Oncogene (2003) 22, 699–709 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc

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

Karel HM 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 Zwarthoff *,1

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

The t(12;22) creates an MN1–TEL fusion leading to TEL gene, also called ETV6, is a member of the ETS . The fusion partner TEL (ETV6) family of transcription factors, and is a partner in many is a member of the ETS family of transcription factors. different translocations leading to leukemia. Examples The nature of the other fusion partner, MN1, has not been of its partner are AML1, ABL, PDGFb-R and investigated in detail until now. We recently described that EVI1. In these fusion proteins TEL contributes its N- MN1 activates the transcription activity of the moloney terminal helix–loop–helix, or pointed domain, that can sarcoma virus long terminal repeat, indicating that this act as an oligomerization domain for the fusion partners protein itself may act as a transcription factor. We show (Golub et al., 1994, 1996). In other translocations, TEL here that MN1 comprises multiple transcription activating contributes its ETS-like DNA binding domain that is domains. A search for a bound DNA sequence revealed located at the C-terminus of the protein. In this case, the that MN1 has affinity for retinoic acid responsive N-terminal part of the fusion is contributed by BTL, elements. A DR5 retinoic acid responsive element was STL, PAX5, HLXB9 or MN1. PAX5 and HLXB9 observed in the LTR. The combination of MN1 and encode transcription factors, leading to the hypothesis ligand-activated leads to a syner- that the formation of a fusion protein results in altered gistic induction of expression directed by the LTR. specificity of both fusion partners (Beverloo et al., 2001; Cotransfection of MN1 with RAC3 or p300, known Cazzaniga et al., 2001). We have shown before that coactivators of retinoic acid receptors, leads to a further MN1–TEL acts as an altered transcription factor, since synergistic induction of transcription. In addition, the it could activate transcription from the moloney effect of MN1 can be inhibited by the wild-type sarcoma virus long terminal repeat (MSV-LTR) and adenovirus ElA protein that inhibits p300 function, but from a reporter construct carrying 5 TEL–responsive not by an E1A mutant lacking the p300-binding site. elements (Buijs et al., 2000). In addition, MN1–TEL was GAL4-MN1-mediated transcription can be enhanced able to transform NIH3T3 cells as deduced from its directly by RAC3 and p300. Taken together, our results ability to stimulate colony growth in soft agar. The indicate that MN1 is a transcription rather MN1 protein contains two proline–glutamine stretches than a sequence-specific transcription factor, and that it plus a glutamine stretch of 28 residues, encoded by a may stimulate RAR/RXR-mediated transcription through reiteration of CAG and CAA triplets. These features interaction with p160 and p300. suggest that the MN1 protein itself may also have a Oncogene (2003) 22, 699–709. doi:10.1038/sj.onc.1206124 function in transcription regulation. This notion was underscored by the finding that MN1 by itself localizes Keywords: translocation; leukemia; retinoic acid; hor- to the nucleus and was also able to activate the MSV- mone receptor; coactivator LTR (Buijs et al., 2000). Recent experiments have addressed the properties of several leukemia-associated fusion proteins, among which are TEL–AML, PML–RARa and AML–ETO Introduction (Gelmetti et al., 1998; Shao et al., 2000; Shurtleff et al., 1995). These fusion proteins not only merely function as The t(12;22)(p13;q11) leads to the formation of an altered transcription factors, but behave as dominant MN1–TEL fusion gene in acute myeloid leukemia negative factors, inhibiting the function of both partner (Lekanne Deprez et al., 1995; Buijs et al., 2000). The proteins encoded by the unaltered alleles (Fenrick et al., 1999; Lin and Evans, 2000; Minucci et al., 2000). Thus, *Correspondence: Dr EC Zwarthoff, Department of Pathology, the balance between inhibition and induction of gene Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The expression by master regulators is affected in leukemias Netherlands; E-mail: [email protected] that are caused by such fusion proteins. One of the key 4The first two authors contributed equally to this paper. Received 6 June 2002; revised 2 October 2002; accepted 9 regulators of proliferation and differentiation of blood October 2002 cell lineages is the retinoic acid receptor RARa.RARa is MN1 synergizes with RAR-RXR KHM van Wely et al 700 a member of the nuclear receptor family and works as a the p160 and p300 coactivators in expression driven by heterodimer with the (RXR). The the MSV-LTR. 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 Results downstream genes is stimulated in the presence of the ligand all-trans retinoic acid (ATRA). Alternatively, MN1 comprises several transcription activating domains the RAR–RXR heterodimers may bind to DR1 elements, a repeat separated by a single random To identify possible transcription activating domains . In this case, transcription of downstream (TADs), we constructed fusion proteins in which genes is repressed rather than induced (Zechel et al., different regions of MN1 are coupled to the yeast 1994). Conversely, RXR–RXR homodimers bind DR1 GAL4 protein DNA binding domain (GAL4-DBD). elements and stimulate transcription when provided Figure 1 shows an outline of the different fusion with their ligand 9-cis retinoic acid. In the presence of proteins used. The nuclear localization and expression ATRA, members from the p160 family of transcription of all constructs was verified using an antibody against coactivators are recruited to RAR–RXR (Leo and the GAL4 moiety (results not shown). The GAL4-DBD- Chen, 2000). These proteins bind to the ligand-binding VP16 construct was used as a positive control. When the domain of nuclear receptors and in turn recruit other entire (AA 1-1319) or almost entire (AA 48-1319) MN1 factors, among which is the coactivator p300/CBP. protein was fused to the GAL4-DBD, a transactivating P300/CBP can positively affect gene expression not only activity similar to the GAL4-DBD-VP16 fusion protein by forming a bridge between the sequence-specific was measured. Deletion mapping of the MN1 protein transcription factors and the general transcription revealed that the region between amino acids 48–139 machinery, but also by acetylation of histones. The harbors the major transactivating activity. A weaker but latter activity is thought to lead to a more open persistent transactivating effect was also observed with configuration of chromatin (Goodman and Smolik, constructs containing amino acids 365–520 and 576– 2000). In the absence of ligand, the DNA-bound 1319. Domains 48–139 and 365–520 are also active in RAR–RXR heterodimer downregulates expression by the induction of transcription in yeast (results not recruiting like NCoR and SMRT. These shown). These results suggest that MN1 indeed has a proteins also bind to the ligand-binding domain of the function in regulation of transcription. receptors, but they in turn recruit the histone deacetylase complex SIN3. MN1 recognizes retinoic acid responsive elements In the leukemias that are caused by altered transcrip- tion factors such as PML–RARa, the balance between In order to identify a putative DNA binding site for inhibition and induction of gene expression is affected. MN1, selection and amplification of high-affinity bind- The transcription downregulating effect of PML–RARa ing sites from a pool of random oligonucleotides was relative to RARa is because of the ability of the PML performed (Blackwell and Weintraub, 1990). Oligonu- moiety in the fusion protein to bind the corepressors cleotides containing a 15 nucleotide random central NCoR and SMRT more tightly than RARa can. Only in region flanked by two constant regions were incubated the presence of nonphysiological levels of ATRA, the with cellular extracts of HtTA–MN1 cells expressing the corepressors can be released from PML–RARa and MN1 cDNA. After five rounds of selection, using the gene expression induced. Indeed, patients with acute MN1-specific monoclonal antibody 2F2, PCR frag- promyelocytic leukemia caused by the PML–RARa ments (only obtained in the experiment with MN1 translocation have been treated successfully with phar- expressing cells) were cloned and 50 plasmid inserts were macological concentrations of ATRA (Warrell et al., sequenced. The sequences revealed a consensus element 1993). Downregulation of RAR responsive genes, for comprising a CACCC sequence that was observed 30 example by PML–RARa, results in a block in differ- times (to be published elsewhere) and a perfect DRl 9-cis entiation and an increase in the number of myelocytic retinoic acid responsive element (RARE) consensus precursor cells (Lin and Evans, 2000; Wang et al., 1998). sequence (AGGTCAAAGGTCA) was found 7 times. Such changes in the balance between active transcription The remaining 13 clones contained apparently random and inhibition of transcription through an altered sequences. Close inspection of the MSV-LTR, a natural recruitment of corepressors and activators are now promoter that is activated by MN1 (Buijs et al., 2000), thought to be the basis of the leukemogenic effects of revealed the presence of two putative DR5 ATRA most translocations in which transcription factors responsive elements related to the DRl sequence. participate. It is therefore not unlikely that the MN1– Probably no DR5 sequence was obtained from the TEL fusion plays a similar role. oligonucleotide selection because of the length of this To gain more insight into the function of this fusion repeat (17 nucleotides), which is more than the random protein in leukemia, we started by studying the core available in the oligonucleotide mixture. Both DR1 biological role of the least well-understood partner, the (AGGTCAAAGGTCA) and DR5 (AGTTCAGAT- MN1 protein. In the work described here, we show that CAAGGTCA) sequences were used for electrophoretic MN1 can synergize with a sequence specific transcrip- mobility shift assays. Band shifts were observed for both tion factor, such as the retinoic acid receptor, and with sequences, suggesting that these elements are genuine

Oncogene MN1 synergizes with RAR-RXR KHM van Wely et al 701

Figure 1 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

transcription factor binding elements (Figure 2a). How- (Figure 2c). These results show that MN1 recognizes ever, no differences were observed in the migration DR1 and DR5 elements. However, the binding of MN1 pattern when cell lysates from MN1-expressing or to these elements most probably is mediated by other nonexpressing cells were used. Also, the addition of an proteins. anti-MN1 monoclonal antibody did not result in a supershift, suggesting that MN1 is not binding to DRl MN1 synergizes with the retinoic acid receptor in and DR5 sequences under the conditions of the mobility transcription from the MSV-LTR shift experiments (results not shown). Very likely, the conditions in this assay are unfavorable for the We observed before that MN1 stimulates transcription necessary interactions. As an alternative method to from the MSV-LTR (Buijs et al., 2000). To investigate determine the binding of MN1 to these sequences, we whether retinoic acid receptors and MN1 work together incubated immobilized oligonucleotides with lysates of on the MSV-LTR, the reporter construct was cotrans- cells expressing HA-MN1 and detected bound MN1 fected to human Hep3B cells with an MN1 cDNA with the antihemagglutinin-specific antibody 12CA5 expression construct in the presence or absence of the (Figure 2b). Identical results were obtained when the RAR ligand ATRA. Note that MN1 is not expressed 2F2 antibody was used for detection. Empty beads and endogenously in the used cell lines (results not shown). beads loaded with a random oligonucleotide were used As can be expected from the presence of RAREs, the as a negative control. Some background binding to these MSV-LTR can also be induced by ATRA, and not only beads was observed, but the immobilized DR1 or DR5 by MN1. Moreover, the effects of both MN1 and oligonucleotides bound significantly more MN1 protein. ATRA on this promoter were synergistic rather than This shows that MN1 indeed is capable of specifically additive (Figure 3a). The control construct for the MSV- recognizing these sequences. Since the DR1 and DR5 LTR is a deletion construct (MSV20), which encom- sequences represent classical binding sites for RXR and passes only the TATA box of the promoter. The RAR, we determined the binding of the latter to the expression from this reporter is very low, and also the immobilized oligonucleotides as a control. RARa, level of induction by MN1 is low when compared to that expressed endogenously in the cells used for lysate of the full-length LTR. To demonstrate the importance preparation, was found to bind to the immobilized DR1 of the RAREs for the effect of MN1 on the LTR, we or DR5 oligonucleotides regardless of MN1 expression have used a number of deletion constructs (Figure 3b).

Oncogene MN1 synergizes with RAR-RXR KHM van Wely et al 702

Figure 2 (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 RARa 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

Figure 3 (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 50 DR5 or the DR5 from the RARb gene were inserted 51 nucleotides upstream of the transcription start site, at the same time deleting the remainder of the LTR

Oncogene MN1 synergizes with RAR-RXR KHM van Wely et al 703 Upon deletion of the first RARE, a reduction in the MN1 may be recruited by p160 or p300 effect of MN1 was observed, although some activity remained. The construct containing only the second Upon binding of ligand, nuclear hormone receptors RARE and the basal promoter (À123) was activated activate gene transcription by recruiting coactivators even more than the somewhat larger constructs. Dele- from the p160 family, such as SRC-1, GRIP1/TIF2 or tion of the second RARE (nucleotide À33 with respect pCIP/RAC3 (Leo and Chen, 2000). In turn, these to the transcription start site) results in a construct that coactivators can recruit secondary coactivators from a is hardly activated by MN1, ATRA, or both. The effect much wider variety of proteins including p300/CBP. A of coexpression of MN1 on two constructs in which the possible explanation for the effect of MN1 on transcrip- major part of the LTR was deleted are shown in tion may be that the protein is recruited to RAR–RXR Figure 3c. In constructs MSV–DR5 and RAR–DR5, the by the p160 family of coactivators or p300/CBP. To test 50 DR5 from the MSV-LTR and the canonical DR5 this hypothesis, cotransfections with increasing amounts from the RARb gene promoter were inserted at of RAC3 or p300 expression plasmids were performed nucleotide À51 of the LTR, thereby deleting almost in the absence or presence of MN1 and/or ATRA. The the entire LTR. Both DR5 elements collaborate results indicate synergy between RAC3 and MN1 and efficiently with MN1, whereas the control containing between RAC3 and RAR–RXR (Figure 5a). A combi- only the TATA-box is not induced. Similar constructs in nation of MN1, ATRA and RAC3 leads to an even which the DR5 elements were inserted in front of higher induction of transcription. In this experiment, another basic promoter, such as the promoter of the RAC3 could be replaced by the other p160 family Herpes Simplex virus TK gene, did not lead to induction member TIF2, but not by SRC1 (results not shown). by MN1 nor did they result in synergy between MN1 Nearly identical results were also obtained when RAC3 and RAR/RXR-induced expression (data not shown). was replaced by p300 (Figure 5b), resulting in compar- These results suggest that collaboration between MN1 able induction levels. Cotransfection of the coactivator and retinoic acid receptors is specific for certain p/CAF had no or only a minor effect on ATRA-induced promoter sequences. expression of the LTR, and no influence of this To investigate the effects of MN1 and ATRA on coactivator on MN1 was noted (results not shown). expression directed by the LTR in more detail, we Since p300 appears to have an important role in the transfected a fixed amount of the MN1 expression effect of MN1 on the MSV-LTR, its inhibition is construct and varied the ATRA concentration in the expected to impair MN1 as well. The multiple inhibitory experiment. A synergistic effect of MN1 on ATRA- effects of the adenovirus E1A oncoprotein on the induced expression is observed over the whole concen- coactivator functions of p300/CBP have been described tration range (Figure 4a). Likewise, increasing amounts extensively before, and include inhibition of histone of the MN1 expression construct synergize with a fixed acetyl transferase activity, of transactivating activity, concentration of ATRA (Figure 4b). The most impor- and of assembly of coactivation complexes (Yang et al., tant conclusion from these experiments is that MN1 can 1996; Chakravarti et al., 1999; Hamamori et al., 1999; synergize efficiently with RAR–RXR in the context of Xu et al., 2000). The product of the 12S wild-type ElA the MSV-LTR. mRNA strongly inhibits activation of the MSV-LTR in

Figure 4 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

Oncogene MN1 synergizes with RAR-RXR KHM van Wely et al 704

Figure 5 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

needs the activity of p300 on the promoter for proper functioning.

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 Figure 6 (a) MN1-stimulated expression from the MSV-LTR in fusion proteins. This effect is greatest for the stronger the presence of adenovirus E1A protein. Wild-type E1A efficiently transactivating domains present in pMN37 and pMN81. inhibits the transcription-stimulating activities of the MN1 protein in the presence or absence of ATRA. In contrast, a deletion mutant However, also the activity of the weaker transactivating E1A-1101 lacking the p300-binding site, is a far less effective domain represented pMN73 can be enhanced to some inhibitor. (b) Western blot showing that the amounts of wild-type extent. These results suggest that the coactivator and mutant E1A protein in the experiment are similar. Transfec- function of MN1 is closely linked to its interaction with tions were performed as described for Figure 3 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 a transfection experiment (Figure 6a). We also used an acids 586–1319 could not be reliably expressed in E. coli. ElA mutant in which part of the N-terminal p300- Sepharose-bound fusion proteins were incubated with in binding domain is deleted. This mutant is no longer able vitro translated RAC3 or Hep3B cell lysates expressing to inhibit the activity of p300 but still inhibits other endogenous p300. Bound material was eluted and target proteins such as the retinoblastoma family analysed by SDS–PAGE followed by autoradiography (Dorsman et al., 1995). In contrast to the wild-type or Western blotting. The fused N-terminal transactivat- E1A, an equal amount of mutant E1A (Figure 6b) ing domain was consistently found to bind RAC3 and inhibited the basal expression level of the LTR far less p300, whereas GST alone shows no or only a very weak efficiently than the wild-type protein. Moreover, in the interaction (Figure 7b). In vitro translation of RAC3 presence of wild-type E1A, the positive effect of ATRA results in two bands of which the lower one binds MN1 or MN1 were almost completely obliterated, whereas in more efficiently than the larger one. At present, we do the presence of mutant E1A, a considerable part of the not know whether these bands are the results of different expression could be restored. In conclusion, these conformations, different translation start sites or pro- experiments strongly suggest that MN1 as a coactivator teolytic activity. The more C-terminal transactivating

Oncogene MN1 synergizes with RAR-RXR KHM van Wely et al 705

Figure 7 (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 domain shows a weaker interaction with RAC3, and no The selection of a sequence from a pool of random apparent interaction with p300, consistent with the oligonucleotides and the binding of MN1 to these observation that the N-terminal part acts as a stronger oligonucleotides show that both DR1 and DR5 repeats, transactivating domain than the more C-terminal classical consensus sequences for retinoic acid receptors domain. These results provide a molecular basis for (Mangelsdorf et al., 1991; Nakshatri and Bhat-Naksha- the effects of MN1 on RAR–RXR. In addition, we tri, 1998), form valid targets for MN1. Still, MN1 does tested the binding of RAR and RXR to these GST not bear any sequence similarity with the otherwise fusion proteins, but did not find any significant binding. conserved family of nuclear hormone receptors to which Also when GST–RAR or GST–RXR fusion proteins the retinoic acid receptors belong. Therefore, we think were used, no binding of in vitro translated MN1 was that it is unlikely that MN1 represents a novel member observed (unpublished results). This suggests that MN1 of this family. Consequently, we favor the hypothesis does not bind RAR–RXR directly, and that p160 and that MN1 is not a sequence-specific DNA binding factor p300 are candidates for intermediary factors. 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 Discussion sequence-specific transcription factors, and results in a drastic inhibition of promoter activity because of We have studied the properties of the MN1 protein, the competition for a limiting number of coactivator fusion partner of TEL in the t(12;22)(pl3;q11) that is complexes. Overexpression of transcription factors that found in a number of cases of myeloid leukemia. do not bind directly to the promoter, such as the p160 Previously, the resulting fusion protein was shown to coactivators, does not result in squelching. For instance activate transcription directed by TEL-responsive ele- GRIP1/TIF2 shows no signs of squelching even when ments, whereas TEL itself is a represser (Buijs et al., expressed to very high levels (Hong et al., 1996). We 2000). By itself, MN1 also seemed to function as a have shown here that MN1 behaves in a similar manner. transcription factor. The protein activated transcription Cotransfection experiments in the presence of ligands from the MSV-LTR, a naturally occurring complex show that MN1 can synergize with RAR–RXR. Not promoter. However, the nature of the interaction only does the addition of higher concentrations of between MN1 and putative target promoters has not ATRA work synergistically with a constant amount of been elucidated so far. In this paper, we provide MN1, but also increasing amounts of MN1 give a evidence that the MN1 protein functions as a transcrip- synergistic effect at one concentration of ATRA. tion coactivator for the retinoic acid receptor, rather The activity of the nuclear hormone receptor super- than being a sequence-specific transcription factor. family is mediated largely through p160 (SRC1-like)

Oncogene MN1 synergizes with RAR-RXR KHM van Wely et al 706 coactivators. These proteins interact with the ligand- MN1TEL, almost the entire MN1 sequence is con- binding domain of the receptors through their conserved served. Therefore, the simplest explanation for the LXXLL binding motifs (Ding et al., 1998; Li and Chen, function of the MN1–TEL fusion protein is that genes 1998; Voegel et al., 1998). The p160 coactivators that are normally repressed by TEL, which is a enhance transcription by way of their intrinsic HAT transcription represser (Chakrabarti and Nucifora, activity and also recruit other cofactors such as p300/ 1999; Lopez et al., 1999), are activated because of the CBP (Li and Chen, 1998; Voegel et al., 1998). MN1 combination of TEL DNA binding and MN1 transacti- shares with some of these coactivators its proline/ vating properties. For transformation of NIH 3T3 cells glutamine-rich domains and its long stretch of glutamine by MN1 TEL, the amino-terminal MN1 transactivating residues. Other structural features, however, are lacking domain is essential (Buijs et al., 2000). In the transfor- in the MN1 protein. There is no evidence for a bHLH/ mation assay, this domain cannot be replaced by other PAS domain and, more importantly, MN1 lacks the TADs such as the VP16 TAD. These findings underline obligatory LXXLL motifs. Clearly, MN1 does not the importance of the p160- and p300-binding domain replace p160 or p300, as cotransfections of MN1 with of MN1 for the activating and transforming properties RAC3 or p300 show a clear synergistic effect on of the MN1–TEL fusion protein. The model of activation of transcription. The binding of RAC3 and activation and transformation by MN1–TEL resembles p300 to the GST fusion protein also shows that this the mechanism of the Ewing sarcoma translocations interaction is of cooperative rather than complementary exemplified by the EWS–FLI fusion (Bailly et al., 1994). nature. On the other hand, it is also plausible that the MN1– The direct stimulating effect of RAC3 and p300 on TEL fusion affects normal MN1 function as well, GAL4-MN1-mediated expression indicates that nuclear similar to the pleiotropic effects of other leukemia hormone receptors are not required for the interaction associated hybrid transcription factors. An indication of between the different coactivators. In these transfec- such a scenario is presented by the finding that an MN1– tions, addition of ATRA does not affect the activity of TEL mutant defective in DNA binding is no longer able the reporter (results not shown). The activity of MN1 on to activate the LTR, even though the fragment of MN1 the MSV-LTR, however, is stimulated significantly by present in the fusion protein is as active on the LTR as the addition of ATRA, corresponding to a model in full-length MN1 (Buijs et al., 2000). A possible which MN1 is recruited to RAR–RXR in a hormone- explanation for this effect is that the TEL moiety dependent way, for example by p160 or p300. As is somehow impedes tethering of MN1 to the promoter obvious from experiments done by us (Figure 5) and by through RAR–RXR or the functional interaction with others (Ding et al., 1998; Li and Chen, 1998; Voegel p160 and p300 coactivators (unpublished results). The et al., 1998), p160 and p300 are capable of stimulating properties of MN1 that are described in this work will RAR–RXR in the absence of MN1. Also, the repression enable a further study in order to identify more of the of MN1 activity by ElA is compatible with MN1 players that participate in its normal activity and its role functioning as a coactivator that is recruited by a p300- in the t(12;22) translocation. containing complex. Whereas the interaction of MN1 with p160 and p300 is easily demonstrated by luciferase in vitro assays and binding, the binding of MN1 to Experimental procedures RAR–RXR has proven to be more elusive. The GST pull-down experiments indicate that MN1 does not bind Plasmids directly to RAR–RXR. Still, MN1 recognizes RAREs, works synergistically with RAR–RXR, and binds to A construct expressing the human p300 protein was well-know coactivators of RAR–RXR. All our findings provided by Dr A Zantema with permission of Dr R are consistent with the classical model in which p160 is Eckner, and an expression construct for RAC3 was the first coactivator to bind to nuclear hormone provided by Dr G Jenster with permission of Dr D receptors (Leo and Chen, 2000; Voegel et al., 1998). Chen. Expression plasmids for wild-type and mutant The collaboration between MN1 and RAR/RXR on the viral ElA proteins were provided by Dr JC Dorsman. very short DR5-promoter constructs, and the finding For expression purposes, the MN1 cDNA (Lekanne that not all DR5-promoter sequences can be stimulated Deprez et al., 1995) was cloned into vector pcDNA3 by the combination of MN1 and RAR/RXR, suggests (Invitrogen, Carlsbad, CA, USA), under control of the that the observed synergy between MN1 and RAR/ CMV promoter, resulting in plasmid pMN50. A vector RXR is brought about by facilitating interaction for the expression of hemagglutinin-tagged MN1 (HA– between specific transcription initiation complexes and MN1) was created by insertion of an oligonucleotide the RAR/RXR–pl60–p300 complex. We are at present coding for the appropriate peptide in pGeneV5A searching for proteins binding to MN1 by mass (Invitrogen) and subsequent insertion of the MN1 spectrometry to address this question. cDNA (amino acids 48–1319). In-frame fusions to the Our experiments show that several domains of the yeast GAL4–DBD were constructed in plasmid pGBT9 MN1 protein efficiently activate transcription when (Clontech, Palo Alto, CA, USA) and subsequently tethered to a promoter through the GAL4–DBD. We transferred to pcDNA3 for expression in mammalian have shown that these transactivating domains most cells. Expression of HA–MN1 and MN1 GAL4–DBD likely function by binding to p160 and p300. In fusions was confirmed by Western blotting. Deletion

Oncogene MN1 synergizes with RAR-RXR KHM van Wely et al 707 constructs of the pGLMSV1 reporter were made by the MN1 protein was selected for further experiments. exonuclease III digestion and confirmed by sequencing. 3T3-Switch cells (Invitrogen) were maintained in The MSV–DR5 and RAR–DR5 contructs were gener- DMEM containing 10% fetal calf serum and hygro- ated by ligating double-stranded oligonucleotides carry- mycin (50 mg/ml). A pGeneV5A-based expression con- ing these sequences to the BssHII site at position À51 struct for HA–MN1 was introduced in these cells, and with respect to the transcription start site of the LTR, at colonies were selected after selection on zeocin (200 mg/ the same time deleting all LTR sequences upstream of ml). A clone with efficient expression of HA–MN1 after this site. A reporter construct bearing the luciferase gene overnight induction with mifepristone (10À8 m) was downstream of five GAL4 upstream activator sequences selected for further experiments. and the E1b TATA box was made by transferring the For transient transfections, 8 Â l04 cells were seeded luciferase gene from pGL2Basic (Clontech) to pGene- per well of a 24 wells tissue culture plate. After 24 h, V5A (Invitrogen). transfections were performed using 1.0 ml FuGENE 6 (Roche, Basel, Switzerland) per 0.5 mg of total plasmid Antibodies DNA, as recommended by the manufacturer. In each experiment, the total amount of transfected DNA as A cDNA fragment encompassing MN1 amino acids 48– well as the molar ratio of CMV promoter were kept 256 was fused in frame with the PinPointtXa-1 vector constant. Cells were harvested, lysed, and expression of (Promega, Madison, WI, USA). The fusion construct the luciferase reporter gene was assayed on a Fluoroscan was expressed in E. coli and the fusion protein was Ascent FL luminometer (Labsystems, Helsinki, Fin- purified as recommended by the manufacturer. Mice land) 24 h after transfection. All transfections were were immunized with the fusion protein and monoclonal performed at least three times and in duplicate or antibodies were isolated as described before (den Bakker triplicate, and the results that were obtained proved et al., 1995). Monoclonal antibody 2F2 is specific for the highly reproducible. The relative amount of protein N-terminal MN1 fragment (amino acids 48–256). The produced by the various MN1 deletion and GAL4- antibody specifically recognizes the MN1 protein fusion constructs was assayed by Western blotting of produced by in vitro transcription–translation and in lysates from the transfected cells, and their expression cell lysates from mammalian cells transfected with MN1 level was found to be similar, showing that transfection cDNA expression vector. Polyclonal antibodies directed efficiencies were reproducible within an experiment. against RARa and p300 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Electrophoretic mobility shift assays

Random oligonucleotide binding selection Band shift experiments were performed using whole-cell extracts. Whole-cell extracts of HtTA–MN1 cells were To identify a DNA binding site for MN1, we used a prepared 48 h after release of tetracyclin repression. modification of the method described by Blackwell and Cells from an 80–90% confluent 10 cm dish were washed Weintraub (Blackwell and Weintraub, 1990). A pool of once with TBS and scraped in 1 ml TBS. The cell pellet oligonucleotides containing a 15-nucleotide random was resuspended in 200 mlof20mm HEPES-KOH, pH central region flanked by two constant regions was 7.9, 1 mm EDTA, 400 mm KC1, 10% glycerol, 10 mm incubated with cellular extracts of HtTA-MN1-25 cells DTT, 1 mm Pefablock SC (Roche). The cells were expressing the MN1 protein. HtTA cells not expressing subjected to four cycles of freezing in liquid nitrogen MN1 served as the negative control. The 2F2 mono- and thawing on ice. Cellular debris was removed by clonal antibody was used to precipitate protein–DNA centrifugation (15 min, 10 000 g). The cellular extract complexes. Bound DNA was amplified and used for was then stored at À801C. For a band shift experiment, consecutive rounds of binding and precipitation. After 2 ml of extract was used in a 20 ml reaction with 10 000 five rounds of selection, PCR fragments were cloned and c.p.m 32P-labeled double-stranded oligonucleotide in sequenced. 10 mm HEPES-KOH pH 7.9, 60 mm KC1, 4% Ficoll, 1mm DTT and 5 mm EDTA, and incubated on ice for Cell culture and transfections 20 min. DNA–protein complexes were separated on a 4% polyacrylamide gel in 0.25 TBE buffer. Hep3B cell were cultured in alpha-MEM supplemented with 5% fetal calf serum and antibiotics. COS and HeLa Oligonucleotide affinity binding assay cells were cultured in DMEM supplemented with 5% fetal calf serum and antibiotics. HtTA cells (Gossen and The creation of DR1 and DR5 oligonucleotide resin and Bujard, 1992) were maintained in DMEM supplemented the binding of HA–MN1 to this affinity resin were with 10% fetal calf serum and tetracyclin (2 mg/ml). An carried out essentially as described by Glass et al. (1988). expression construct in which MN1 is under control of A control resin containing a random oligonucleotide the tetracyclin operator, was introduced in these cells was prepared in the same way. Briefly, a biotinylated using calcium phosphate precipitation. After selection double-stranded oligonucleotide, which contains DR1 on puromycin (0.5 mg/ml), colonies were picked, cul- (AGGTCAAAGGTCA) or DR5 (AGTTCAGAT- tured for 64 h in the absence of tetracyclin, and checked CAAGGTCA) recognition sequences (100 pmol), was for MN1 expression. A clone with efficient expression of incubated with 75 ml of streptavidin-coated beads

Oncogene MN1 synergizes with RAR-RXR KHM van Wely et al 708 (Roche) at room temperature for 15 min in affinity resin protein was purified as recommended by the manufac- binding buffer (15 mm Tris-HCl pH 7.5, 60 mm KCl, turer. Approximately, 2 Â l07 Hep3B cells were resus- 7.5% glycerol, 1 mm DTT, 4 mm spermidine, 0.01% NP- pended in binding buffer (20 mm Tris-HCl pH 7.9, 40, 0.25% BSA). Remaining free streptavidin groups 180 mm KCl, 0.2 mm EDTA, 1 mm DTT, 1 mm Pefa- were blocked for 15 min with binding buffer containing block SC, 0.05% NP-40, 0.1% BSA) and lysed by four 1 mm biotin, followed by three washes with affinity resin freeze–thaw cycles. After clearing the debris, 750 mgof binding buffer. Approximately, 5 Â l06 3T3-Switch cells total cell lysate was incubated for 3 h at 41Cwith overexpressing HA–MN1 were resuspended in the glutathione sepharose columns containing fusion pro- affinity resin buffer and lysed by four rounds of freezing teins. Columns containing nonfused GST were used for and thawing. After clearing the debris, 250 mg of total controls. Alternatively, 35S-methionine labeled in vitro cell lysate was incubated in the presence of 0.05 mg/ml transcription–translation products were used in the dIodC (Pharmacia, Uppsala, Sweden) with either the binding experiment. After binding, columns were DR1 or DR5 resin or control resin at room temperature washed six times with wash buffer (binding buffer for 30 min. The samples were then washed three times without BSA) and eluted with wash buffer containing with the affinity resin buffer and once with phosphate- 0.2% N-lauryl-sarkosine. The eluates were separated by buffered saline (PBS). The retained proteins were 8% SDS–PAGE, transferred to PVDF membranes and released by adding SDS–PAGE loading buffer and analysed by Western blotting. Labeled in vitro transcrip- boiling the mixture for 5 min. The supernatant was tion–translation products were detected by autoradio- separated by 8% SDS–PAGE, transferred to PVDF graphy. membranes and analyzed by Western blot using 12CA5 monoclonal antibodies (Roche) directed against the hemagglutinin tag. Acknowledgments GST fusion protein pull-downs We thank Nicole Groen and Lydia van den Andel-Thijssen for Binding of cellular proteins to immobilized GST fusion their excellent technical assistance. We thank Drs D Chen, R Eckner, R Evans and JC Dorsman for permission to use proteins was carried out essentially according to Rachez RAC3, p300 and E1A expression constructs. This work was et al. (1998). Two cDNA fragments encompassing MN1 supported by Dutch Cancer Society grants EUR 94-653 and amino acids 48–256 and 365–520 were fused in frame 98-1778, and in part by NCI Grant CA72996-04 and the with the pGEX3X vector (Pharmacia). The fusion American Lebanese Syrian Associated Charities (ALSAC) of constructs were expressed in E. coli and the fusion St Jude Children’s Hospital.

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