RESUMO
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.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas E2 de Adenovirus/genética , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Sequência de Bases , Sequência Conservada/genética , Análise Mutacional de DNA , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Camundongos , Dados de Sequência Molecular , Mutagênese , Oligopeptídeos/síntese química , Oligopeptídeos/genética , Mutação Puntual , Fator de Transcrição 1 de Leucemia de Células Pré-BRESUMO
E2A-PBX1 is the oncogene produced at the t(1;19) chromosomal breakpoint of pediatric pre-B-cell leukemia. Expression of E2A-Pbx1 induces fibroblast transformation and myeloid and T-cell leukemia in mice and arrests differentiation of granulocyte macrophage colony-stimulating factor-dependent myeloblasts in cultured marrow. Recently, the Drosophila melanogaster protein Exd, which is highly related to Pbx1, was shown to bind DNA cooperatively with the Drosophila homeodomain proteins Ubx and Abd-A. Here, we demonstrate that the normal Pbx1 homeodomain protein, as well as its oncogenic derivative, E2A-Pbx1, binds the DNA sequence ATCAATCAA cooperatively with the murine Hox-A5, Hox-B7, Hox-B8, and Hox-C8 homeodomain proteins, which are themselves known oncoproteins, as well as with the Hox-D4 homeodomain protein. Cooperative binding to ATCAATCAA required the homeodomain-dependent DNA-binding activities of both Pbx1 and the Hox partner. In cotransfection assays, Hox-B8 suppressed transactivation by E2A-Pbx1. These results suggest that (i) Pbx1 may participate in the normal regulation of Hox target gene transcription in vivo and therein contribute to aspects of anterior-posterior patterning and structural development in vertebrates, (ii) that E2A-Pbx1 could abrogate normal differentiation by altering the transcriptional regulation of Hox target genes in conjunction with Hox proteins, and (iii) that the oncogenic mechanism of certain Hox proteins may require their physical interaction with Pbx1 as a cooperating, DNA-binding partner.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Genes Homeobox , Proteínas de Homeodomínio/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Oncogenes , Proteínas Proto-Oncogênicas/metabolismo , Animais , Sequência de Bases , Western Blotting , Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/genética , Metilação , Camundongos , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/genética , Fator de Transcrição 1 de Leucemia de Células Pré-B , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico , Relação Estrutura-Atividade , Transcrição Gênica , Ativação Transcricional , TransfecçãoRESUMO
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.
Assuntos
Sítio Alostérico , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3 , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada/genética , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Dimerização , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Proteína Meis1 , Fatores de Regulação Miogênica/química , Fatores de Regulação Miogênica/genética , Fatores de Regulação Miogênica/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B , Elementos de Resposta/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ativação Transcricional , Triptofano/genética , Triptofano/metabolismoRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Genes Homeobox , Proteínas de Homeodomínio/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ativação Transcricional , Sequência de Bases , Sítios de Ligação , Cromossomos Humanos Par 1 , Cromossomos Humanos Par 19 , DNA/isolamento & purificação , Proteínas de Ligação a DNA/química , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/farmacologia , Humanos , Proteínas de Fusão Oncogênica/química , Fosfoproteínas/farmacologia , Fator de Transcrição 1 de Leucemia de Células Pré-B , Testes de Precipitina , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Recombinantes/metabolismo , Sequências Reguladoras de Ácido Nucleico , Translocação Genética , Células Tumorais CultivadasRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/fisiologia , Células Progenitoras Mieloides/citologia , Proteínas Proto-Oncogênicas/metabolismo , Motivos de Aminoácidos , Animais , Diferenciação Celular , Núcleo Celular/metabolismo , Células Cultivadas , DNA/metabolismo , Feminino , Interleucina-6/farmacologia , Leucemia Mieloide Aguda , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/citologia , Células Progenitoras Mieloides/metabolismo , Neutrófilos/citologia , Fator de Transcrição 1 de Leucemia de Células Pré-B , Transcrição Gênica , Células Tumorais CultivadasRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Tretinoína/farmacologia , Animais , Carcinoma Embrionário , Diferenciação Celular , Núcleo Celular/metabolismo , Dexametasona/farmacologia , Estrogênios/farmacologia , Humanos , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Células PC12 , Fator de Transcrição 1 de Leucemia de Células Pré-B , Ratos , Ativação Transcricional , Transfecção , Células Tumorais CultivadasRESUMO
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.
