Meis1a suppresses differentiation by G-CSF and promotes proliferation by SCF: potential mechanisms of cooperativity with Hoxa9 in myeloid leukemia.

Hoxa9 and Meis1a are homeodomain transcription factors that heterodimerize on DNA and are down-regulated during normal myeloid differentiation. Hoxa9 and Meis1a cooperate to induce acute myeloid leukemia (AML) in mice, and are coexpressed in human AML. Despite their cooperativity in leukemogenesis, we demonstrated previously that retroviral expression of Hoxa9 alone--in the absence of coexpressed retroviral Meis1 or of expression of endogenous Meis genes--blocks neutrophil and macrophage differentiation of primary myeloid progenitors cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression of Meis1 alone did not immortalize any factor-dependent marrow progenitor. Because HoxA9-immortalized progenitors still execute granulocytic differentiation in response to granulocyte CSF (G-CSF) and monocyte differentiation in response to macrophage CSF (M-CSF), we tested the possibility that Meis1a cooperates with Hoxa9 by blocking viable differentiation pathways unaffected by Hoxa9 alone. Here we report that Meis1a suppresses G-CSF-induced granulocytic differentiation of Hoxa9-immortalized progenitors, permitting indefinite self-renewal in G-CSF. Meis1a also reprograms Hoxa9-immortalized progenitors to proliferate, rather than die, in response to stem cell factor (SCF) alone. We propose that Meis1a and Hoxa9 are part of a molecular switch that regulates progenitor abundance by suppressing differentiation and maintaining self-renewal in response to different subsets of cytokines during myelopoiesis. The independent differentiation pathways targeted by Hoxa9 and Meis1a prompt a "cooperative differentiation arrest" hypothesis for a subset of leukemia, in which cooperating transcription factor oncoproteins block complementary subsets of differentiation pathways, establishing a more complete differentiation block in vivo.

Estrogen-dependent E2a/Pbx1 myeloid cell lines exhibit conditional differentiation that can be arrested by other leukemic oncoproteins.

The molecular pathways of normal myeloid differentiation, as well as the mechanisms by which oncogenes disrupt this process, remain poorly understood. A major limitation in approaching this problem has been the lack of suitable cell lines that exhibit normal, terminal, and synchronous differentiation in the absence of endogenous oncoproteins and in response to physiologic cytokines, and whose differentiation can be arrested by ectopically expressed human oncoproteins. This report describes clonal, granulocyte-macrophage colony-stimulating factor-dependent myeloid cell lines that exhibit these properties. The cell lines were established by conditional immortalization of primary murine marrow progenitors with an estrogen-regulated E2a/Pbx1-estrogen receptor fusion protein. Clones were identified that proliferated as immortalized blasts in the presence of estrogen, and that exhibited granulocytic, monocytic, or bipotential (granulocytic and monocytic) differentiation on estrogen withdrawal. Differentiation was normal and terminal as evidenced by morphology, cell surface markers, gene expression, and functional assays. The differentiation of the cells could be arrested by heterologous oncoproteins including AML1/ETO, PML/RARalpha, PLZF/RARalpha, Nup98/HoxA9, and other Hox proteins. Furthermore, the study examined the effects of cooperating oncoproteins such as Ras or Bcr/Abl, which allowed for both factor-independent proliferation and differentiation, or Bcl-2, which permitted factor-independent survival but not proliferation. These myeloid cell lines provide tools for examining the biochemical and genetic pathways that accompany normal differentiation as well as a system in which to dissect how other leukemic oncoproteins interfere with these pathways.

HoxB8 requires its Pbx-interaction motif to block differentiation of primary myeloid progenitors and of most cell line models of myeloid differentiation.

HoxB8 was the first homeobox gene identified as a cause of leukemia. In murine WEHI3B acute myeloid leukemia (AML) cells, proviral integration leads to the expression of both HoxB8 and Interleukin (IL-3). Enforced expression of HoxB8 blocks differentiation of factor-dependent myeloid progenitors, while IL-3 co-expression induces autocrine proliferation and overt leukemogenicity. Previously, we demonstrated that HoxB8 binds DNA cooperatively with members of the Pbx family of transcription factors, and that HoxB8 makes contact with the Pbx homeodomain through a hexameric sequence designated the Pbx-interaction motif (PIM). E2a-Pbx1, an oncogenic derivative of Pbx1, both retains its ability to heterodimerize with Hox proteins and arrest myeloid differentiation. This observation prompts the question of whether E2a-Pbx1 and Hox oncoproteins use endogenous Hox and Pbx proteins, respectively, to target a common set of cellular genes. Here, we use four different models of neutrophil and macrophage differentiation to determine whether HoxB8 needs to bind DNA or Pbx cofactors in order to arrest myeloid differentiation. The ability of HoxB8 to bind DNA or to bind Pbx was essential (1) to block differentiation of factor-dependent myeloid progenitors from primary marrow; (2) to block IL-6-induced monocytic differentiation of M1-AML cells; and (3) to block granulocytic differentiation of GM-CSF-dependent ECoM-G cells. However, while DNA-binding was required, the HoxB8 Pbx-interaction motif was unnecessary for preventing macrophage differentiation of ECoM-M cells. We conclude that HoxB8 prevents differentiation by directly influencing cellular gene expression, and that the genetic context within a cell dictates whether the effect of HoxB8 is dependent on a physical interaction with Pbx proteins.

Hoxa9 immortalizes a granulocyte-macrophage colony-stimulating factor-dependent promyelocyte capable of biphenotypic differentiation to neutrophils or macrophages, independent of enforced meis expression.

The genes encoding Hoxa9 and Meis1 are transcriptionally coactivated in a subset of acute myeloid leukemia (AML) in mice. In marrow reconstitution experiments, coexpression of both genes produces rapid AML, while neither gene alone generates overt leukemia. Although Hoxa9 and Meis1 can bind DNA as heterodimers, both can also heterodimerize with Pbx proteins. Thus, while their coactivation may result from the necessity to bind promoters as heterodimers, it may also result from the necessity of altering independent biochemical pathways that cooperate to generate AML, either as monomers or as heterodimers with Pbx proteins. Here we demonstrate that constitutive expression of Hoxa9 in primary murine marrow immortalizes a late myelomonocytic progenitor, preventing it from executing terminal differentiation to granulocytes or monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3. This immortalized phenotype is achieved in the absence of endogenous or exogenous Meis gene expression. The Hoxa9-immortalized progenitor exhibited a promyelocytic transcriptional profile, expressing PU.1, AML1, c-Myb, C/EBP alpha, and C/EBP epsilon as well as their target genes, the receptors for GM-CSF, G-CSF, and M-CSF and the primary granule proteins myeloperoxidase and neutrophil elastase. G-CSF obviated the differentiation block of Hoxa9, inducing neutrophilic differentiation with accompanying expression of neutrophil gelatinase B and upregulation of gp91phox. M-CSF also obviated the differentiation block, inducing monocytic differentiation with accompanying expression of the macrophage acetyl-low-density lipoprotein scavenger receptor and F4/80 antigen. Versions of Hoxa9 lacking the ANWL Pbx interaction motif (PIM) also immortalized a promyelocytic progenitor with intrinsic biphenotypic differentiation potential. Therefore, Hoxa9 evokes a cytokine-selective block in differentiation by a mechanism that does not require Meis gene expression or interaction with Pbx through the PIM.

Direct interaction of two homeoproteins, homothorax and extradenticle, is essential for EXD nuclear localization and function.

The Drosophila Homothorax (HTH) and Extradenticle (EXD) are two homeoproteins required in a number of developmental processes. EXD can function as a cofactor to Hox proteins. Its nuclear localization is dependent on HTH. In this study we present evidence of in vivo physical interaction between HTH and EXD, mediated primarily through an evolutionarily conserved MH domain in HTH. This interaction is essential for the mutual stabilization of both proteins, for EXD nuclear localization, and for the cooperative DNA binding of the EXD-HTH heterodimer. Some in vivo functions require both EXD and HTH in the nucleus, suggesting that the EXD-HTH complex may function as a transcriptional regulator.

mAngiogenin-3, a target gene of oncoprotein E2a-Pbx1, encodes a new angiogenic member of the angiogenin family.

Angiogenins are proteins in the pancreatic ribonuclease superfamily that utilize their ribonuclease activity to induce formation of new blood vessels. Recently we identified a new member of the angiogenin gene family, mouse angiogenin-3, by virtue of its transcriptional activation in NIH3T3 fibroblasts coincident with transformation by the chimeric leukemia oncogene, E2a-Pbx1. Here we have isolated the cDNA encoding mouse angiogenin-3 and used it to produce the protein in E. coli. We demonstrate that mouse angiogenin-3 is a ribonuclease whose activity and specificity towards tRNA and dinucleotide substrates differ from those of mouse angiogenin or of mouse angiogenin-related protein, a non-angiogenic factor. Mouse angiogenin-3 induced angiogenesis in both the chicken embryo chorioallantoic membrane assay and the rat cremaster muscle. Electron microscopy revealed that endothelial cells within vessels induced by both mouse angiogenin-3 and mouse angiogenin contain fenestrations similar to those observed in endothelial cells from neovasculature induced by vascular endothelial growth factor and basic fibroblast growth factor. Mouse angiogenin-3 also induced other molecular events typical of rapidly proliferating endothelial cells, such as increases in rough endoplasmic reticulum, polysomes, and mitochondria.

A conserved motif N-terminal to the DNA-binding domains of myogenic bHLH transcription factors mediates cooperative DNA binding with pbx-Meis1/Prep1.

The t(1;19) chromosomal translocation of pediatric pre-B cell leukemia produces chimeric oncoprotein E2a-Pbx1, which contains the N-terminal transactivation domain of the basic helix-loop-helix (bHLH) transcription factor, E2a, joined to the majority of the homeodomain protein, Pbx1. There are three Pbx family members, which bind DNA as heterodimers with both broadly expressed Meis/Prep1 homeo-domain proteins and specifically expressed Hox homeodomain proteins. These Pbx heterodimers can augment the function of transcriptional activators bound to adjacent elements. In heterodimers, a conserved tryptophan motif in Hox proteins binds a pocket on the surface of the Pbx homeodomain, while Meis/Prep1 proteins bind an N-terminal Pbx domain, raising the possibility that the tryptophan-interaction pocket of the Pbx component of a Pbx-Meis/Prep1 complex is still available to bind trypto-phan motifs of other transcription factors bound to flanking elements. Here, we report that Pbx-Meis1/Prep1 binds DNA cooperatively with heterodimers of E2a and MyoD, myogenin, Mrf-4 or Myf-5. As with Hox proteins, a highly conserved tryptophan motif N-terminal to the DNA-binding domains of each myogenic bHLH family protein is required for cooperative DNA binding with Pbx-Meis1/Prep1. In vivo, MyoD requires this tryptophan motif to evoke chromatin remodeling in the Myogenin promoter and to activate Myogenin transcription. Pbx-Meis/Prep1 complexes, therefore, have the potential to cooperate with the myogenic bHLH proteins in regulating gene transcription.

EB-1, a tyrosine kinase signal transduction gene, is transcriptionally activated in the t(1;19) subset of pre-B ALL, which express oncoprotein E2a-Pbx1.

The t(1;19) translocation of pre-B cell acute lymphocytic leukemia (ALL) produces E2a-Pbx1, a chimeric oncoprotein containing the transactivation domains of E2a joined to the homeodomain protein, Pbx1. E2a-Pbx1 causes T cell and myeloid leukemia in mice, blocks differentiation of cultured myeloid progenitors, and transforms fibroblasts through a mechanism accompanied by aberrant expression of tissue-specific and developmentally-regulated genes. Here we investigate whether aberrant gene expression also occurs specifically in the t(1;19)-containing subset of pre-B cell ALL in man. Two new genes, EB-1 and EB-2, as well as Caldesmon were transcriptionally activated in each of seven t(1;19) cell lines. EB-1 expression was extremely low in marrow from patients having pre-B ALL not associated with the t(1;19), and elevated more than 100-fold in marrow from patients with pre-B ALL associated with the t(1;19). Normal EB-1 expression was strong in brain and testis, the same tissues exhibiting the highest levels of PBX1 expression. EB-1 encodes a signaling protein containing a phosphotyrosine binding domain homologous to that of dNumb developmental regulators and two SAM domains homologous to those in the C-terminal tail of Eph receptor tyrosine kinases. We conclude that aberrant expression of tissue-specific genes is a characteristic of t(1;19) pre-B ALL, as was previously found in fibroblasts transformed by E2a-Pbx1. Potentially, EB-1 overexpression could interfere with normal signaling controlling proliferation or differentiation.

An inhibitory switch derepressed by pbx, hox, and Meis/Prep1 partners regulates DNA-binding by pbx1 and E2a-pbx1 and is dispensable for myeloid immortalization by E2a-pbx1.

The Pbx/Exd family of homeodomain (HD) proteins contribute to the transcriptional and developmental roles of other Hox and Meis/Prep1/Hth HD proteins through heterodimer formation. E2a-Pbx1 is an oncogenic derrivative of Pbx1 produced by the t(1;19) translocation in pediatric pre-B cell acute lymphoblastic leukemia. E2a-Pbx1 heterodimerizes with Hox but not with Meis/Prep1 proteins, produces acute myeloid leukemia in mice, and blocks differentiation of cultured murine myeloid progenitors. Here, we characterize negative and positive regulatory sequences that flank the Pbx1 HD and determine their importance for myeloid immortalization by E2a-Pbx1. A 25 residue predicted alpha helix preceding the Pbx1 HD bound the HD and prevented both its binding to DNA and its ability to heterodimerize with Hox proteins. Addition of 39 residues N-terminal to this inhibitory helix exposed a Pbx dimerization interface that orchestrated cooperative DNA-binding of E2a-Pbx1 and all Pbx proteins as homodimers and heterdimers. Sequences inhibiting DNA-binding and mediating Pbx dimerization coincided with those reported to have nuclear export function. An additional 103 residues N-terminal to the Pbx dimerization interface restored heterodimerization with Hox and Meis1/Prep1 proteins. This negative switch domain - comprised of the inhibitory helix and N-terminal regions required for its partner-mediated derepression - was dispensable for myeloid immortalization by E2a-Pbx1. While stabilizing the heterodimer, the 310 helix C-terminal to the Pbx1 HD was also dispensable for the ability of E2a-Pbx1 to heterodimerize with Hox proteins and immortalize myeloblasts. Retention of myeloid immortalization by E2a-Pbx1 proteins lacking all Pbx1 sequences N- or C-terminal to the HD indicates that Hox proteins, or a yet undefined factor that binds the Pbx1 HD and derepresses DNA-binding by the HD, cooperate with E2a-Pbx1 in myeloid immortalization.

The highest affinity DNA element bound by Pbx complexes in t(1;19) leukemic cells fails to mediate cooperative DNA-binding or cooperative transactivation by E2a-Pbx1 and class I Hox proteins - evidence for selective targetting of E2a-Pbx1 to a subset of Pbx-recognition elements.

Oncoprotein E2a-Pbx1 contains the N-terminal transactivation domains of E2a and the majority of the homeodomain protein, Pbx1. Using recombinant proteins, both Pbx1 and E2a-Pbx1 heterodimerize with Hox proteins on bipartite elements, Pbx1 binding a 5' TGAT core and Class I Hox proteins binding adjacent 3' TAAT, TTAT, or TGAT cores. In contrast to these in vitro results, nuclear extracts from E2a-Pbx1-transformed cells assemble an abundant Pbx-containing complex on TGATTGAT that excludes E2a-Pbx1, suggesting that an uncharacterized in vivo partner discriminates between E2a-Pbx1 and Pbx proteins, distinguishing it from Hox proteins. Here, we describe the DNA-binding properties of this complex, and identify TGATTGAC (PCE; Pbx Consensus Element) as its optimal recognition motif. In vitro, the PCE fails to bind heterodimers of Class I Hox proteins plus either Pbx1 or E2a-Pbx1. Likewise, in vivo, the PCE fails to mediate cooperative transactivation by E2a-Pbx1 plus Class I Hox proteins. Thus, the PCE binds a Pbx dimer partner that behaves unlike Class I Hox proteins. Competition analysis indicates that the Pbx-containing complex that binds the PCE also binds the TGATTGAT Pbx-Hox element and binds promoter elements required for tissue-specific expression of a number of cellular genes. Thus, different Pbx partners dictate targetting of Pbx heterodimers to related DNA motifs that differ in the sequence of their 3' half-sites, and E2a-Pbx1 heterodimerizes with only a subset of Pbx partners, restricting its potential DNA targets.

The Pbx family of proteins is strongly upregulated by a post-transcriptional mechanism during retinoic acid-induced differentiation of P19 embryonal carcinoma cells.

Retinoic acid (RA) induces expression of genes encoding the Hox family of transcription factors, whose differential expression orchestrates developmental programs specifying anterior-posterior structures during embryogenesis. Hox proteins bind DNA as monomers and heterodimers with Pbx proteins. Here we show that RA upregulates Pbx protein abundance coincident with transcriptional activation of Hox genes in P19 embryonal carcinoma cells undergoing neuronal differentiation. However, in contrast to Hox induction, Pbx upregulation is predominantly a result of post-transcriptional mechanisms. Interestingly, Pbx1, Pbx2, and Pbx3 exhibit different profiles of upregulation, suggesting possible functional divergence. The parallel upregulation of Pbx and Hox proteins in this model suggests an important role for transcriptional control by Pbx-Hox heterodimers during neurogenesis, and argues for precise control by RA.

E2a-Pbx1 induces aberrant expression of tissue-specific and developmentally regulated genes when expressed in NIH 3T3 fibroblasts.

The E2a-Pbx1 oncoprotein contains the transactivation domain of E2a joined to the DNA-binding homeodomain (HD) of Pbx1. In mice, E2a-Pbx1 transforms T lymphoblasts and fibroblasts and blocks myeloblast differentiation. Pbx1 and E2a-Pbx1 bind DNA as heterodimers with other HD proteins whose expression is tissue specific. While the transactivation domain of E2a is required for all forms of transformation, DNA binding by the Pbx1 HD is essential for blocking myeloblast differentiation but dispensable for fibroblast or T-lymphoblast transformation. These properties suggest (i) that E2a-Pbx1 causes cellular transformation by activating gene transcription, (ii) that transcription of E2a-Pbx1 target genes is normally regulated by ubiquitous Pbx proteins and tissue-specific partners, and (iii) that DNA-binding mutants of E2a-Pbx1 activate a subset of all gene targets. To test these predictions, genes induced in NIH 3T3 fibroblasts by E2a-Pbx1 were identified and examined for tissue- and stage-specific expression and their differential abilities to be upregulated by E2a-Pbx1 in NIH 3T3 fibroblasts and myeloblasts and by a DNA-binding mutant of E2a-Pbx1 in NIH 3T3 cells. Of 12 RNAs induced by E2a-Pbx1, 4 encoded known proteins (a J-C region of the immunoglobulin kappa light chain, natriuretic peptide receptor C, mitochondrial fumarase, and the 3',5'-cyclic nucleotide phosphodiesterase, PDE1A) and 5 encoded new proteins related to angiogenin, ion channels, villin, epidermal growth factor repeat proteins, and the human 2.19 gene product. Expression of many of these genes was tissue specific or developmentally regulated, and most were not expressed in fibroblasts, indicating that E2a-Pbx1 can induce ectopic expression of genes associated with lineage-specific differentiation.

Heterodimerization of Hox proteins with Pbx1 and oncoprotein E2a-Pbx1 generates unique DNA-binding specifities at nucleotides predicted to contact the N-terminal arm of the Hox homeodomain--demonstration of Hox-dependent targeting of E2a-Pbx1 in vivo.

Hox proteins control genetic programs that orchestrate development, and a large subset of Hox proteins can bind DNA elements as heterodimers with the Pbx family of homeodomain proteins. A transcriptionally activated version of Pbx1, E2a-Pbx1, is an oncoprotein in human pre-B cell leukemia that strongly suppresses differentiation and retains its ability to heterodimerize with Hox proteins. Because monomeric Hox proteins bind very similar DNA motifs, it is unclear how they activate diverse developmental programs. Here we demonstrate that heterodimers containing different Hox proteins and a common Pbx1 or E2a-Pbx1 partner bind different DNA motifs. Structural models suggest that the specificity of the Hox protein is altered by a conformation change involving residues in the N-terminal arm of the Hox homeodomain. Mutational analysis also supported the hypothesis that unique sequences in the N-terminal arm of the Hox homeodomain are at least partially responsible for mediating this specificity. In vivo, Hox proteins directed E2a-Pbx1-mediated transactivation with moderate specificity to cognate Hox-Pbx motifs. Thus, the development specificity of individual Hox proteins may be mediated, in part, by differential targeting of cellular genes by Pbx1-Hox complexes. Likewise, through its function as a common heterodimer partner, oncoprotein E2a-Pbx1 may be able to interfere with multiple programs of development that are induced by the sequential or simultaneous expression of Hox proteins during hematopoiesis.

Meis1 and pKnox1 bind DNA cooperatively with Pbx1 utilizing an interaction surface disrupted in oncoprotein E2a-Pbx1.

E2a-Pbx1 is a chimeric transcription factor oncoprotein produced by the t(1;19) translocation in human pre-B cell leukemia. Class I Hox proteins bind DNA cooperatively with both Pbx proteins and oncoprotein E2a-Pbx1, suggesting that leukemogenesis by E2a-Pbx1 and Hox proteins may alter transcription of cellular genes regulated by Pbx-Hox motifs. Likewise, in murine myeloid leukemia, transcriptional coactivation of Meis1 with HoxA7/A9 suggests that Meis1-HoxA7/9 heterodimers may evoke aberrant gene transcription. Here, we demonstrate that both Meis1 and its relative, pKnox1, dimerize with Pbx1 on the same TGATTGAC motif selected by dimers of Pbx proteins and unidentified partner(s) in nuclear extracts, including those from t(1;19) pre-B cells. Outside their homeodomains, Meis1 and pKnox1 were highly conserved only in two motifs required for cooperativity with Pbx1. Like the unidentified endogenous partner(s), both Meis1 and pKnox1 failed to dimerize significantly with E2a-Pbx1. The Meis1/pKnox1-interaction domain in Pbx1 resided predominantly in a conserved N-terminal Pbx domain deleted in E2a-Pbx1. Thus, the leukemic potential of E2a-Pbx1 may require abrogation of its interaction with members of the Meis and pKnox families of transcription factors, permitting selective targeting of genes regulated by Pbx-Hox complexes. In addition, because most motifs bound by Pbx-Meis1/pKnox1 were not bound by Pbx1-Hox complexes, the leukemic potential of Meis1 in myeloid leukemias may involve shifting Pbx proteins from promoters containing Pbx-Hox motifs to those containing Pbx-Meis motifs.

Pbx-1 Hox heterodimers bind DNA on inseparable half-sites that permit intrinsic DNA binding specificity of the Hox partner at nucleotides 3' to a TAAT motif.

Heterodimers between the Pbx/Exd and Hox/HOM-C classes of homeodomain proteins bind regulatory elements in tissue-specific and developmentally regulated genes. In this work, we characterize the half-site bound by both Pbx1 and Hox proteins on a prototypic element (TGATTAAT) and determine how the orientation of the Hox protein contributes to the DNA binding specificity of Pbx-Hox heterodimers. We demonstrate that the Hox protein binds the 3' TAAT sequence as its recognition core and exhibits sequence-specific binding at positions 3' to the TAAT core. Unfavored sequences at this position, such as two cytosines, abrogate binding to the element. The upstream Pbx1 core sequence, TGAT, must immediately juxtapose the Hox core. This geometry maintains the preference of Hox/HOM-C proteins for a T base at position -1, as T represents the fourth position of the Pbx1 core, and suggests that this T base is bound by both Pbx1 and Hox proteins, Pbx1 binding in the major grove and the Hox protein binding in the minor grove. Pbx1 also exhibits base selectivity 5' to its TGAT recognition sequence.

Structural determinants within Pbx1 that mediate cooperative DNA binding with pentapeptide-containing Hox proteins: proposal for a model of a Pbx1-Hox-DNA complex.

Genetic studies have identified a family of divergent homeodomain proteins, including the human protooncoprotein Pbx1 and its drosophila homolog extradenticle (Exd), which function as cofactors with a subset of Hox and HOM-C proteins, and are essential for specific target gene expression. Pbx1/Exd binds DNA elements cooperatively with a large subset of Hox/HOM-C proteins containing a conserved pentapeptide motif, usually YPWMR, located just N terminally to their homeodomains. The pentapeptide is essential for cooperative DNA binding with Pbx1. In this study, we identify structural determinants of Pbx1 that are required for cooperative DNA binding with the pentapeptide-containing Hox protein HoxA5. We demonstrate that the homeodomain of Pbx1 contains a surface that binds the pentapeptide motif and that the Pbx1 homeodomain is sufficient for cooperative DNA binding with a Hox protein. A sequence immediately C terminal to the Pbx1 homeodomain, which is highly conserved in Pbx2 and Pbx3 and predicted to form an alpha-helix, enhances monomeric DNA binding by Pbx1 and also contributes to maximal cooperativity with Hox proteins. Binding studies with chimeric HoxA5-Pbx1 fusion proteins suggest that the homeodomains of Pbx1 and HoxA5 are docked on the representative element, TTGATTGAT, in tandem, with Pbx1 recognizing the 5' TTGAT core motif and the Hox protein recognizing the 3' TGAT core. The proposed binding orientation permits Hox proteins to exhibit further binding specificity on the basis of the identity of the four residues 3' to their core binding motif.

DNA-binding by oncoprotein E2a-Pbx1 is important for blocking differentiation but dispensable for fibroblast transformation.

The t(1;19) chromosomal translocation of pediatric pre-B cell lymphoblastic leukemia produces the E2A-PBX1 oncogene, which can transform fibroblasts, induce acute myeloid leukemia and T cell lymphomas in mice, and immortalize factor-dependent myeloid progenitors in cultured marrow. The homeodomain of Pbx1 binds ATCAATCAA, and while Pbx1 does not activate transcription through this motif, E2A-Pbx1 induces constitutive transactivation. Here, we investigate whether DNA-binding by Pbx1 or transcriptional activation by E2A are essential for the transforming abilities of E2A-Pbx1. Elimination of DNA-binding in E2A-Pbx1 by point mutations in the Pbx1 homeodomain or by large deletions that removed the Pbx1 homeodomain and carboxyl terminus did not alter ability of E2A-Pbx1 to induce focus-formation in fibroblast, even though these mutations completely eliminated its ability to activate transcription through the PRS. These same DNA-binding mutations, however, severely impaired or eliminated the ability of E2A-Pbx1 to immortalize factor-dependent myeloid progenitors in marrow cultures. Elimination of the first transcriptional activation domain of E2A abolished both fibroblast and myeloid transforming activities while elimination of the second altered neither of these activities. We conclude that DNA-binding is important for the ability of E2A-Pbx1 to disrupt differentiation, as evidenced in myeloblast immortalization, but dispensable for its ability to induce focus-formation, and that the aminoterminal domain of E2A, which strongly activates transcription, is essential for both transforming activities.

Selective repression of transcriptional activators by Pbx1 does not require the homeodomain.

PBX1 is a homeobox-containing gene identified as the chromosome 1 participant of the t(1;19) chromosomal translocation of childhood pre-B-cell acute lymphoblastic leukemia. This translocation produces a fusion gene encoding the chimeric oncoprotein E2A-Pbx1, which can induce both acute myeloid and T-lymphoid leukemia in mice. The binding of Pbx1 to DNA is weak; however, both Pbx1 and E2A-Pbx1 exhibit tight binding to specific DNA motifs in conjunction with certain other homeodomain proteins, and E2A-Pbx1 activates transcription through these motifs, whereas Pbx1 does not. In this report, we investigate potential transcriptional functions of Pbx1, using transient expression assays. While no segments of Pbx1 activated transcription, an internal domain of Pbx1 repressed transcription induced by the activation domain of Sp1, but not by the activation domains of VP16 or p53. This Pbx1 domain, which lies upstream of the homeodomain and is highly conserved among Pbx proteins, is thus predicted to bind a specific transcription factor. Surprisingly, the repression activity of Pbx1 did not require homeodomain-dependent DNA binding. Thus, Pbx1 may be able to alter gene transcription by both DNA-binding-dependent and DNA-binding-independent mechanisms.

The pentapeptide motif of Hox proteins is required for cooperative DNA binding with Pbx1, physically contacts Pbx1, and enhances DNA binding by Pbx1.

The vertebrate Hox genes, which represent a subset of all homeobox genes, encode proteins that regulate anterior-posterior positional identity during embryogenesis and are cognates of the Drosophila homeodomain proteins encoded by genes composing the homeotic complex (HOM-C). Recently, we demonstrated that multiple Hox proteins bind DNA cooperatively with both Pbx1 and its oncogenic derivative, E2A-Pbx1. Here, we show that the highly conserved pentapeptide motif F/Y-P-W-M-R/K, which occurs in numerous Hox proteins and is positioned 8 to 50 amino acids N terminal to the homeodomain, is essential for cooperative DNA binding with Pbx1 and E2A-Pbx1. Point mutational analysis demonstrated that the tryptophan and methionine residues within the core of this motif were critical for cooperative DNA binding. A peptide containing the wild-type pentapeptide sequence, but not one in which phenylalanine was substituted for tryptophan, blocked the ability of Hox proteins to bind cooperatively with Pbx1 or E2A-Pbx1, suggesting that the pentapeptide itself provides at least one surface through which Hox proteins bind Pbx1. Furthermore, the same peptide, but not the mutant peptide, stimulated DNA binding by Pbx1, suggesting that interaction of Hox proteins with Pbx1 through the pentapeptide motif raises the DNA-binding ability of Pbx1.