with HoxA10 or Drosophila even-skipped. Site-directed mutagenesis showed that the hexapeptide motif (IYPWMK) upstream of the Hox homeo domain was essential for HoxB6 and B7 to cooperatively bind DNA with Pbx proteins. Engraftment of the HoxB7 hexapeptide onto HoxA10 endowed it with robust cooperative properties, demonstrating a functional role for the highly conserved hexapeptide element as one of the molecular determinants delimiting Hox-Pbx cooperativity. The Pbx homeo domain was necessary but not sufficient for cooperativity, which required conserved amino acids carboxy-terminal of the homeo domain. These findings demonstrate that interactions between Hox and Pbx proteins modulate their DNA-binding properties, suggesting that Pbx and Hox proteins act in parallel as heterotypic complexes to regulate expression of specific subordinate genes.

Recent studies show that Hox homeodomain proteins from paralog groups 1 to 10 gain DNA binding specificity and affinity through cooperative binding with the divergent homeodomain protein Pbx1. However, the AbdB-like Hox proteins from paralogs 11, 12, and 13 do not interact with Pbx1a, raising the possibility of different protein partners. The Meis1 homeobox gene has 44% identity to Pbx within the homeodomain and was identified as a common site of viral integration in myeloid leukemias arising in BXH-2 mice. These integrations result in constitutive activation of Meis1. Furthermore, the Hoxa-9 gene is frequently activated by viral integration in the same BXH-2 leukemias, suggesting a biological synergy between these two distinct classes of homeodomain proteins in causing malignant transformation. We now show that the Hoxa-9 protein physically Although the vertebrate homeobox genes play a critical role in embryonic development (30), there has been little progress in understanding how homeodomain proteins function. Despite the assumption that they are transcription factors (24), few natural regulatory targets have been identified (13, 16), and little data exist showing an in vivo transcriptional role for the mammalian homeodomain proteins. Vertebrate homeobox genes can be divided into the Hox family of clustered genes which contain homeobox sequences related to the Drosophila HOM-C homeobox genes and the numerous subfamilies of non-Hox genes which possess more distantly related homeobox sequences. The 39 Hox genes are arranged in four separate loci (A, B, C, and D), and the genes in different clusters can be aligned on the basis of homology within the homeobox to form so-called paralog groups, which have limited homology with the Drosophila HOM-C genes as well (1). A significant advance in understanding how Hox proteins function has been the demonstration that they form cooperative DNA binding complexes with the non-Hox homeodomain protein Pbx1a (10, 27, 42). We have recently reported that Hox proteins from paralog groups 1 through 10 gain DNA binding specificity through cooperative binding with Pbx1a, but proteins from the remaining three paralog groups, 11, 12, and 13, do not appear to interact with Pbx1a (8, 47). These findings suggest that the Hox proteins from paralog groups 11 through 13 might cooperatively bind to DNA with other non-Hox homeodomain protein partners.In recent studies, we demonstrated that a novel homeobox gene, Meis1, is a common site of viral integration in myeloid leukemias occurring in BXH-2 mice (33). Two forms of the Meis1 transcript, which differ in the amino acid sequence Cterminal to the homeodomain, were identified in all tissues examined (33). The homeodomain of Meis1 has homology with that of Pbx1, and both proteins contain a 3-amino-acid insertion which is a feature of the TALE class of homeobox genes (2). With the exception of the recently described Meisrelated genes (34,48), the N-terminal and C-terminal flanking regions of Meis1 have no homology to known homeodomain proteins. A...

Aberrant activation of the HOX, MEIS, and PBX homeodomain protein families is associated with leukemias, and retrovirally driven coexpression of HOXA9 and MEIS1 is sufficient to induce myeloid leukemia in mice. Previous studies have demonstrated that HOX-9 and HOX-10 paralog proteins are unique among HOX homeodomain proteins in their capacity to form in vitro cooperative DNA binding complexes with either the PBX or MEIS protein. Furthermore, PBX and MEIS proteins have been shown to form in vivo heterodimeric DNA binding complexes with each other. We now show that in vitro DNA site selection for MEIS1 in the presence of HOXA9 and PBX yields a consensus PBX-HOXA9 site. MEIS1 enhances in vitro HOXA9-PBX protein complex formation in the absence of DNA and forms a trimeric electrophoretic mobility shift assay (EMSA) complex with these proteins on an oligonucleotide containing a PBX-HOXA9 site. Myeloid cell nuclear extracts produce EMSA complexes which appear to contain HOXA9, PBX2, and MEIS1, while immunoprecipitation of HOXA9 from these extracts results in coprecipitation of PBX2 and MEIS1. In myeloid cells, HOXA9, MEIS1, and PBX2 are all strongly expressed in the nucleus, where a portion of their signals are colocalized within nuclear speckles. However, cotransfection of HOXA9 and PBX2 with or without MEIS1 minimally influences transcription of a reporter gene containing multiple PBX-HOXA9 binding sites. Taken together, these data suggest that in myeloid leukemia cells MEIS1 forms trimeric complexes with PBX and HOXA9, which in turn can bind to consensus PBX-HOXA9 DNA targets.

There is increasing evidence that HOX homeobox genes play a role in leukemogenesis. Recent studies have demonstrated that enforced co-expression of HOXA9 and MEIS1 in murine marrow leads to rapid development of myeloid leukemia, and that these proteins exhibit cooperative DNA binding. However, it is unclear whether co-activation of HOXA9 and MEIS genes is a common occurrence in human leukemias. We surveyed expression of HOXA9 and MEIS1 in 24 leukemic cell lines and 80 patient samples, using RNase protection analyses and immunohistochemistry. We demonstrate that the expression of HOXA9 and MEIS1 in leukemia cells is uniquely myeloid, and that these genes are commonly co-expressed in myeloid cell lines and in samples of acute myelogenous leukemia (AML) of all subtypes except in promyelocytic leukemia. While HOXA9 is expressed in most cases of chronic myelogenous leukemia, MEIS1 is weakly expressed or not at all. Immunohistochemical staining of selected AML samples showed moderate to high levels of HOXA9 protein, primarily cytoplasmic, in leukemic myeloblasts, with weaker and primarily nuclear staining for MEIS1. These data support the concept that co-activation of HOXA9 and MEIS1 is a common event in AML, and may represent a common pathway of many different oncogenic mutations.

Previous studies showed that the Hox homeodomain proteins from paralog groups 1-8 display cooperative DNA binding with the non-Hox homeodomain protein Pbx, mediated by a canonical YPWM. Although the Abd-B-like Hox proteins in paralogs 9 -13 lack this sequence, Hoxb-9 and Hoxa-10 were reported to bind with Pbx1a to DNA. We show that these interactions require a tryptophan 6 amino acids N-terminal to the homeodomain. Binding site selection for Hoxb-9 with Pbx1a yielded ATGATTTACGAC, containing a novel TTAC Hox-binding site adjacent to a Pbx site. In the presence of Pbx1a, Hoxb-9 and Hoxa-10 bound to targets containing either TTAC or TTAT. These data extend previous findings that interactions with Pbx define a Hox protein binding code for different DNA sequences across paralog groups 1 through 10. Members of the 11, 12, and 13 paralogs do not cooperatively bind DNA with Pbx1a, despite the presence of tryptophan residues N-terminal to the homeodomain in Hoxd-12 and Hoxd-13. Hoxa-11, Hoxd-12, or Hoxd-13, in the presence of Pbx1a, selected a TTAC Hox site but lacking a Pbx1a site. These data suggest that Abd-B-like Hox proteins bind to a novel TTAC site and can be divided by their cooperative binding to DNA with Pbx1a.