The MN1 oncoprotein synergizes with coactivators RAC3 and p300 in RAR-RXR-mediated transcription Karel HM van Wely 1,4 , Anco C Molijn 1,4 , Arjan Buijs 2 , Magda A Meester-Smoor 1 , Albert Jan Aarnoudse 1 , Anita Hellemons 1 , Pim den Besten 1 , Gerard C Grosveld 3 and Ellen C Zwarthoff * ,1 1 Department of Pathology, Erasmus University, PO Box 1738, 3000 DR Rotterdam, The Netherlands; 2 Department of Hematology, University Hospital Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands; 3 Department of Genetics, St. Jude’s Children’s Research Hospital, Memphis, TN 38101-0318, USA 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 syner- gistic 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. Oncogene (2003) 22, 699–709. doi:10.1038/sj.onc.1206124 Keywords: translocation; leukemia; retinoic acid; hor- mone receptor; coactivator 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, PDGFb-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–RARa 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 RARa. RARa is Received 6 June 2002; revised 2 October 2002; accepted 9 October 2002 *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 4 The first two authors contributed equally to this paper. Oncogene (2003) 22, 699–709 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc