibits other target proteins such as the retinoblastoma family (Dorsman et al., 1995). In contrast to the wild-type E1A, an equal amount of mutant E1A (Figure 6b) inhibited the basal expression level of the LTR far less efficiently than the wild-type protein. Moreover, in the presence of wild-type E1A, the positive effect of ATRA or MN1 were almost completely obliterated, whereas in the presence of mutant E1A, a considerable part of the expression could be restored. In conclusion, these experiments strongly suggest that MN1 as a coactivator needs the activity of p300 on the promoter for proper functioning.

Figure 6.
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(a) MN1-stimulated expression from the MSV-LTR in the presence of adenovirus E1A protein. Wild-type E1A efficiently inhibits the transcription-stimulating activities of the MN1 protein in the presence or absence of ATRA. In contrast, a deletion mutant E1A-1101 lacking the p300-binding site, is a far less effective inhibitor. (b) Western blot showing that the amounts of wild-type and mutant E1A protein in the experiment are similar. Transfections were performed as described for Figure 3

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Physical interaction between MN1 and p160 or p300

Since MN1 appears to bear several transcription-activating domains, the possibility arises that these work through the p160 or p300 coactivators. To test this hypothesis, we have analysed the ability of RAC3 and p300 to enhance expression directed by the GAL4–DBD–MN1 fusion proteins (Figure 7a). In analogy to the synergy with MN1 on the MSV-LTR, RAC3 and p300 were able to stimulate the GAL4–DBD–MN1 fusion proteins. This effect is greatest for the stronger transactivating domains present in pMN37 and pMN81. However, also the activity of the weaker transactivating domain represented pMN73 can be enhanced to some extent. These results suggest that the coactivator function of MN1 is closely linked to its interaction with p160 and p300. To show physical interaction, we have used the two strongest transactivating domains, amino acids 48–256 and 365–520, for the construction and purification of glutathione-S-transferase (GST) fusion proteins. Unfortunately, fusion proteins bearing amino acids 586–1319 could not be reliably expressed in E. coli. Sepharose-bound fusion proteins were incubated with in vitro translated RAC3 or Hep3B cell lysates expressing endogenous p300. Bound material was eluted and analysed by SDS–PAGE followed by autoradiography or Western blotting. The fused N-terminal transactivating domain was consistently found to bind RAC3 and p300, whereas GST alone shows no or only a very weak interaction (Figure 7b). In vitro translation of RAC3 results in two bands of which the lower one binds MN1 more efficiently than the larger one. At present, we do not know whether these bands are the results of different conformations, different translation start sites or proteolytic activity. The more C-terminal transactivating domain shows a weaker interaction with RAC3, and no apparent interaction with p300, consistent with the observation that the N-terminal part acts as a stronger transactivating domain than the more C-terminal domain. These results provide a molecular basis for the effects of MN1 on RAR–RXR. In addition, we tested the binding of RAR and RXR to these GST fusion proteins, but did not find any significant binding. Also when GST–RAR or GST–RXR fusion proteins were used, no binding of in vitro translated MN1 was observed (unpublished results). This suggests that MN1 does not bind RAR–RXR directly, and that p160 and p300 are candidates for intermediary factors.

Figure 7.
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(a) Transcription activating domains of MN1 can be activated further by p160 and p300. Luciferase reporters were cotransfected with fixed amounts of GAL4–MN1 fusion constructs and increasing amounts of RAC3 or p300 constructs. In the presence of endogenous levels of coactivators, pMN35, 31, 37 and 81 activate transcription. RAC3 and p300 further enhance expression driven by the GAL4–MN1 hybrid proteins bearing functional TADs. (b) Physical interaction between MN1 and p160/p300. GST fusion bearing amino acids 48–256 or 365–520 were incubated with 35S-labeled RAC3 or endogenously expressed p300. Eluates were separated by SDS–PAGE and detected by autoradiography or Western blotting, respectively. The control, GST alone, bound very little or no p160/p300 proteins, whereas a fusion bearing amino acid 48–256 of MN1 binds a low but significant amount of both. A fusion bearing amino acids 365–520 only binds to p160

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Discussion

We have studied the properties of the MN1 protein, the fusion partner of TEL in the t(12;22)(pl3;q11) that is found in a number of cases of myeloid leukemia. Previously, the resulting fusion protein was shown to activate transcription directed by TEL-responsive elements, whereas TEL itself is a represser (Buijs et al., 2000). By itself, MN1 also seemed to function as a transcription factor. The protein activated transcription from the MSV-LTR, a naturally occurring complex promoter. However, the nature of the interaction between MN1 and putative target promoters has not been elucidated so far. In this paper, we provide evidence that the MN1 protein functions as a transcription coactivator for the retinoic acid receptor, rather than being a sequence-specific transcription factor.

The selection of a sequence from a pool of random oligonucleotides and the binding of MN1 to these oligonucleotides show that both DR1 and DR5 repeats, classical consensus sequences for retinoic acid receptors (Mangelsdorf et al., 1991; Nakshatri and Bhat-Nakshatri, 1998), form valid targets for MN1. Still, MN1 does not bear any sequence similarity with the otherwise conserved family of nuclear hormone receptors to which the retinoic acid receptors belong. Therefore, we think that it is unlikely that MN1 represents a novel member of this family. Consequently, we favor the hypothesis that MN1 is not a sequence-specific DNA binding factor but works by binding to other proteins. This hypothesis is also supported by the absence of the so-called squelching phenomenon (Shemshedini et al., 1992; Shibata et al., 1997). This effect is caused by many sequence-specific transcription factors, and results in a drastic inhibition of promoter activity because of competition for a limiting number of coactivator complexes. Overexpression of transcription factors that do not bind directly to the promoter, such as the p160 coactivators, does not result in squelching. For instance GRIP1/TIF2 shows no signs of squelching even when expressed to very high levels (Hong et al., 1996). We have shown here that MN1 behaves in a similar manner.

Cotransfection experiments in the presence of ligands show that MN1 can synergize with RAR–RXR. Not only does the addition of higher concentrations of ATRA work synergistically with a constant amount of MN1, but also increasing amounts of MN1 give a synergistic effect at one concentration of ATRA.

The activity of the nuclear hormone receptor superfamily is mediated largely through p160 (SRC1-like) coactivators. These proteins interact with the ligand-binding domain of the receptors through their conserved LXXLL binding motifs (Ding et al., 1998; Li and Chen, 1998; Voegel et al., 1998). The p160 coactivators enhance transcription by way of their intrinsic HAT activity and also recruit other cofactors such as p300/CBP (Li and Chen, 1998; Voegel et al., 1998). MN1 shares with some of these coactivators its proline/glutamine-rich domains and its long stretch of glutamine residues. Other structural features, however, are lacking in the MN1 protein. There is no evidence for a bHLH/PAS domain and, more importantly, MN1 lacks the obligatory LXXLL motifs. Clearly, MN1 does not replace p160 or p300, as cotransfections of MN1 with RAC3 or p300 show a clear synergistic effect on activation of transcription. The binding of RAC3 and p300 to the GST fusion protein also shows that this interaction is of cooperative rather than complementary nature.

The direct stimulating effect of RAC3 and p300 on GAL4-MN1-mediated expression indicates that nuclear hormone receptors are not required for the interaction between the different coactivators. In these transfections, addition of ATRA does not affect the activity of the reporter (results not shown). The activity of MN1 on the MSV-LTR, however, is stimulated significantly by the addition of ATRA, corresponding to a model in which MN1 is recruited to RAR–RXR in a hormone-dependent way, for example by p160 or p300. As is obvious from experiments done by us (Figure 5) and by others (Ding et al., 1998; Li and Chen, 1998; Voegel et al., 1998), p160 and p300 are capable of stimulating RAR–RXR in the absence of MN1. Also, the repression of MN1 activity by ElA is compatible with MN1 functioning as a coactivator that is recruited by a p300-containing complex. Whereas the interaction of MN1 with p160 and p300 is easily demonstrated by luciferase assays and in vitro binding, the binding of MN1 to RAR–RXR has proven to be more elusive. The GST pull-down experiments indicate that MN1 does not bind directly to RAR–RXR. Still, MN1 recognizes RAREs, works synergistically with RAR–RXR, and binds to well-know coactivators of RAR–RXR. All our findings are consistent with the classical model in which p160 is the first coactivator to bind to nuclear hormone receptors (Leo and Chen, 2000; Voegel et al., 1998). The collaboration between MN1 and RAR/RXR on the very short DR5-promoter constructs, and the finding that not all DR5-promoter sequences can be stimulated by the combination of MN1 and RAR/RXR, suggests that the observed synergy between MN1 and RAR/RXR is brought about by facilitating interaction between specific transcription initiation complexes and the RAR/RXR–pl60–p300 complex. We are at present searching for proteins binding to MN1 by mass spectrometry to address this question.

Our experiments show that several domains of the MN1 protein efficiently activate transcription when tethered to a promoter through the GAL4–DBD. We have shown that these transactivating domains most likely function by binding to p160 and p300. In MN1TEL, almost the entire MN1 sequence is conserved. Therefore, the simplest explanation for the function of the MN1–TEL fusion protein is that genes that are normally repressed by TEL, which is a transcription represser (Chakrabarti and Nucifora, 1999; Lopez et al., 1999), are activated because of the combination of TEL DNA binding and MN1 transactivating properties. For transformation of NIH 3T3 cells by MN1 TEL, the amino-terminal MN1 transactivating domain is essential (Buijs et al., 2000). In the transformation assay, this domain cannot be replaced by other TADs such as the VP16 TAD. These findings underline the importance of the p160- and p300-binding domain of MN1 for the activating and transforming properties of the MN1–TEL fusion protein. The model of activation and transformation by MN1–TEL resembles the mechanism of the Ewing sarcoma translocations exemplified by the EWS–FLI fusion (Bailly et al., 1994). On the other hand, it is also plausible that the MN1–TEL fusion affects normal MN1 function as well, similar to the pleiotropic effects of other leukemia associated hybrid transcription factors. An indication of such a scenario is presented by the finding that an MN1–TEL mutant defective in DNA binding is no longer able to activate the LTR, even though the fragment of MN1 present in the fusion protein is as active on the LTR as full-length MN1 (Buijs et al., 2000). A possible explanation for this effect is that the TEL moiety somehow impedes tethering of MN1 to the promoter through RAR–RXR or the functional interaction with p160 and p300 coactivators (unpublished results). The properties of MN1 that are described in this work will enable a further study in order to identify more of the players that participate in its normal activity and its role in the t(12;22) translocation.

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Experimental procedures

Plasmids

A construct expressing the human p300 protein was provided by Dr A Zantema with permission of Dr R Eckner, and an expression construct for RAC3 was provided by Dr G Jenster with permission of Dr D Chen. Expression plasmids for wild-type and mutant viral ElA proteins were provided by Dr JC Dorsman.

For expression purposes, the MN1 cDNA (Lekanne Deprez et al., 1995) was cloned into vector pcDNA3 (Invitrogen, Carlsbad, CA, USA), under control of the CMV promoter, resulting in plasmid pMN50. A vector for the expression of hemagglutinin-tagged MN1 (HA–MN1) was created by insertion of an oligonucleotide coding for the appropriate peptide in pGeneV5A (Invitrogen) and subsequent insertion of the MN1 cDNA (amino acids 48–1319). In-frame fusions to the yeast GAL4–DBD were constructed in plasmid pGBT9 (Clontech, Palo Alto, CA, USA) and subsequently transferred to pcDNA3 for expression in mammalian cells. Expression of HA–MN1 and MN1 GAL4–DBD fusions was confirmed by Western blotting. Deletion constructs of the pGLMSV1 reporter were made by exonuclease III digestion and confirmed by sequencing. The MSV–DR5 and RAR–DR5 contructs were generated by ligating double-stranded oligonucleotides carrying these sequences to the BssHII site at position -51 with respect to the transcription start site of the LTR, at the same time deleting all LTR sequences upstream of this site. A reporter construct bearing the luciferase gene downstream of five GAL4 upstream activator sequences and the E1b TATA box was made by transferring the luciferase gene from pGL2Basic (Clontech) to pGeneV5A (Invitrogen).

Antibodies

A cDNA fragment encompassing MN1 amino acids 48–256 was fused in frame with the PinPoint™Xa-1 vector (Promega, Madison, WI, USA). The fusion construct was expressed in E. coli and the fusion protein was purified as recommended by the manufacturer. Mice were immunized with the fusion protein and monoclonal antibodies were isolated as described before (den Bakker et al., 1995). Monoclonal antibody 2F2 is specific for the N-terminal MN1 fragment (amino acids 48–256). The antibody specifically recognizes the MN1 protein produced by in vitro transcription–translation and in cell lysates from mammalian cells transfected with MN1 cDNA expression vector. Polyclonal antibodies directed against RARalpha and p300 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Random oligonucleotide binding selection

To identify a DNA binding site for MN1, we used a modification of the method described by Blackwell and Weintraub (Blackwell and Weintraub, 1990). A pool of oligonucleotides containing a 15-nucleotide random central region flanked by two constant regions was incubated with cellular extracts of HtTA-MN1-25 cells expressing the MN1 protein. HtTA cells not expressing MN1 served as the negative control. The 2F2 monoclonal antibody was used to precipitate protein–DNA complexes. Bound DNA was amplified and used for consecutive rounds of binding and precipitation. After five rounds of selection, PCR fragments were cloned and sequenced.

Cell culture and transfections

Hep3B cell were cultured in alpha-MEM supplemented with 5% fetal calf serum and antibiotics. COS and HeLa cells were cultured in DMEM supplemented with 5% fetal calf serum and antibiotics. HtTA cells (Gossen and Bujard, 1992) were maintained in DMEM supplemented with 10% fetal calf serum and tetracyclin (2 mug/ml). An expression construct in which MN1 is under control of the tetracyclin operator, was introduced in these cells using calcium phosphate precipitation. After selection on puromycin (0.5 mug/ml), colonies were picked, cultured for 64 h in the absence of tetracyclin, and checked for MN1 expression. A clone with efficient expression of the MN1 protein was selected for further experiments. 3T3-Switch cells (Invitrogen) were maintained in DMEM containing 10% fetal calf serum and hygromycin (50 mug/ml). A pGeneV5A-based expression construct for HA–MN1 was introduced in these cells, and colonies were selected after selection on zeocin (200 mug/ml). A clone with efficient expression of HA–MN1 after overnight induction with mifepristone (10-8 M) was selected for further experiments.

For transient transfections, 8timesl04 cells were seeded per well of a 24 wells tissue culture plate. After 24 h, transfections were performed using 1.0 mul FuGENE 6 (Roche, Basel, Switzerland) per 0.5 mug of total plasmid DNA, as recommended by the manufacturer. In each experiment, the total amount of transfected DNA as well as the molar ratio of CMV promoter were kept constant. Cells were harvested, lysed, and expression of the luciferase reporter gene was assayed on a Fluoroscan Ascent FL luminometer (Labsystems, Helsinki, Finland) 24 h after transfection. All transfections were performed at least three times and in duplicate or triplicate, and the results that were obtained proved highly reproducible. The relative amount of protein produced by the various MN1 deletion and GAL4-fusion constructs was assayed by Western blotting of lysates from the transfected cells, and their expression level was found to be similar, showing that transfection efficiencies were reproducible within an experiment.

Electrophoretic mobility shift assays

Band shift experiments were performed using whole-cell extracts. Whole-cell extracts of HtTA–MN1 cells were prepared 48 h after release of tetracyclin repression. Cells from an 80–90% confluent 10 cm dish were washed once with TBS and scraped in 1 ml TBS. The cell pellet was resuspended in 200 mul of 20 mM HEPES-KOH, pH 7.9, 1 mM EDTA, 400 mM KC1, 10% glycerol, 10 mM DTT, 1 mM Pefablock SC (Roche). The cells were subjected to four cycles of freezing in liquid nitrogen and thawing on ice. Cellular debris was removed by centrifugation (15 min, 10 000 g). The cellular extract was then stored at -80°C. For a band shift experiment, 2 mul of extract was used in a 20 mul reaction with 10 000 c.p.m 32P-labeled double-stranded oligonucleotide in 10 mM HEPES-KOH pH 7.9, 60 mM KC1, 4% Ficoll, 1 mM DTT and 5 mM EDTA, and incubated on ice for 20 min. DNA–protein complexes were separated on a 4% polyacrylamide gel in 0.25 TBE buffer.

Oligonucleotide affinity binding assay

The creation of DR1 and DR5 oligonucleotide resin and the binding of HA–MN1 to this affinity resin were carried out essentially as described by Glass et al. (1988). A control resin containing a random oligonucleotide was prepared in the same way. Briefly, a biotinylated double-stranded oligonucleotide, which contains DR1 (AGGTCAAAGGTCA) or DR5 (AGTTCAGATCAAGGTCA) recognition sequences (100 pmol), was incubated with 75 mul of streptavidin-coated beads (Roche) at room temperature for 15 min in affinity resin binding buffer (15 mM Tris-HCl pH 7.5, 60 mM KCl, 7.5% glycerol, 1 mM DTT, 4 mM spermidine, 0.01% NP-40, 0.25% BSA). Remaining free streptavidin groups were blocked for 15 min with binding buffer containing 1 muM biotin, followed by three washes with affinity resin binding buffer. Approximately, 5timesl06 3T3-Switch cells overexpressing HA–MN1 were resuspended in the affinity resin buffer and lysed by four rounds of freezing and thawing. After clearing the debris, 250 mug of total cell lysate was incubated in the presence of 0.05 mug/ml dIodC (Pharmacia, Uppsala, Sweden) with either the DR1 or DR5 resin or control resin at room temperature for 30 min. The samples were then washed three times with the affinity resin buffer and once with phosphate-buffered saline (PBS). The retained proteins were released by adding SDS–PAGE loading buffer and boiling the mixture for 5 min. The supernatant was separated by 8% SDS–PAGE, transferred to PVDF membranes and analyzed by Western blot using 12CA5 monoclonal antibodies (Roche) directed against the hemagglutinin tag.

GST fusion protein pull-downs

Binding of cellular proteins to immobilized GST fusion proteins was carried out essentially according to Rachez et al. (1998). Two cDNA fragments encompassing MN1 amino acids 48–256 and 365–520 were fused in frame with the pGEX3X vector (Pharmacia). The fusion constructs were expressed in E. coli and the fusion protein was purified as recommended by the manufacturer. Approximately, 2timesl07 Hep3B cells were resuspended in binding buffer (20 mM Tris-HCl pH 7.9, 180 mM KCl, 0.2 mM EDTA, 1 mM DTT, 1 mM Pefablock SC, 0.05% NP-40, 0.1% BSA) and lysed by four freeze–thaw cycles. After clearing the debris, 750 mug of total cell lysate was incubated for 3 h at 4°C with glutathione sepharose columns containing fusion proteins. Columns containing nonfused GST were used for controls. Alternatively, 35S-methionine labeled in vitro transcription–translation products were used in the binding experiment. After binding, columns were washed six times with wash buffer (binding buffer without BSA) and eluted with wash buffer containing 0.2% N-lauryl-sarkosine. The eluates were separated by 8% SDS–PAGE, transferred to PVDF membranes and analysed by Western blotting. Labeled in vitro transcription–translation products were detected by autoradiography.

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Acknowledgements

We thank Nicole Groen and Lydia van den Andel-Thijssen for their excellent technical assistance. We thank Drs D Chen, R Eckner, R Evans and JC Dorsman for permission to use RAC3, p300 and E1A expression constructs. This work was supported by Dutch Cancer Society grants EUR 94-653 and 98-1778, and in part by NCI Grant CA72996-04 and the American Lebanese Syrian Associated Charities (ALSAC) of St Jude Children's Hospital.