These related genes fall into 13 paralogous groups. The products of groups 1 to 8 are more similar to the fly HOX protein Antennapedia (ANTP class), while 9 to 13 are related to the fly Abdominal-B (ABD-B class).Hox gene products function as sequence-specific DNA-binding transcription factors, as evidenced by their ability to regulate natural and artificial promoters in cell culture (18,48,49,53,61,63) and in the animal embryo (3,12,21,33,45,47,51,64).Homeodomain-DNA interactions are facilitated through residues in the flexible N-terminal arm and the recognition helix in the homeodomain (20), with the majority of HOX proteins binding a TAAT core motif. Asparagine 51 in the recognition helix plays a key role in the specificity of target site recognition, both in monomeric and in heterodimeric complexes (7,8,25,41,46,62). The size of the HOX family and their relatively poor discrimination in target site recognition suggest that they may interact with cofactors.Recently, it has been shown that the affinity and specificity of DNA binding by HOX proteins is indeed augmented by cofactor interactions. HOX cofactors in mammals include PBX (15,32,39,44,47) and MEIS (37, 57), members of the TALE family (9) of homeodomain proteins. The Drosophila homologs of mammalian PBX and MEIS are Extradenticle (EXD) (50) and Homothorax (HTH) (52), respectively. In mammals, while the majority of HOX proteins interact with PBX (HOX paralogs 1 to 10) (13), only the group 9 and 10 ABD-B class HOX proteins complex with MEIS (57).We and others have shown that a conserved motif present N terminal to the homeodomain of HOX proteins (11,15,23,27,39,43,44,54,55) and residues in the homeodomain of PBX (14,15,22,31,46) contact each other within the PBX-HOX cooperative complex. These results have been confirmed and extended through the resolution of the crystal structure of the cooperative complex (41,46). Systematic deletions carried out in HOXA9 and MEIS proteins have mapped amino acids (aa) 1 to 61 in HOXA9 and a region C terminal to the homeodomain of MEIS as responsible for mediating .In addition to their interaction with HOX proteins, MEIS and PBX form stable heterodimers that cooperatively bind DNA (16). The MEIS-related protein PREP1 interacts with PBX in a similar fashion (6). This interaction requires a portion of the first 89 aa in PBX and conserved N-terminal regions of MEIS or PREP. The chimeric oncoprotein E2A-PBX (24, 40) lacks the first 89 residues of PBX1 and is unable to interact with MEIS (16).Recently, a number of groups have reported on the formation of trimeric complexes involving proteins of the HOX, PBX, and MEIS extended families. In each case, a DNAbound PBX-HOX (or HOX-like) heterodimer tethers a member of the MEIS/PREP family via protein-protein interactions. Thus, PREP1 associates with DNA-bound HOXB1 and PBX, thereby modulating transcriptional activity (6). Mutation of the PREP1 homeodomain actually improves formation of the tri-* Corresponding author. Mailing address:

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HOX proteins are dependent upon cofactors of the PBX family for specificity of DNA binding. Two regions that have been implicated in HOX/PBX cooperative interactions are the YPWM motif, found N-terminal to the HOX homeodomain, and the GKFQ domain (also known as the Hox cooperativity motif) immediately C-terminal to the PBX homeodomain. Using derivatives of the E2A-PBX oncoprotein, we find that the GKFQ domain is not essential for cooperative interaction with HOXA1 but contributes to the stability of the complex. By contrast, the YPWM motif is strictly required for cooperative interactions in vitro and in vivo, even with mutants of E2A-PBX lacking the GKFQ domain. Using truncated PBX proteins, we show that the YPWM motif contacts the PBX homeodomain. The presence of the GKFQ domain increases monomer binding by the PBX homeodomain 5-fold, and the stability of the HOXA1⅐E2A-PBX complex 2-fold. These data suggest that the GKFQ domain acts mainly to increase DNA binding by PBX, rather than providing a primary contact site for the YPWM motif of HOXA1. We have identified 2 residues, Glu-301 and Tyr-305, required for GKFQ function and suggest that this is dependent on ␣-helical character.Hox genes are widely conserved regulators of anteroposterior patterning during animal development (1). The 39 mammalian Hox genes are expressed in overlapping domains and confer positional identity along the body axes (1, 2). The Hox genes encode homeodomain-containing transcription factors that bind a degenerate site with a TAAT core (3). Whereas modest binding-site preferences exist, these appear insufficient to account for specificity of action in vivo. The PBC (PBX and CEH-20) subfamily of homeodomain proteins (4), including PBX (pre-B-cell transformation-related gene) (5) and EXD (extradenticle) (6), are cofactors that greatly increase the specificity of HOX proteins. PBX is the mammalian homolog of Drosophila exd. The latter was first identified in a genetic screen for patterning defects in the fly (6, 7). The homeodomain proteins encoded by PBC genes cooperatively interact with HOX proteins to bind an extended site on DNA and regulate transcription in vivo (8 -17). PBC proteins improve HOX specificity due to the increased size of the cooperative binding site and the strength of DNA binding and by modulating recognition of cooperative binding sites by different groups of HOX proteins (15, 16, 18 -24).The human PBX family comprises three genes, PBX1, PBX2, and PBX3 (25). PBX1 was initially discovered through the study of the t(1:19) chromosomal translocation causing 25% of all acute pre-B cell leukemia (26,27). This translocation fuses the powerful transcriptional activation domains of E2A to the homeodomain and C terminus of PBX1, creating the novel transcription factor, E2A-PBX. The oncogenicity of E2A-PBX has been demonstrated in focus-forming assays, myeloid differentiation assays, and in mice (5,28,29). Studies done to uncover the regions of E2A-PBX required for transformation have identified the transcriptional activation dom...

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