Oncogene (2001) 20, 5440 ± 5448 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc HoxB8 requires its Pbx-interaction motif to block dierentiation of primary myeloid progenitors and of most cell line models of myeloid dierentiation Paul S Knoep¯er2,3, David B Sykes1,3, Martina Pasillas1 and Mark P Kamps*,1 1Department of Pathology, University of California, San Diego, School of Medicine, 9500 Gilman Drive, La Jolla, California, CA 92093, USA; 2Department of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N., Seattle, Washington, WA 98109, USA HoxB8 was the ®rst homeobox gene identi®ed as a cause Introduction of leukemia. In murine WEHI3B acute myeloid leukemia (AML) cells, proviral integration leads to the expression Homeodomain (HD) proteins of the Hox, Pbx, and of both HoxB8 and Interleukin (IL-3). Enforced Meis families regulate gene expression in normal expression of HoxB8 blocks dierentiation of factor- development, and mutations that alter their expres- dependent myeloid progenitors, while IL-3 co-expression sion or function can lead to cancer. The mammalian induces autocrine proliferation and overt leukemogeni- Class I Hox genes are homologs of the Drosophila city. Previously, we demonstrated that HoxB8 binds homeobox genes located in the homeotic complex, DNA cooperatively with members of the Pbx family of and encode transcription factors that orchestrate transcription factors, and that HoxB8 makes contact anterior-posterior pattern formation (McGinnis and with the Pbx homeodomain through a hexameric Krumlauf, 1992), determine segmental identity during sequence designated the Pbx-interaction motif (PIM). embryogenesis (Krumlauf, 1994; Lewis, 1978) and E2a-Pbx1, an oncogenic derivative of Pbx1, both retains contribute to lineage-speci®c proliferation and/or its ability to heterodimerize with Hox proteins and arrest dierentiation of hematopoietic progenitors (Law- myeloid dierentiation. This observation prompts the rence et al., 1996). Class I Hox proteins can in¯uence question of whether E2a-Pbx1 and Hox oncoproteins use transcription through their binding to DNA as endogenous Hox and Pbx proteins, respectively, to monomers, and most are also capable of hetero- target a common set of cellular genes. Here, we use dimerizing on more complex sites with members of four dierent models of neutrophil and macrophage the three amino acid loop extension (TALE) family dierentiation to determine whether HoxB8 needs to of transcription factors, which include the products of bind DNA or Pbx cofactors in order to arrest myeloid the Pbx and Meis genes (Chang et al., 1996, 1997; dierentiation. The ability of HoxB8 to bind DNA or to Knoep¯er et al., 1996, 1997). Pbx1 is involved in the bind Pbx was essential (1) to block dierentiation of t(1;19) chromosomal translocation of pediatric pre-B factor-dependent myeloid progenitors from primary cell leukemia, which fuses the transcriptional activa- marrow; (2) to block IL-6-induced monocytic dierentia- tion domains of E2a to the HD of Pbx, producing a tion of M1-AML cells; and (3) to block granulocytic chimeric transcriptional activator that transforms dierentiation of GM-CSF-dependent ECoM-G cells. NIH3T3 ®broblasts (Kamps et al., 1991), prevents However, while DNA-binding was required, the HoxB8 dierentiation of myeloid progenitors (Kamps and Pbx-interaction motif was unnecessary for preventing Wright, 1994), and causes both AML (Kamps and macrophage dierentiation of ECoM-M cells. We Baltimore, 1993) and T-cell ALL in mice (Dedera et conclude that HoxB8 prevents dierentiation by directly al., 1993). Biochemical evidence (Lu and Kamps, in¯uencing cellular gene expression, and that the genetic 1996), later con®rmed by X-ray crystallography context within a cell dictates whether the eect of (Passner et al., 1999; Piper et al., 1999), revealed HoxB8 is dependent on a physical interaction with Pbx that a conserved tryptophan motif positioned N- proteins. Oncogene (2001) 20, 5440 ± 5448. terminal to the HD of Class I Hox proteins binds the Pbx1 HD. When the Hox tryptophan consensus Keywords: HoxB8; Pbx; myeloid; dierentiation motif (IYPWMR or ANWL; designated the Pbx- interaction motif or PIM) binds the Pbx HD, it alters the conformation of the Pbx HD so as to stabilize DNA-binding by the Pbx HD (Knoep¯er and Kamps, 1995; Lu and Kamps, 1996). Heterodimer- ization of Hox proteins with Pbx or E2a-Pbx1 on TGATTNAT (Pbx-Hox consensus motifs) also alters *Correspondence: MP Kamps; E-mail: [email protected] 3These authors contributed equally to this manuscript the conformation of the Hox HD, shifting the Received 1 March 2001; revised 1 June 2001; accepted 8 June 2001 binding speci®city for its 3' nucleotide core from The Pbx-interaction motif is required for myeloid immortalization by HoxB8 PS Knoepfler et al 5441 TAAT to TGAT or TTAT (Chang et al., 1996; sequences bind Pbx, and maintaining the possibility Knoep¯er et al., 1996; Lu and Kamps, 1997). that interaction with Pbx is essential for oncogenesis Subsets of Hox, Pbx,andMeis genes are expressed (Calvo et al., 2000). In contrast, mutations in the Pbx- during dierentiation of all hematopoietic lineages that interaction motif of Nup98-HoxA9 clearly abolish have been examined to date, raising the question of transcriptional cooperativity with Pbx and also prevent whether their function as oncogenes requires the the formation of foci on NIH3T3 monolayers (Kasper activity of endogenous heterodimer partners. One et al., 1999), providing direct evidence that Pbx oncogenic phenotype elicited by both Hox and E2a- proteins are important for transformation by Nup98- Pbx1 oncoproteins is blocking hematopoietic dieren- HoxA9. tiation: enforced expression of Hox11 (Hatano et al., HoxB8 was the ®rst Hox protein found to be 1991; Hawley et al., 1994), HoxA9 (Calvo et al., 2000), transcriptionally activated in AML (in WEHI3B mouse HoxA10 (Thorsteinsdottir et al., 1997), HoxB3 (Sau- myeloid leukemia cells). Persistent expression of vageau et al., 1997), HoxB4 (Sauvageau et al., 1995), HoxB8 prevents dierentiation of factor-dependent HoxB7 (Care et al., 1999), or HoxB8 (Blatt et al., 1988; myeloid progenitors (Blatt et al., 1988), and cooperates Perkins et al., 1990) prevents the dierentiation of a with mutations that establish autocrine proliferation to variety of myeloid and lymphoid progenitors. Among generate overt AML (Perkins et al., 1990). Here we these Hox proteins, the importance of interaction with examine whether the biochemical abilities of HoxB8 to Pbx and Meis partners for oncogenic function has been bind DNA and to bind Pbx proteins through its addressed for HoxB3, HoxB4, HoxB7, and HoxA9, as conserved Pbx-interaction motif are required for its well as Nup98-HoxA9, a transcriptionally activated cellular functions of blocking dierentiation of primary version of HoxA9 formed by the t(7;11) translocation marrow progenitors, as well as of three distinct cell line that fuses N-terminal sequences of Nucleoporin 98 models of neutrophil or macrophage dierentiation. (Nup98) to sequences just upstream of the HoxA9 Pbx- We demonstrate that DNA-binding is essential for all interaction motif and HD (Borrow et al., 1996; models of dierentiation arrest and that an intact Pbx- Nakamura et al., 1996). In the case of HoxB3 and interaction motif of HoxB8 is required to block HoxB4, the magnitude of their oncogenic function in myeloid dierentiation of all but one cell line. These Rat-1 ®broblast (number and size of colonies in soft data suggest that HoxB8 prevents myeloid dierentia- agar) is augmented by co-expression of Pbx1 (Krosl et tion in most cell types by a mechanism that requires al., 1998), providing indirect evidence that hetero- interactions with endogenous Pbx proteins on target dimerization with endogenous Pbx may be essential for gene promoters. their transforming properties. In the case of HoxB7, its ability to prevent G-CSF-induced granulocytic dierentiation of 32D cells requires its hexapeptide Results Pbx-interaction motif (Yaron et al., 2001). The question of whether transformation by HoxA9 The ability of HoxB8 to bind both Pbx and DNA is and NUP98-HoxA9 requires interaction with Pbx required to prevent differentiation of marrow-derived proteins has also been addressed by mutation of their myelomonocytic progenitors Pbx-interaction motifs. HoxA9 is required for normal myelopoiesis and lymphopoiesis as evidenced in HoxA9 A form of HoxB8 containing the conservative sub- knockout mice which are de®cient in their production stitution of phenylalanine for tryptophan in the Pbx- of mature granulocytes and lymphocytes (Lawrence et interaction motif (LFPWMR), designated al., 1997) and show both increased apoptosis and HoxB8W137F, was previously shown to be defective impaired T-cell dierentiation (Izon et al., 1998). in heterodimerization on DNA with Pbx proteins Enforced expression of HoxA9 blocks the dierentia- (Knoep¯er and Kamps, 1995). The ability of tion of myeloid progenitors capable of biphenotypic HoxB8W137F to bind DNA monomerically is equiva- dierentiation to either neutrophils in response to G- lent to that of wild-type HoxB8 (Figure 1, lane 5 vs 4) CSF or macrophages in response to M-CSF (Calvo et while its ability to heterodimerize with Pbx proteins al., 2000). Coactivation of HoxA9 and Meis1 tran- (Pbx1 or Pbx3) is reduced more than 20-fold (lanes 8 scription causes rapid AML in mice (Nakamura et al., and 11 vs 7 and 10). A form of HoxB8 predicted to be 1996; Kroon et al., 1998). HoxA9 is normally expressed incapable of binding DNA was produced by substitu- in CD34+ progenitors, downregulated during dier- tion of serine for asparagine at position 51 of the entiation, and expressed in all human AML (with the homeodomain (designated HoxB8N196S). Asp51 forms exception of promyelocytic leukemia), prompting an essential hydrogen bond with the third adenine speculation that persistent HoxA9 expression may residue in the core TAAT DNA element bound by Hox contribute to the dierentiation arrest in human proteins, while serine is incapable of forming this AML (Golub et al., 1999).