Homeoprotein products of theVertebrate embryos are patterned along multiple trunk and limb axes by the Hox gene complex (42). This family encodes homeodomain-containing transcription factors that confer positional identity through the differential regulation of target gene expression. The 38 Hox genes found in mice and humans are arranged in four genomic clusters, Hoxa to Hoxd, related to the HOM complex of Drosophila melanogaster. These complexes can be aligned such that more closely related genes (paralogs) occupy the same position in each complex, suggesting that they arose by duplications of an ancestral complex (42). Recent phylogenetic comparisons indicate that all animals share related Hox/HOM-type gene clusters (76). Expression of the Hox and HOM genes largely follows a colinear pattern, such that the more 3Ј a gene is located in a cluster, the more anteriorly it is expressed. Thus, more closely related HOX proteins from the same paralogous group are expressed in similar fashions. Paralogous genes have both distinct and overlapping functions manifested in their most anterior domain of expression (7,30).DNA binding by HOX and HOM proteins is mediated by the homeodomain, a conserved 60-amino-acid sequence that consists of a flexible N-terminal arm followed by three alpha helices (25). Base-specific contacts are made by the N-terminal arm and helix 3 via the minor and major grooves, respectively. Structural studies have demonstrated that even highly diverged homeodomains interact with DNA in a conserved fashion (83). HOX/HOM homeodomains can be divided into major classes based on sequence homology. This division also serves to classify the proteins according to their DNA-binding preferences. The majority of HOX/HOM homeodomains belong to the Antennapedia (Antp) class (HOX paralogous groups 2 to 8), which includes a number of proteins shown to bind and activate through elements of 6 to 9 bp containing a TAAT core motif. All of the Antp class homeodomains contain arginine at position 3 in the N-terminal arm, which contacts the second position in the TAAT core. Abdominal-B (Abd-B) class homeodomains have a lysine at position 3, and Abd-B exhibits a preference for a TTAT core (17). The members of the labial subfamily (HOX paralogous group 1) are unique since none of their homeodomains encode basic residues at positions 2 and 3. At least for HOXA-1, this results in a decreased affinity for DNA rather than an alteration in DNA-binding specificity (63). This may necessitate cooperation with a cofactor for site-specific DNA-binding by HOXA-1.Three other aspects of DNA-binding by the HOX/HOM family suggest that cofactor interactions may be generally employed. First, inspection of gene regulatory sequences reveals a high proportion of presumptive HOX binding sites, suggesting that not all of these sites are used. Second, most HOX or HOM proteins recognize similar binding sites with only modest preferences, suggesting that the HOX homeodomain alone could not effectively discriminate between targets (8, 13, 18, 61). Th...

HOX proteins are sequence-specific DNA-binding transcription factors that play a crucial role in the specification of anteroposterior identity in the animal embryo (20, 54). Conservation within the DNA-binding homeodomains results in different HOX proteins recognizing similar regulatory elements with only modest preferences (reviewed in reference 27). High-affinity DNA binding is achieved when HOX proteins are heterodimerized with partners of the PBC family (mammalian PBX, Drosophila Extradenticle [EXD], and Caenorhabditis elegans CEH-20) (55). Mammalian MEIS1 has been shown to independently dimerize with HOX proteins and with PBX (11,57,78). Recently, trimeric complexes encompassing all three homeoproteins, HOX-PBX-MEIS, have also been characterized (77, 79). The MEIS-related protein PREP1, also known as PKNOX1, can additionally form a dimer with PBX, as well as a trimeric complex with HOX and PBX partners (6,7,15,34). While the majority of HOX monomers recognize a DNA core motif of TAAT (23), HOX-PBX, HOX-MEIS, and PBX-MEIS heterodimers recognize larger motifs resulting in a higher affinity and specificity of DNA binding by these homeoproteins (49).A conserved motif with the consensus YPWM is found N terminal to the homeodomain of HOX proteins from paralogous groups 1 to 8. The YPWM motif contacts the PBX homeodomain and is strictly required for cooperative DNA binding by PBX and HOX partners (49,50). A conserved W in HOX proteins from groups 9 and 10 performs a similar function (12).The downstream targets of mammalian HOX proteins have been poorly characterized. The best-characterized targets are some Hox genes known to be positively autoregulated by their own products or cross-regulated by the products of other Hox genes (26,68,69). In these instances, HOX-PBX complexes act as activators of transcription. For example, the Hoxb1 autoregulatory element (ARE) contains three binding sites for HOX-PBX complexes. These sites are required to direct expression of a Hoxb1 transgene in rhombomere 4 (r4) of the developing hindbrain (68).Genetic and molecular studies have provided evidence supporting a negative regulatory role for HOX proteins (43). In the case of decapentaplegic (dpp) regulation in Drosophila, repression by HOX proteins dominates over activation (9). This implies active transcriptional repression by HOX proteins (9,25,46). In addition, in vitro mapping studies have characterized repression domains in different HOX proteins, as well as in the PBX partner (13,45,75). Therefore, HOX proteins may be activators or repressors in a context-dependent manner.By analogy to nuclear receptors, HOX-PBX complexes are likely to achieve transcriptional repression or activation through differential association with coactivators and corepressors (81). One class of coregulators are the histone acetyltransferases (HATs) and the histone deacetylases (HDACs), which modify chromatin as well as nonhistone proteins. The HATs include GCN5, PCAF, CREB-binding protein (CBP)/p300, the steroid receptor coactivator class, and the MY...

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: