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Mark P Kamps - One of the best experts on this subject based on the ideXlab platform.

  • meis1 and pknox1 bind dna cooperatively with PBX1 utilizing an interaction surface disrupted in oncoprotein e2a PBX1
    Proceedings of the National Academy of Sciences of the United States of America, 1997
    Co-Authors: Paul S. Knoepfler, Katherine R Calvo, Haiming Chen, Stylianos E Antonarakis, Mark P Kamps
    Abstract:

    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.

  • E2a-PBX1 induces aberrant expression of tissue-specific and developmentally regulated genes when expressed in NIH 3T3 fibroblasts.
    Molecular and cellular biology, 1997
    Co-Authors: Mark P Kamps
    Abstract:

    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 specificities at nucleotides predicted to contact the n terminal arm of the hox homeodomain demonstration of hox dependent targeting of e2a PBX1 in viv
    Oncogene, 1997
    Co-Authors: Mark P Kamps
    Abstract:

    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.

  • structural determinants within PBX1 that mediate cooperative dna binding with pentapeptide containing hox proteins proposal for a model of a PBX1 hox dna complex
    Molecular and Cellular Biology, 1996
    Co-Authors: Qiang Lu, Mark P Kamps
    Abstract:

    : 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.

  • structural determinants within PBX1 that mediate cooperative dna binding with pentapeptide containing hox proteins proposal for a model of a PBX1 hox dna complex
    Molecular and Cellular Biology, 1996
    Co-Authors: Qiang Lu, Mark P Kamps
    Abstract:

    : 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.

Michael L. Cleary - One of the best experts on this subject based on the ideXlab platform.

  • Oligomeric self-association contributes to E2A-PBX1-mediated oncogenesis
    Nature Publishing Group, 2019
    Co-Authors: Chiou-hong Lin, Zhong Wang, Jesús Duque-afonso, Stephen Hon-kit Wong, Janos Demeter, Alexander V. Loktev, Tim C. P. Somervaille, Peter K. Jackson, Michael L. Cleary
    Abstract:

    Abstract The PBX1 homeodomain transcription factor is converted by t(1;19) chromosomal translocations in acute leukemia into the chimeric E2A-PBX1 oncoprotein. Fusion with E2A confers potent transcriptional activation and constitutive nuclear localization, bypassing the need for dimerization with protein partners that normally stabilize and regulate import of PBX1 into the nucleus, but the mechanisms underlying its oncogenic activation are incompletely defined. We demonstrate here that E2A-PBX1 self-associates through the PBX1 PBC-B domain of the chimeric protein to form higher-order oligomers in t(1;19) human leukemia cells, and that this property is required for oncogenic activity. Structural and functional studies indicate that self-association facilitates the binding of E2A-PBX1 to DNA. Mutants unable to self-associate are transformation defective, however their oncogenic activity is rescued by the synthetic oligomerization domain of FKBP, which confers conditional transformation properties on E2A-PBX1. In contrast to self-association, PBX1 protein domains that mediate interactions with HOX DNA-binding partners are dispensable. These studies suggest that oligomeric self-association may compensate for the inability of monomeric E2A-PBX1 to stably bind DNA and circumvents protein interactions that otherwise modulate PBX1 stability, nuclear localization, DNA binding, and transcriptional activity. The unique dependence on self-association for E2A-PBX1 oncogenic activity suggests potential approaches for mechanism-based targeted therapies

  • oncogenic role for the lck zap70 plcg2 signaling pathway in pre b all pathogenesis
    Blood, 2015
    Co-Authors: Jesus Duqueafonso, Jue Feng, Corina Buechele, Stephen H.k. Wong, Michael C. Bassik, Michael L. Cleary
    Abstract:

    Although the treatment and prognosis of patients with pediatric acute lymphoblastic leukemia (ALL) have improved during the last decades, there is still a clinical need for more effective/selective and less toxic therapies. To address this, we have interrogated various signaling pathways in human ALL cells and mouse strains that express E2A-PBX1, which is present in 5-7% of pediatric ALL. Phospho-flow analysis revealed basal hyper-phosphorylation levels of PLCγ2 in mouse E2A-PBX1 leukemias, consistent with hyper-activation of upstream signaling pathways. Efficient shRNA-mediated depletion of PLCγ2 reduced colony formation of mouse E2A-PBX1+ leukemias in vitro and increased disease-free survival after secondary bone marrow transplantation in vivo. Furthermore, PLCγ2-depleted human ALL cell lines including E2A-PBX1+ cells, showed reduced proliferation. These data suggest a pathogenic role of hyperactivated PLCγ2 in pre-B-ALL. Bioinformatics analysis of E2A-PBX1 target genes in human ALLs revealed an enrichment of B- and T-cell activation pathways, which include the SRC-family kinase LCK and the cytoplasmic kinase ZAP70, upstream of PLCγ2. Comparative analyses of global transcriptional profiles in human primary and mouse leukemias and preleukemias induced by the E2A-PBX1 oncogene identified the signaling kinase ZAP70 as one of the earliest and most consistently up-regulated genes in E2A-PBX1 leukemias. Using a candidate gene approach, we identified LCK with increased expression levels in E2A-PBX1 leukemia cells compared to normal B-cell progenitors. Mouse and human E2A-PBX1 leukemia cells were dependent on the E2A-PBX1 target genes ZAP70 and LCK for proliferation and survival as confirmed by shRNA knock-down experiments. Hence, efficient depletion of these genes resulted in a decrease of phosphorylated PLCγ2, suggesting therapeutic targets in E2A-PBX1 leukemias. Combined suppression of ZAP70 and LCK using double-shRNA experiments showed an additive effect on inhibition of cell proliferation and decrease of phosphorylated PLCγ2. These results provide a rationale for combination therapy to block this hyper-activated signaling pathway at different levels. Several small molecule inhibitors were evaluated for their effects on PLCγ2 upstream pathways in E2A-PBX1 leukemia cells. SRC-family kinase inhibitors including dasatinib were most effective in reducing phosphorylation of PLCγ2 and inhibiting cell proliferation. Furthermore, dasatinib showed promising preclinical efficacy in vitro in colony forming assays and in vivo after secondary bone marrow transplantation of leukemias. In summary, our studies demonstrate that the proliferation and survival of E2A-PBX1 leukemias are dependent on PLCγ2 and upstream signaling pathways, which are suitable for pharmacological inhibition. Disclosures No relevant conflicts of interest to declare.

  • comparative genomics reveals multistep pathogenesis of e2a PBX1 acute lymphoblastic leukemia
    Journal of Clinical Investigation, 2015
    Co-Authors: Jesus Duqueafonso, Jue Feng, Stephen H.k. Wong, Chiou-hong Lin, Zhong Wang, Masayuki Iwasaki, Florian Scherer, Michael L. Cleary
    Abstract:

    Acute lymphoblastic leukemia (ALL) is the most common childhood cancer; however, its genetic diversity limits investigation into the molecular pathogenesis of disease and development of therapeutic strategies. Here, we engineered mice that conditionally express the E2A-PBX1 fusion oncogene, which results from chromosomal translocation t(1;19) and is present in 5% to 7% of pediatric ALL cases. The incidence of leukemia in these mice varied from 5% to 50%, dependent on the Cre-driving promoter (Cd19, Mb1, or Mx1) used to induce E2A-PBX1 expression. Two distinct but highly similar subtypes of B cell precursor ALLs that differed by their pre-B cell receptor (pre-BCR) status were induced and displayed maturation arrest at the pro-B/large pre-B II stages of differentiation, similar to human E2A-PBX1 ALL. Somatic activation of E2A-PBX1 in B cell progenitors enhanced self-renewal and led to acquisition of multiple secondary genomic aberrations, including prominent spontaneous loss of Pax5. In preleukemic mice, conditional Pax5 deletion cooperated with E2A-PBX1 to expand progenitor B cell subpopulations, increasing penetrance and shortening leukemia latency. Recurrent secondary activating mutations were detected in key signaling pathways, most notably JAK/STAT, that leukemia cells require for proliferation. These data support conditional E2A-PBX1 mice as a model of human ALL and suggest targeting pre-BCR signaling and JAK kinases as potential therapeutic strategies.

  • PBX1 restrains myeloid maturation while preserving lymphoid potential in hematopoietic progenitors
    Journal of Cell Science, 2013
    Co-Authors: Francesca Ficara, Laura Crisafulli, Chenwei Lin, Masayuki Iwasaki, Kevin S Smith, Luca Zammataro, Michael L. Cleary
    Abstract:

    The capacity of the hematopoietic system to promptly respond to peripheral demands relies on adequate pools of progenitors able to transiently proliferate and differentiate in a regulated manner. However, little is known about factors that may restrain progenitor maturation to maintain their reservoirs. Conditional knockout mice for the PBX1 proto-oncogene have a significant reduction in lineage-restricted progenitors in addition to a profound defect in hematopoietic stem cell (HSC) self-renewal. Through analysis of purified progenitor proliferation, differentiation capacity and transcriptional profiling, we demonstrate that PBX1 regulates the lineage-specific output of multipotent and oligopotent progenitors. In the absence of PBX1 multipotent progenitor (MPP) and common myeloid progenitor (CMP) pools are reduced due to aberrantly rapid myeloid maturation. This is associated with premature expression of myeloid differentiation genes and decreased maintenance of proto-oncogene transcriptional pathways, including reduced expression of Meis1, a PBX1 dimerization partner, and its subordinate transcriptional program. Conversely, PBX1 maintains the lymphoid differentiation potential of lymphoid-primed MPPs (LMPPs) and common lymphoid progenitors (CLPs), whose reduction in the absence of PBX1 is associated with a defect in lymphoid priming that is also present in CMPs, which persistently express lymphoid and HSC genes underlying a previously unappreciated lineage promiscuity that is maintained by PBX1. These results demonstrate a role for PBX1 in restraining myeloid maturation while maintaining lymphoid potential to appropriately regulate progenitor reservoirs.

  • PBX1 a novel stage specific regulator of adipocyte development
    Stem Cells, 2011
    Co-Authors: Michael L. Cleary, Mrinmoy Sanyal, Miguel Caetano Monteiro, Coralie Sengenes, Anne Bouloume, Christian Dani, Nathalie Billon
    Abstract:

    Although adipocyte terminal differentiation has been extensively studied, the early steps of adipocyte development and the embryonic origin of this lineage remain largely unknown. Here we describe a novel role for the pre-B-cell leukemia transcription factor one (PBX1) in adipocyte development using both mouse embryonic stem cells (mESCs) and human multipotent adipose-derived stem (hMADS) cells. We show that PBX1(-/-) mESCs are unable to generate adipocytes, despite normal expression of neuroectoderm and neural crest (NC) markers. Early adipocyte lineage markers are not induced in PBX1(-/-) mESCs, suggesting that PBX1 controls the generation and/or the maintenance of adipocyte progenitors (APs) from the NC. We further characterize the function of PBX1 in postnatal adipogenesis and show that silencing of PBX1 expression in hMADS cells reduces their proliferation by preventing their entry in the S phase of the cell cycle. Furthermore, it promotes differentiation of hMADS cells into adipocytes and partially substitutes for glucocorticoids and rosiglitazone, two key proadipogenic agents. These effects involve direct modulation of PPARγ activity, most likely through regulation of the biosynthesis of PPARγ natural endogenous ligand(s). Together, our data suggest that PBX1 regulates adipocyte development at multiple levels, promoting the generation of NC-derived APs during embryogenesis, while favoring APs proliferation and preventing their commitment to the adipocyte lineage in postnatal life.

Licia Selleri - One of the best experts on this subject based on the ideXlab platform.

  • abstract 1966 a novel small molecule compound targeting PBX1 dna interaction impedes cancer cell survival and carboplatin resistance
    Cancer Research, 2018
    Co-Authors: Yao An Shen, Licia Selleri, Ie Ming Shih, Jin Jung, Yohan Suryo Rahmanto, Chimu Chuang, Tian-li Wang
    Abstract:

    Pre-B-cell leukemia homeobox-1 (PBX1), a transcriptional factor and downstream effector of Notch signaling pathway, plays pivotal roles in a wide spectrum of tumors, as well as developmental, inflammatory, autoimmune, and neurodegenerative disorders. Exploiting the crystal structure of the PBX1-DNA complex, we developed a novel small-molecular inhibitor T417 that can directly block PBX1-binding to DNA, unlike other existing compounds that interfere with protein-protein interactions. When T417 docks into the hydrophobic pocket of PBX1 protein, the small molecule can dampen PBX1 transcription activity by hindering its binding to the promoter regions of PBX1 downstream target genes. Intriguingly, the amount of PBX1 expression in cells can dictate its response to T417. Increment of PBX1 expression is found in the high-grade serous carcinoma (HGSC) and carboplatin-resistant (CR) cells, and its expression is generally low in normal tissues as compared to the transformed tissues, making it such an arguably unique therapeutic target in ovarian cancer cells. This expression pattern illustrates the very minimal toxicity of T417 on normal tissues and organs in animal models, while it imposed in vitro and in vivo detrimental effects toward HGSC and CR cells. Besides, T417 holds synergistic cytotoxic effects with DNA damage-related drugs including PARP inhibitor and platinum-based drug. As PBX1 was shown to participate in maintaining cancer stem cell (CSC)-like phenotypes and promoting resistance to antitumor drugs, T417 is able to hammer out the stemness traits of CR cells to revert to a differentiated status through tacking PBX1 signaling cascade. The novel PBX1-targeting compound selectively interferes with PBX1-binding to DNA, which potentially points to powerful therapeutics and broad applications for the treatment of different human malignancies and stem cell therapy. Citation Format: Yao-An Shen, Jin Jung, Yohan Suryo Rahmanto, Licia Selleri, Ie-Ming Shih, Chi-Mu Chuang, Tian-Li Wang. A novel small-molecule compound targeting PBX1-DNA interaction impedes cancer cell survival and carboplatin resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1966.

  • Ovarian Cancer Chemoresistance Relies on the Stem Cell Reprogramming Factor PBX1.
    Cancer research, 2016
    Co-Authors: Jin-gyoung Jung, Joon Tae Park, Ie Ming Shih, Emily Gerry, Tae Hoen Kim, Ayse Ayhan, Karen Handschuh, Ben Davidson, Amanda Nickles Nickles Fader, Licia Selleri
    Abstract:

    The evolution of chemoresistance is a fundamental characteristic of cancer that ultimately hampers its clinical management. However, it may be possible to improve patient outcomes significantly by a better understanding of resistance mechanisms, which cancers rely upon during the evolution to an untreatable state. Here we report an essential role of the stem cell reprogramming factor, PBX1, in mediating chemoresistance in ovarian carcinomas. In the clinical setting, high levels of PBX1 expression correlated with shorter survival in post-chemotherapy ovarian cancer patients. In tumor cells with low endogenous levels of PBX1, its enforced expression promoted cancer stem cell-like phenotypes, including most notably an increase in resistance to platinum-based therapy used most commonly for treating this disease. Conversely, silencing PBX1 in platinum-resistant cells that overexpressed PBX1 sensitized them to platinum treatment and reduced their stem-like properties. An analysis of published genome-wide chromatin immunoprecipitation data indicated that PBX1 binds directly to promoters of genes involved in stem cell maintenance and the response to tissue injury. We confirmed direct regulation of one of these genes, STAT3, demonstrating that the PBX1 binding motif at its promoter acted to positively regulate STAT3 transcription. We further demonstrated that a STAT3/JAK2 inhibitor could potently sensitize platinum-resistant cells to carboplatin and suppress their growth in vivo Our findings offer a mechanistic rationale to target the PBX1/STAT3 axis to antagonize a key mechanism of chemoresistance in ovarian cancers and possibly other human cancers. Cancer Res; 76(21); 6351-61. ©2016 AACR.

  • PBX1 is required for adult subventricular zone neurogenesis
    Development, 2016
    Co-Authors: Britta Moyo Grebbin, Matthew Koss, Licia Selleri, Annchristin Hau, Anja Gros, Marie Andersmaurer, Jasmine Schramm, Christoph Wille, Michel Mittelbronn
    Abstract:

    TALE-homeodomain proteins function as components of heteromeric complexes that contain one member each of the PBC and MEIS/PREP subclasses. We recently showed that MEIS2 cooperates with the neurogenic transcription factor PAX6 in the control of adult subventricular zone (SVZ) neurogenesis in rodents. Expression of the PBC protein PBX1 in the SVZ has been reported, but its functional role(s) has not been investigated. Using a genetic loss-of-function mouse model, we now show that PBX1 is an early regulator of SVZ neurogenesis. Targeted deletion of PBX1 by retroviral transduction of Cre recombinase into Pbx2-deficient SVZ stem and progenitor cells carrying floxed alleles of PBX1 significantly reduced the production of neurons and increased the generation of oligodendrocytes. Loss of PBX1 expression in neuronally committed neuroblasts in the rostral migratory stream in a Pbx2 null background, by contrast, severely compromised cell survival. By chromatin immunoprecipitation from endogenous tissues or isolated cells, we further detected PBX1 binding to known regulatory regions of the neuron-specific genes Dcx and Th days or even weeks before the respective genes are expressed during the normal program of SVZ neurogenesis, suggesting that PBX1 might act as a priming factor to mark these genes for subsequent activation. Collectively, our results establish that PBX1 regulates adult neural cell fate determination in a manner beyond that of its heterodimerization partner MEIS2.

  • epigenetic regulation of early osteogenesis and mineralized tissue formation by a hoxa10 PBX1 associated complex
    Cells Tissues Organs, 2011
    Co-Authors: Jonathan A R Gordon, Matthew Koss, Licia Selleri, Mohammad Q Hassan, Martin Montecino, Janet L Stein, Gary S Stein, Jane B Lian
    Abstract:

    Homeodomain-containing (HOX) factors such as the abdominal class homeodomain protein HOXA10 and the TALE-family protein PBX1 form coregulatory complexes and are potent transcriptional and epigenetic regulators of tissue morphogenesis. We have identified that HOXA10 and PBX1 are expressed in osteoprogenitors; however, their role in osteogenesis has not been established. To determine the mechanism of HOXA10-PBX-mediated regulation of osteoblast commitment and the related gene expression, PBX1 or HOX10 were depleted (shRNA or genetic deletion, respectively) or exogenously expressed in C3H10T1/2, bone marrow stromal progenitors, and MC3T3-E1 (preosteoblast) cells. Overexpression of HOXA10 increased the expression of osteoblast-related genes, osteoblast differentiation and mineralization; expression of PBX1 impaired osteogenic commitment of pluripotent cells and the differentiation of osteoblasts. In contrast, the targeted depletion of PBX1 by shRNA increased the expression of bone marker genes (osterix, alkaline phosphatase, BSP, and osteocalcin). Chromatin-associated PBX1 and HOXA10 were present at osteoblast-related gene promoters preceding gene expression, but PBX1 was absent from promoters during the transcription of bone-related genes, including osterix (Osx). Further, PBX1 complexes were associated with histone deacetylases normally linked with chromatin inactivation. Loss of PBX1 but not of HOXA10 from the Osx promoter was associated with increases in the recruitment of histone acetylases (p300), as well as decreased H3K9 methylation, reflecting transcriptional activation. We propose PBX1 plays a central role in attenuating the activity of HOXA10 as an activator of osteoblast-related genes and functions to establish the proper timing of gene expression during osteogenesis, resulting in proper matrix maturation and mineral deposition in differentiated osteoblasts.

  • Scapula development is governed by genetic interactions of PBX1 with its family members and with Emx2 via their cooperative control of Alx1.
    Development (Cambridge England), 2010
    Co-Authors: Giulia Vaccari, James Sharpe, Massimo Pellegrini, Elisabetta Ferretti, Sebastian Fantini, Laura Quintana, Licia Selleri
    Abstract:

    The genetic pathways underlying shoulder blade development are largely unknown, as gene networks controlling limb morphogenesis have limited influence on scapula formation. Analysis of mouse mutants for Pbx and Emx2 genes has suggested their potential roles in girdle development. In this study, by generating compound mutant mice, we examined the genetic control of scapula development by Pbx genes and their functional relationship with Emx2 . Analyses of Pbx and PBX1 ; Emx2 compound mutants revealed that Pbx genes share overlapping functions in shoulder development and that PBX1 genetically interacts with Emx2 in this process. Here, we provide a biochemical basis for PBX1 ; Emx2 genetic interaction by showing that PBX1 and Emx2 can bind specific DNA sequences as heterodimers. Moreover, the expression of genes crucial for scapula development is altered in these mutants, indicating that Pbx genes act upstream of essential pathways for scapula formation. In particular, expression of Alx1 , an effector of scapula blade patterning, is absent in all compound mutants. We demonstrate that PBX1 and Emx2 bind in vivo to a conserved sequence upstream of Alx1 and cooperatively activate its transcription via this potential regulatory element. Our results establish an essential role for PBX1 in genetic interactions with its family members and with Emx2 and delineate novel regulatory networks in shoulder girdle development.

Jun Wang - One of the best experts on this subject based on the ideXlab platform.

  • e2a PBX1 functions as a coactivator for runx1 in acute lymphoblastic leukemia
    Blood, 2020
    Co-Authors: Jun Wang, Huimin Geng, Miho Shimada, Jia Wei Lin, Yuling Lee, Gang Greg Wang, Robert G Roeder, Weiyi Chen
    Abstract:

    E2A, a basic helix-loop-helix transcription factor, plays a crucial role in determining tissue-specific cell fate, including differentiation of B-cell lineages. In 5% of childhood acute lymphoblastic leukemia (ALL), the t(1,19) chromosomal translocation specifically targets the E2A gene and produces an oncogenic E2A-PBX1 fusion protein. Although previous studies have shown the oncogenic functions of E2A-PBX1 in cell and animal models, the E2A-PBX1-enforced cistrome, the E2A-PBX1 interactome, and related mechanisms underlying leukemogenesis remain unclear. Here, by unbiased genomic profiling approaches, we identify the direct target sites of E2A-PBX1 in t(1,19)-positive pre-B ALL cells and show that, compared with normal E2A, E2A-PBX1 preferentially binds to a subset of gene loci cobound by RUNX1 and gene-activating machineries (p300, MED1, and H3K27 acetylation). Using biochemical analyses, we further document a direct interaction of E2A-PBX1, through a region spanning the PBX1 homeodomain, with RUNX1. Our results also show that E2A-PBX1 binding to gene enhancers is dependent on the RUNX1 interaction but not the DNA-binding activity harbored within the PBX1 homeodomain of E2A-PBX1. Transcriptome analyses and cell transformation assays further establish a significant RUNX1 requirement for E2A-PBX1-mediated target gene activation and leukemogenesis. Notably, the RUNX1 locus itself is also directly activated by E2A-PBX1, indicating a multilayered interplay between E2A-PBX1 and RUNX1. Collectively, our study provides the first unbiased profiling of the E2A-PBX1 cistrome in pre-B ALL cells and reveals a previously unappreciated pathway in which E2A-PBX1 acts in concert with RUNX1 to enforce transcriptome alterations for the development of pre-B ALL.

  • overexpression of lipid metabolism genes and PBX1 in the contralateral breasts of women with estrogen receptor negative breast cancer
    International Journal of Cancer, 2017
    Co-Authors: Jun Wang, Ali Shidfar, Liannian Liu, Miran Choi, David Ivancic, Megan E Sullivan, Demirkan B Gursel, Manish Ranjan, Vamsi Parimi, Matthew S Najor
    Abstract:

    Risk biomarkers for estrogen receptor (ER)-negative breast cancer have clear value for breast cancer prevention. We previously reported a set of lipid metabolism (LiMe) genes with high expression in the contralateral unaffected breasts (CUBs) of ER-negative cancer cases. We now further examine LiMe gene expression in both tumor and CUB, and investigate the role of Pre-B-cell leukemia homeobox-1 (PBX1) as a candidate common transcription factor for LiMe gene expression. mRNA was extracted from laser-capture microdissected epithelium from tumor and CUB of 84 subjects (28 ER-positive cases, 28 ER-negative cases, 28 healthy controls). Gene expression was quantitated by qRT-PCR. Logistic regression models were generated to predict ER status of the contralateral cancer. Protein expression of HMGCS2 and PBX1 was measured using immunohistochemistry. The effect of PBX1 on LiMe gene expression was examined by overexpressing PBX1 in MCF10A cells with or without ER, and by suppressing PBX1 in MDA-MB-453 cells. The expression of DHRS2, HMGCS2, UGT2B7, UGT2B11, ALOX15B, HPGD, UGT2B28 and GLYATL1 was significantly higher in ER-negative versus ER-positive CUBs, and predicted ER status of the tumor in test and validation sets. In contrast, LiMe gene expression was significantly lower in ER-negative than ER-positive tumors. PBX1 overexpression in MCF10A cells up-regulated most LiMe genes, but not in MCF10A cells overexpressing ER. Suppressing PBX1 in MDA-MB-453 cells resulted in decrease of LiMe gene expression. Four binding sites of PBX1 and cofactor were identified in three lipid metabolism genes using ChIP-qPCR. These data suggest a novel role for PBX1 in the regulation of lipid metabolism genes in benign breast, which may contribute to ER-negative tumorigenesis.

  • abstract 1985 PBX1 regulated lipid metabolism gene expression and epithelial mesenchymal transition independent of estrogen receptor
    Cancer Research, 2015
    Co-Authors: Ali Shidfar, Liannian Liu, Vamsi Parini, Miran Choi, David Ivancic, Megan E Sullivan, Demirkan B Gursel, Seema A Khan, Jun Wang
    Abstract:

    Introduction: Pre-B-cell leukemia homeobox-1 (PBX1) is a member of the three amino acid loop extension (TALE) family of homeodomain proteins that bind to DNA and regulate gene transcription by forming heterodimeric transcription complexes with Meis and Prep1. PBX1 is involved in cell fate determination during organogenesis and contributes to oncogenic activity in breast cancer. As a pioneer factor, PBX1 was found to drive ER signaling in ER+ breast cancer by remodeling the chromatin and increasing DNA accessibility. But the role of PBX1 in benign breast and ER- cancer cells is not clear. In our previous studies, we identified and validated that the expression of a set of lipid metabolism genes was higher in the contralateral breast of ER- tumor. Bioinformatic analysis on lipid metabolism gene promoter regions and revealed that PBX1 may act as a potential transcription factor to co-regulate those genes. In this study, we further investigate the function of PBX1 in ER- cells. Methods: Among the ER- cell lines, we infected cell lines expressing low endogenous PBX1 (MCF10A and MDA-MB-231) with PBX1 gene in lentiviral vector. We also infected cell lines expressing high endogenous PBX1 (MDA-MB-453 and SK-BR-3) with PBX1-shRNA to knockdown PBX1. The expression of lipid metabolism genes was detected by qRT-PCR. Markers for epithelial-to-mesenchymal transition (EMT) including E-cadherin, vimentin, β-catenin and α-SMA were detected using Western blot. The effects of overexpression or knock-down of PBX1 on proliferation, migration, and invasion were measured using IncuCyte live cell imaging system. The expression of PBX1 protein was measured in benign contralateral breast and in the matching tumor using mmnunohistochemistry. Results: Over-expression of PBX1 in ER- cell lines (MCF10A and MDA-MB-231) up-regulated lipid metabolism genes and promoted cell migration and invasion by inducing EMT (increased vimentin and decreased E-cadherin and β-catenin). In contrast, knocking-down PBX1 using shRNA in ER- cell lines (MDA-MB-453 and SK-BR-3) suppressed lipid metabolism gene expression. PBX1 was more highly expressed in benign tissues associated with ER- tumors compared to ER+ tumors. In tumor tissue, on the contrary, ER+ tumors shower higher PBX1 expression levels than ER- tumors. Conclusion: PBX1 is a master regulator of lipid metabolism genes. PBX1 promoted cell migration and invasion by inducing EMT. PBX1 may play different roles and interact with different co-factors in ER+ tumors and in benign tissues associated with ER- tumors. Citation Format: Ali Shidfar, Liannian Liu, Vamsi Parini, MiRan Choi, David Ivancic, Megan E. Sullivan, Demirkan B. Gursel, Seema A. Khan, Jun Wang. PBX1 regulated lipid metabolism gene expression and epithelial-mesenchymal transition independent of estrogen receptor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1985. doi:10.1158/1538-7445.AM2015-1985

Guy Sauvageau - One of the best experts on this subject based on the ideXlab platform.

  • the oncoprotein e2a PBX1a collaborates with hoxa9 to acutely transform primary bone marrow cells
    Molecular and Cellular Biology, 1999
    Co-Authors: Unnur Thorsteinsdottir, Evert Kroon, Jana Krosl, Andre Haman, Trang Hoang, Guy Sauvageau
    Abstract:

    Homeodomain (HD)-containing Hox gene products, regulators of pattern formation and tissue identity during embryogenesis (15), have also been identified as potential regulators of hemopoietic cell proliferation and differentiation (17). Several lines of evidence now also directly implicate Hox genes in human and murine leukemias. These include (i) the expression of Hoxa9 as a fusion protein Hoxa9-NUP98, in a subset of human myeloid leukemias (2, 21); (ii) the activation of Hoxa7 and Hoxa9 by retroviral insertional mutagenesis in myeloid leukemias in BXH-2 mice (22), and (iii) the development of leukemias in mice transplanted with bone marrow cells engineered to retrovirally overexpress Hoxb8, Hoxb3, Hoxa10, or Hoxa9 (13, 25, 32, 39). A number of studies have demonstrated that Hox proteins collaborate in the in vitro DNA binding with a group of HD-containing proteins comprising Pbx and Meis families (34–36). This cooperative interaction between Pbx and Hox proteins is important since genetic and molecular studies in mice and in Drosophila have shown that PBX1 (or its Drosophila homolog exd) is required for some of the biological functions of Hox proteins (1, 4, 19, 29, 30). The relevance of this Hox-Pbx interaction for malignant transformation has been demonstrated, as we recently showed that Hoxb3- or Hoxb4-induced transformation of Rat-1 fibroblasts is dependent on the presence of endogenous PBX1 (14). An oncogenic collaboration between Hox and Meis proteins has also been established both by proviral insertion (22) and by retroviral overexpression studies (13). The Hox-Pbx interacting surfaces have been the focus of a number of studies and include, in addition to the HDs of both proteins, a tryptophan-containing motif located N terminal to the HD of Hox (found in several Hox proteins) and a region of 20 to 25 conserved amino acids called HCM (Hox cooperativity motif) located C terminal to the HD of PBX1 (6, 24, 26, 27). The structure of the Hoxb1-PBX1 complex bound to DNA was recently solved by crystallographic studies and confirmed the importance of these motives for Hox-PBX1 interactions (28). Interestingly, these studies also demonstrated that the HCM motif of PBX1 is part of its HD giving rise to a fourth α helix (28). One member of the Pbx family, PBX1, is also involved in human malignancy. In the t(1;19)(q23;p13.3) chromosomal translocation (11, 23), found in 10 to 20% of human pediatric pre-B acute lymphoblastic leukemias (3), most of the PBX1 coding sequence, including the segment encoding the HD, is fused to the 5′ half of the E2A gene, which encodes two transcription activation domains but lacks both the DNA binding and dimerization domains of E2A (11, 23). In addition to the involvement of the E2A-PBX1 fusion gene in human acute lymphoblastic leukemia, various transformation assays have clearly demonstrated that the E2A-PBX1 fusion protein is oncogenic. Transgenic mice expressing the E2A-PBX1 cDNA in lymphoid cells developed T-cell lymphoblastic lymphomas (8), and mice reconstituted with bone marrow cells engineered by retrovirus-mediated gene transfer to overexpress E2A-PBX1 developed growth factor-dependent acute myeloid leukemias (AML) (10). The in vitro cooperative DNA binding properties of PBX1 and E2A-PBX1 with Hox proteins are not significantly different (18). This has lead to one current hypothesis, that at least in part, the transforming capacity of E2A-PBX1 could be mediated by Hox gene products. In agreement with this possibility, deletion of all of the PBX1 sequence from the E2A-PBX1 fusion protein completely abrogates transformation of NIH 3T3 cells, indicating that the PBX1 half of the fusion protein is essential for its transforming abilities (12, 20). Furthermore, the HCM of the PBX1 half of the E2A-PBX1 fusion protein is essential for cellular transformation induced by E2A-PBX1 (5). This finding is very interesting in light of the recent crystallographic studies mentioned above which have redefined the HCM as part of an extended HD in PBX1 (28). The concept that E2A-PBX1-induced transformation is Hox dependent was challenged by studies which showed that the helices 1 to 3 of the HD in E2A-PBX1 are dispensable for the capacity of this fusion protein to transform NIH 3T3 cells and T lymphocytes (12, 20). However, it was recently shown that the HD of E2A-PBX1 is necessary to block cellular differentiation of myeloid progenitor cells, a process central to leukemic transformation (12). Together, these studies thus suggest that cellular transformation induced by E2A-PBX1 may involve more than one pathway (mechanisms). In contrast to E2A-PBX1, the Pbx proteins lack inherent transforming potential (12–14, 20), and fusion with E2A is essential for the transforming ability of E2A-PBX1. Structure-function and mutagenesis experiments have demonstrated that the two transcriptional activation domains in E2A are essential for mediating both the malignant transformation of NIH 3T3 cells (20) and blocking the differentiation of myeloid progenitor cells (12). Furthermore, using a Pbx-responsive sequence which allows cooperative DNA binding between Hox and PBX1 (or E2A-PBX1), it was shown in a reporter assay that E2A-PBX1, but not PBX1, could induce significant transcriptional activity of the reporter gene and that Hox proteins had the capacity to modulate the transactivating activity of E2A-PBX1 (18). Together, these observations suggest that the oncogenic potential of E2A-PBX1 is directly linked to the transcriptional activating function of the chimeric protein, and that this activity can be modulated by Hox gene products. To examine whether Hox proteins and the E2A-PBX1a fusion protein could collaborate to transform primary hematopoietic cells, we engineered mouse bone marrow cells, by retroviral gene transfer, to cooverexpress E2A-PBX1a with Hoxa9, and the oncogenic collaboration between these two genes was directly tested in vitro and in vivo following transplantation of retrovirally transduced cells.

  • hoxa9 transforms primary bone marrow cells through specific collaboration with meis1a but not PBX1b
    The EMBO Journal, 1998
    Co-Authors: Evert Kroon, Arthur M Buchberg, Jana Krosl, Unnur Thorsteinsdottir, Soheyl Baban, Guy Sauvageau
    Abstract:

    Hoxa9, Meis1 and PBX1 encode homeodomaincontaining proteins implicated in leukemic transformation in both mice and humans. Hoxa9, Meis1 and PBX1 proteins have been shown to physically interact with each other, as Hoxa9 cooperatively binds consensus DNA sequences with Meis1 and with PBX1, while Meis1 and PBX1 form heterodimers in both the presence and absence of DNA. In this study, we sought to determine if Hoxa9 could transform hemopoietic cells in collaboration with either PBX1 or Meis1. Primary bone marrow cells, retrovirally engineered to overexpress Hoxa9 and Meis1a simultaneously, induced growth factor-dependent oligoclonal acute myeloid leukemia in <3 months when transplanted into syngenic mice. In contrast, overexpression of Hoxa9, Meis1a or PBX1b alone, or the combination of Hoxa9 and PBX1b failed to transform these cells acutely within 6 months post-transplantation. Similar results were obtained when FDC-P1 cells, engineered to overexpress these genes, were transplanted to syngenic recipients. Thus, these studies demonstrate a selective collaboration between a member of the Hox family and one of its DNA-binding partners in transformation of hemopoietic cells.