Oncoprotein E2a-Pbx1 Research Articles

Translocation t(1;19) in acute precursor B-cell lymphoblastic Leukaemia in paediatric patients- Pakistani population.

To determine the impact of translocation t(1;19) in paediatric patients diagnosed with precursor B-cell acute lymphoblastic leukaemia. The retrospective study was conducted at the Shaukat Khanum Memorial Cancer Hospital and Research Centre, Lahore, Pakistan, and comprised data from January 2012 to January 2018 of paediatric patients diagnosed with precursor B-cell acute lymphoblastic leukaemia. Data of patients having t(1;19) translocation with or without complex karyotype formed group A, while data of patients without any cytogenetic abnormality formed the control group B. Relapse and event-free survival were calculated and the outcomes were compared between the groups. Data was analysed using SPSS 20. Of the 450 subjects whose data was analysed, 84(18.7%) were included; 25(30%) in group A and 59(70%) were in group B. There were 21(84%) males and 4(16%) females in group A with mean age on presentation 3.68±0.6 years compared to 41(69.4%) males and 18(30.5%) females with mean age on presentation 4.0±0.92 (p>0.05). There were no differences between the groups in terms of baseline markers (p>0.05). The relapse and event-free survival rates were also not significantly different between the groups (p>0.05). There was no significant difference related to outcomes of precursor B-cell acute lymphoblastic leukaemia patients having t(1;19) translocation with or without complex karyotype and those without any cytogenetic abnormality, indicating that translocation t(1;19)-positive patients do not need treatment intensification.

Functional redundancy between the transcriptional activation domains of E2A is mediated by binding to the KIX domain of CBP/p300

The E-protein transcription factors play essential roles in lymphopoiesis, with E12 and E47 (hereafter called E2A) being particularly important in B cell specification and maturation. The E2A gene is also involved in a chromosomal translocation that results in the leukemogenic oncoprotein E2A-PBX1. The two activation domains of E2A, AD1 and AD2, display redundant, independent, and cooperative functions in a cell-dependent manner. AD1 of E2A functions by binding the transcriptional co-activator CBP/p300; this interaction is required in oncogenesis and occurs between the conserved ϕ-x-x-ϕ-ϕ motif in AD1 and the KIX domain of CBP/p300. However, co-activator recruitment by AD2 has not been characterized. Here, we demonstrate that the first of two conserved ϕ-x-x-ϕ-ϕ motifs within AD2 of E2A interacts at the same binding site on KIX as AD1. Mutagenesis uncovered a correspondence between the KIX-binding affinity of AD2 and transcriptional activation. Although AD2 is dispensable for oncogenesis, experimentally increasing the affinity of AD2 for KIX uncovered a latent potential to mediate immortalization of primary hematopoietic progenitors by E2A-PBX1. Our findings suggest that redundancy between the two E2A activation domains with respect to transcriptional activation and oncogenic function is mediated by binding to the same surface of the KIX domain of CBP/p300.

Rational design of an immunoconjugate for selective knock-down of leukemia-specific E2A–PBX1 fusion gene expression in human Pre-B leukemia

The t(1;19)(q23;p13) is one of the most common chromosomal translocations in acute lymphoblastic leukemia (ALL) and results in production of the transforming oncoprotein E2A-PBX1. Here we first report a novel, biomarker-guided biotherapy strategy for personalized treatment of t(1;19)(+) ALL. A supervised interrogation of the gene expression profiles of primary leukemic cells from a cohort of 207 children with high risk B-lineage ALL identified up-regulated CD19 gene expression as a biomarker for t(1;19)(+) ALL. A disulfide-linked immunoconjugate of a 5-amino-modified 24 mer phosphorothioate anti-sense E2A-PBX1 oligonucleotide (AON) with a mAb specific for a CD19 receptor (αCD19-AON) was prepared as a CD19-directed and leukemia-specific biotherapeutic agent against E2A-PBX1(+) B-lineage ALL. Treatment of E2A-PBX1(+) leukemia cells with low nanomolar concentrations of αCD19-AON resulted in selective depletion of E2A-PBX1 transcripts and caused apoptotic destruction and abrogation of clonogenic growth. Subcutaneously administered αCD19-AON at a total dose level of 93 nmol kg(-1) delivered over 14 days using a micro-osmotic pump more than doubled the leukemia-free survival time of SCID mice in a xenograft model of E2A-PBX1(+) human B-lineage ALL (82.0 ± 1.9 days vs. 37.0 ± 0.1 days, P < 0.0001). Both the AON moiety and the targeting CD19-specific mAb moiety were required for the in vitro as well as in vivo anti-leukemic activity of αCD19-AON. The observed in vitro and in vivo anti-leukemic potency of the αCD19-AON immunoconjugate provides the first preclinical proof-of-principle that t(1;19)(+) high risk B-lineage ALL can be treated with leukemia-specific biotherapeutic agents that knock-down E2A-PBX1 expression.

GCN5 acetylates and regulates the stability of the oncoprotein E2A-PBX1 in acute lymphoblastic leukemia

The t(1;19) translocation in pediatric pre-B-cell acute lymphoblastic leukemia (ALL) fuses the genes, which encode the transcriptional activator E2A and homeobox pre-B-cell leukemia transcription factor 1 (PBX1), resulting in expression of the chimeric transcription factor E2A-PBX1. E2A-PBX1 can promote cell transformation both in vitro and in vivo; however, the mechanisms by which E2A-PBX1 contributes to malignancy merit further investigation. In the current work we report, for the first time, a physical and functional interaction between the SPT3-TAFII31-GCN5L acetylase (STAGA) complex and E2A-PBX1. STAGA, and its acetyltransferase subunit GCN5, directly interacted with the E2A portion of E2A-PBX1. GCN5 acetylated E2A-PBX1 and increased the stability of E2A-PBX1 protein in cells. Moreover, the GCN5 inhibitor α-methylene-γ-butyrolactone 3 (MB-3) decreased E2A-PBX1 acetylation and E2A-PBX1 protein levels in leukemic cells, indicating that GCN5 inhibitors have potential value as therapeutic agents for ALL. In addition, we show that the E3 ubiquitin ligase HDM2 potentiates the degradation of E2A-PBX1. We suggest that dynamic regulation of E2A-PBX1 protein levels in vivo has a fundamental role in ALL.

Ectopic Expression of the Leukemogenic Protein E2A-PBX1 in Early Hematopoietic Progenitors Blocks B-Lymphoid Commitment

Abstract 1393The oncoprotein E2A-PBX1 is produced by t(1;19)(q23;p13) in pre-B acute lymphoblastic leukemia (ALL). Perplexingly, in vitro and in vivo models of E2A-PBX1 leukemogenesis to date generate either myeloid or T-lymphoblasts, neither of which are representative of the human disease. Furthermore, we have observed recently that lineage-depleted (lin-) murine bone marrow cells infected with an E2A-PBX1 retrovirus selectively fail to repopulate the B-lymphoid compartment on transplantation into irradiated recipients. Therefore, we have now begun to investigate the mechanism by which enforced expression of E2A-PBX1 antagonizes B-lymphopoiesis.We show that E2A-PBX1-transduced lin- fetal liver progenitor cells (FLPs) fail to differentiate to committed CD45R+ pro-B-cells when cultured with IL-7 in the presence of OP9 stromal cells. Instead these cells manifest a relatively immature immunophenotype (CD45R-, CD11b-, CD117+), are murine stem cell factor (SCF) dependent, and undergo myeloid differentiation upon stimulation with granulocyte macrophage colony-stimulating factor (GM-CSF) or transplantation into irradiated mice. Amino acid substitutions in E2A-PBX1 that impair co-activator recruitment or DNA binding completely rescue the differentiation block, implying a critical role for target-gene regulation by E2A-PBX1. E2A-PBX1-transduced cells fail to induce the B-lymphoid commitment genes Ebf1 and Pax5, and chromatin immunoprecipitation (ChIP) analysis shows aberrant persistence of the Polycomb silencing mark H3K27me3 at these promoters. Previous studies have identified the Polycomb gene Bmi1 as a candidate E2A-PBX1 target gene. However, enforced expression of Bmi1 neither blocked induction of Ebf1 or Pax5 nor impaired B-lymphopoiesis, indicating that Bmi1 is not sufficient to mediate blocked differentiation by E2A-PBX1. Gene expression microarray analysis identified cytokines (ex., Csf2, IL1a, Il6) and transcription factors (HoxA9, Tal1) up-regulated in E2A-PBX1-transduced FLPs that are known to antagonize B-lymphoid development.In summary, our results support the notion that enforced expression of E2A-PBX1 in early hematopoietic progenitors blocks B-lymphopoiesis, to an important degree, by inducing the transcription of genes whose products antagonize early steps of B-lymphoid commitment, including induction of Ebf1 and Pax5. HoxA9 is particularly interesting as a mediator of E2A-PBX1 effects in view of its extreme induction ratio in our system (at least 1000-fold relative to controls), its propensity to promote myeloid over lymphoid differentiation, and its well-established role in leukemogenesis. Studies to investigate a potential requirement for HoxA9 and other candidates in mediating the E2A-PBX1-driven B-lymphoid differentiation block are ongoing. Disclosures:No relevant conflicts of interest to declare.

Histone H3 Lysine 79 Methyltransferase Dot1 Is Required for Immortalization by MLL Oncogenes

Chimeric oncoproteins resulting from fusion of MLL to a wide variety of partnering proteins cause biologically distinctive and clinically aggressive acute leukemias. However, the mechanism of MLL-mediated leukemic transformation is not fully understood. Dot1, the only known histone H3 lysine 79 (H3K79) methyltransferase, has been shown to interact with multiple MLL fusion partners including AF9, ENL, AF10, and AF17. In this study, we utilize a conditional Dot1l deletion model to investigate the role of Dot1 in hematopoietic progenitor cell immortalization by MLL fusion proteins. Western blot and mass spectrometry show that Dot1-deficient cells are depleted of the global H3K79 methylation mark. We find that loss of Dot1 activity attenuates cell viability and colony formation potential of cells immortalized by MLL oncoproteins but not by the leukemic oncoprotein E2a-Pbx1. Although this effect is most pronounced for MLL-AF9, we find that Dot1 contributes to the viability of cells immortalized by other MLL oncoproteins that are not known to directly recruit Dot1. Cells immortalized by MLL fusions also show increased apoptosis, suggesting the involvement of Dot1 in survival pathways. In summary, our data point to a pivotal requirement for Dot1 in MLL fusion protein-mediated leukemogenesis and implicate Dot1 as a potential therapeutic target.

Abstract 3097: The leukemogenic protein E2A-PBX1 blocks B-lymphoid commitment in early hematopoietic progenitors

Abstract The oncoprotein E2A-PBX1 is produced by the t(1;19) chromosomal translocation in pre-B cell acute lymphoblastic leukemia (ALL). We and others have shown that retrovirus-enforced expression of E2A-PBX1 in mouse bone marrow results in a lethal leukemia with short latency. Suprisingly, almost all animals die of myeloid leukemia rather than the B-lymphoid disease observed clinically. The E-protein E47, a product of the wild-type E2A gene, is required in the earliest stages of B-lymphoid commitment. E47 and E2A-PBX1 are identical in their amino-terminal 483 residues and this region includes transcriptional activation domains capable of recruiting CBP/p300 and presumably other co-regulators. Therefore, we hypothesized that E2A-PBX1 may block B-lymphoid commitment by exerting dominant-negative effects on E-proteins including E47. Bone marrow cells transduced with E2A-PBX1 failed to repopulate the B-lymphoid compartment at two weeks post-reconstitution of sub-lethally-irradiated mice, whereas there was exuberant proliferation of transduced myeloid progenitors suggesting selective intereference with B-lymphopoiesis. This phenomenon was examined further using a more experimentally tractable in vitro system. “Lineage negative” hematopoietic progenitors from fetal livers differentiate rapidly to committed B-lymphoid progenitors (B220+, CD19+, Mac-1-, Gr-1-) on co-culture with OP9 stromal cells in the presence of interleukin-7 (IL-7). However, enforced expression of E2A-PBX1 produced a myeloid immunophenotype (B220-, CD19-, Mac-1+, Gr-1+). Furthermore, these cells are dependent on myeloid cytokines, display no IL-7 responsiveness, and have a high forward scatter/side scatter profile. RT-PCR analysis shows severely reduced levels of lymphoid-associated transcripts (E2A, Ebf1, Pax5 and Irf8) and substantially elevated levels of myeloid associated transcripts (Csfr1, Id1 and Pu.1) relative to control lymphoid cells. All of these properties are characteristic of macrophage progenitors, a fate adopted by E2A−/− cells under similar culture conditions. Our findings showing that E2A-PBX1 is capable of altering the lineage fate of early hematopoietic progenitors in a manner similar to gene targeting of E2A provide support, albeit circumstantial, for a mechanism that involves dominant-negative effects on E-proteins and perhaps competition for limiting co-regulators such as CBP/p300. Consistent with the latter possibility, murine bone marrow cells expressing an E2A-PBX1 mutant impaired in CBP/p300 recruitment retain a minor propensity to repopulate the B-lymphoid compartment of irradiated transplant recipients. The results of experiments to evaluate the impact of E2A-PBX1 on transcriptional induction by E47 will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3097.

Acetylation of a Conserved Lysine Residue within Activation Domain One of E2A Transcription Factors Plays a Role in Transcriptional Regulation.

The E2A locus encodes transcription factors, called E12 and E47, involved in lineage-specific cellular differentiation. The locus is also involved in chromosomal translocations associated with acute lymphoblastic leukemia, the most common of which results in expression of the oncoprotein E2A-PBX1. We showed recently that direct interaction between E2A and the histone acetyl-transferase (AT) and transcriptional co-activator proteins CBP/p300 is required in leukemogenesis by E2A-PBX1. E2A proteins have also been shown to interact with another AT/co-activator, p/CAF. Interaction with these AT proteins results in acetylation of E2A proteins themselves. Here we map the acetylated lysine residues within the oncogenic portion of E2A proteins and begin to elucidate some functional correlates of E2A acetylation. Our results indicate that the isolated AT domain of p/CAF as well as full-length p/CAF were capable of acetylating E2A. Interestingly, full-length p300 was capable of acetylating E2A while an isolated AT domain was unable to acetylate E2A, suggesting that additional domains of CBP/p300 are required to mediate E2A acetylation. We demonstrate that both p300 and p/CAF can interact directly with E2A, independent of the known interaction between p300 and p/CAF. These co-activators do, however, appear to co-operate to achieve maximal E2A acetylation. Mutagenesis-based mapping studies indicate that several lysines are substrates for acetylation. Of particular interest, a conserved lysine residue (K34) located within the N-terminal transcriptional activation domain (AD1) is acetylated in vitro by p/CAF. K34 is located within a GKXXP consensus sequence, suggested to be a recognition motif for acetylation by several AT enzymes including CBP/p300 and p/CAF. Conservative replacement of K34 with arginine (i.e., K43R) substantially impairs transcriptional activation of a luciferase reporter gene by E2A, suggesting that post-translational modification of this residue may play an important functional role. Consistent with a role for acetylation, relative to some other lysine-dependent modification, we were unable to demonstrate sumoylation or ubiquitination of the N-terminus of E2A. Therefore, we have found that acetylation by AT/co-activator proteins contributes to transcriptional regulation by the functionally critical N-terminal activation domain (AD1) of E2A proteins. The mechanisms by which this acetylation event is regulated and how it contributes to target gene induction by E2A are not clear. It seems plausible that the acetylation status of AD1 could be determined by upstream signaling events and acetylation of E2A could modulate interactions with transcriptional co-regulators, DNA or chromatin. Further studies to investigate these possibilities are underway. In particular, results using an amino acid substitution that mimics acetylation of AD1 (K34Q) will be presented.

Interaction with the Na,K-ATPase and Tissue Distribution of FXYD5 (Related to Ion Channel)

FXYD5 (related to ion channel, dysadherin) is a member of the FXYD family of single span type I membrane proteins. Five members of this group have been shown to interact with the Na,K-ATPase and to modulate its properties. However, FXYD5 is structurally different from other family members and has been suggested to play a role in regulating E-cadherin and promoting metastasis (Ino, Y., Gotoh, M., Sakamoto, M., Tsukagoshi, K., and Hirohashi, S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 365-370). The goal of this study was to determine whether FXYD5 can modulate the Na,K-ATPase activity, establish its cellular and tissue distribution, and characterize its biochemical properties. Anti-FXYD5 antibodies detected a 24-kDa polypeptide that was preferentially expressed in kidney, intestine, spleen, and lung. In kidney, FXYD5 resides in the basolateral membrane of the connecting tubule, the collecting tubule, and the intercalated cells of the collecting duct. However, there is also labeling of the apical membrane in long thin limb of Henle's loop. FXYD5 was effectively immunoprecipitated by antibodies to the alpha subunit of Na,K-ATPase and the anti-FXYD5 antibody immunoprecipitates alpha. Co-expressing FXYD5 with the alpha1 and beta1 subunits of the Na,K-ATPase in Xenopus oocytes elicited a more than 2-fold increase in pump activity, measured either as ouabain-blockable outward current or as ouabain-sensitive (86)Rb(+) uptake. Thus, as found with other FXYD proteins, FXYD5 interacts with the Na,K-ATPase and modulates its properties.

Bmi-1 Regulation of INK4A-ARF Is a Downstream Requirement for Transformation of Hematopoietic Progenitors by E2a-Pbx1

Loss-of-function alterations of INK4A are commonly observed in lymphoid malignancies, but are consistently absent in pre-B cell leukemias induced by the chimeric oncoprotein E2a-Pbx1 created by t(1;19) chromosomal translocations. We report here that experimental induction of E2a-Pbx1 enhances expression of BMI-1, a lymphoid oncogene whose product functions as a transcriptional repressor of the INK4A-ARF tumor suppressor locus. Bmi-1-deficient hematopoietic progenitors are resistant to transformation by E2a-Pbx1; however, the requirement for Bmi-1 is alleviated in cells deficient for both Bmi-1 and INK4A-ARF. Furthermore, the adverse effects of E2a-Pbx1 on pre-B cell survival and differentiation are partially bypassed by forced expression of p16Ink4a. These results link E2a-Pbx1 with Bmi-1 on an oncogenic pathway that is likely to play a role in the pathogenesis of human lymphoid leukemias through downregulation of the INK4A-ARF gene.

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.

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.

M Angiogenin-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.

The gene encoding the granulocyte colony-stimulating factor receptor is a target for deregulation in pre-B ALL by the t(1;19)-specific oncoprotein E2A-Pbx1.

Approximately 25-30% of childhood pre-B cell acute lymphoblastic leukemias (pre-B ALL) is characterized by the presence of a (1;19)(q23;p13.3) translocation. The presence of this translocation is generally accompanied by a poor prognosis. The chimeric gene resulting from this chromosomal rearrangement encodes a hybrid transcription factor, E2A-Pbx1. In an attempt to delineate the genetic cascade initiated by E2A-Pbx1, we sought to identify genes that are deregulated by this transcription factor in t(1;19) pre-B ALL. We show here that the gene encoding the granulocyte colony-stimulating factor receptor (G-CSFr) is specifically upregulated in pre-B cells expressing E2A-Pbx1. G-CSFr is also expressed in cell lines established from t(1;19) pre-B cell leukemia and on primary t(1;19) tumor cells, but not on control cells. These data indicate that G-CSFr gene is a target for deregulation by E2A-Pbx1.

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.

Meis proteins are major in vivo DNA binding partners for wild-type but not chimeric Pbx proteins.

The Pbx1 and Meis1 proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively, and implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate their DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. In the present study, we demonstrate that Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. We conclude that Meis proteins are preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1.

A novel fibroblast growth factor gene expressed in the developing nervous system is a downstream target of the chimeric homeodomain oncoprotein E2A-Pbx1

Pbx1 is a homeodomain transcription factor that has the ability to form heterodimers with homeodomain proteins encoded by the homeotic selector (Hox) gene complexes and increase their DNA-binding affinity and specificity. A current hypothesis proposes that interactions with Pbx1 are necessary for Hox proteins to regulate downstream target genes that in turn control growth, differentiation and morphogenesis during development. In pre B cell leukemias containing the t(1;19) chromosome translocation, Pbx1 is converted into a strong transactivator by fusion to the activation domain of the bHLH transcription factor E2A. The E2A-Pbx1 fusion protein should therefore activate transcription of genes normally regulated by Pbx1. We have used the subtractive process of representational difference analysis to identify targets of E2A-Pbx1. We show that E2A-Pbx1 can directly activate transcription of a novel member of the fibroblast growth factor family of intercellular signalling molecules, FGF-15. The FGF-15 gene is expressed in a regionally restricted pattern in the developing nervous system, suggesting that FGF-15 may play an important role in regulating cell division and patterning within specific regions of the embryonic brain, spinal cord and sensory organs.

Chimeric oncoprotein E2a-Pbx1 induces apoptosis of hematopoietic cells by a p53-independent mechanism that is suppressed by Bcl-2.

The chimeric oncoprotein E2a-Pbx1 results from fusion of the E2A and PBX1 genes following t(1;19) chromosomal translocations in B cell precursor acute leukemias. Experimentally B cell progenitors do not tolerate constitutive expression of E2a-Pbx1 which contrasts with transformation of several other cell types following its stable expression both in vitro and in vivo. To further investigate the effects of E2a-Pbx1 on the B cell progenitors, we conditionally expressed E2a-Pbx1 under control of a metal response element in hematopoietic precursor cell lines in vitro. Inducible expression of E2a-Pbx1 resulted in cell death with the morphologic and molecular features of apoptosis. A structure-function analysis demonstrated that induction of apoptosis was not a dominant-negative effect of the E2a moiety but, rather, required the DNA-binding homeodomain of Pbx1. E2a-Pbx1-induced apoptosis proceeded through a BCL2-responsive checkpoint eventuating in PARP inactivation but did require p53. Constitutive expression of E2a-Pbx1 did not induce apoptosis or continued cycling of Rat-1 fibroblasts in low serum conditions. These studies demonstrate that E2a-Pbx1 initiates programmed cell death of hematopoietic precursers by a mechanism that requires its chimeric transcriptional properties, but, unlike other nuclear oncoproteins, is independent of p53.

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.

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.