Assuntos
Proteínas de Drosophila , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , Sequência Conservada , DNA/metabolismo , Drosophila/genética , Drosophila/metabolismo , Drosophila/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Humanos , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
One of the biggest roadblocks to using stem cells as the basis for regenerative medicine therapies is the tumorigenicity of stem cells. Unfortunately, the unique abilities of stem cells to self-renew and differentiate into a variety of cell types are also mechanistically linked to their tumorigenic behaviors. Understanding the mechanisms underlying the close relationship between stem cells and cancer cells has therefore become a primary goal in the field. In addition, knowledge gained from investigating the striking parallels between mechanisms orchestrating normal embryogenesis and those that invoke tumorigenesis may well serve as the foundation for developing novel cancer treatments. Emerging discoveries have demonstrated that epigenetic regulatory machinery has important roles in normal stem cell functions, cancer development and cancer stem cell (CSC) identity. These studies provide valuable insights into both the shared and distinct mechanisms by which pluripotency and oncogenicity are established and regulated. In this review, the cancer-related epigenetic mechanisms found in pluripotent stem cells and cancer cells will be discussed, focusing on both the similarities and the differences.
Assuntos
Epigênese Genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Transformação Celular Neoplásica/genética , Metilação de DNA , Humanos , Células-Tronco Neoplásicas/patologia , Células-Tronco Pluripotentes/patologiaRESUMO
Deregulated expression of members of the myc oncogene family has been linked to the genesis of a wide range of cancers, whereas their normal expression is associated with growth, proliferation, differentiation, and apoptosis. Myc proteins are transcription factors that function within a network of transcriptional activators (Myc) and repressors (Mxd/Mad and Mnt), all of which heterodimerize with the bHLHZ protein Mad and bind E-box sequences in DNA. These transcription factors recruit coactivator or corepressor complexes that in turn modify histones. Myc, Mxd/Max, and Mnt proteins have been thought to act on a specific subset of genes. However, expression array studies and, most recently, genomic binding studies suggest that these proteins exhibit widespread binding across the genome. Here we demonstrate by immunostaining of Drosophila polytene chromosome that Drosophila Myc (dMyc) is associated with multiple euchromatic chromosomal regions. Furthermore, many dMyc-binding regions overlap with regions containing active RNA polymerase II, although dMyc can also be found in regions lacking active polymerase. We also demonstrate that the pattern of dMyc expression in nuclei overlaps with histone markers of active chromatin but not pericentric heterochromatin. dMyc binding is not detected on the X chromosome rDNA cluster (bobbed locus). This is consistent with recent evidence that in Drosophila cells dMyc regulates rRNA transcription indirectly, in contrast to mammalian cells where direct binding of c-Myc to rDNA has been observed. We further show that the dMyc antagonist dMnt inhibits rRNA transcription in the wing disc. Our results support the view that the Myc/Max/Mad network influences transcription on a global scale.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Cromatina/genética , Cromatina/metabolismo , Cromossomos/genética , Cromossomos/metabolismo , Genes de Insetos , Transcrição GênicaRESUMO
We have previously demonstrated that collateral development takes place in a swine model of coronary artery occlusion. In this report we have examined the effect of coronary artery occlusion on urokinase and tissue plasminogen activator activity in the myocardium. Urokinase activity was increased four-fold in the ischemic heart compared to sham and unoperated controls. In contrast, the level of tissue plasminogen activator activity remained relatively constant. The increase in urokinase activity was associated with an upregulation of urokinase RNA levels and of the RNAs corresponding to the plasminogen activator inhibitors, PAI I and II. Urokinase has been shown to be an important angiogenic protease both in vivo and in cultured cells. Its increase during collateral development suggests that urokinase may play a role in angiogenesis in the ischemic heart.
Assuntos
Doença das Coronárias/metabolismo , Miocárdio/metabolismo , Ativadores de Plasminogênio/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Velocidade do Fluxo Sanguíneo , Circulação Colateral , Doença das Coronárias/etiologia , Doença das Coronárias/fisiopatologia , Modelos Animais de Doenças , Expressão Gênica , Neovascularização Patológica/etiologia , Ativadores de Plasminogênio/genética , Suínos , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Bases , Sítios de Ligação , Sondas de DNA , Modelos Moleculares , Dados de Sequência Molecular , Fator de Transcrição 1 de Leucemia de Células Pré-B , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico , Relação Estrutura-AtividadeRESUMO
The homeodomain protein TG-interacting factor (TGIF) represses transcription by histone deacetylase-dependent and -independent means. Heterozygous mutations in human TGIF result in holoprosencephaly, a severe genetic disorder affecting craniofacial development, suggesting that TGIF is critical for normal development. After transforming growth factorbeta (TGFbeta) stimulation, Smad proteins enter the nucleus and form transcriptional activation complexes or interact with TGIF, which functions as a corepressor. The relative levels of Smad corepressors and coactivators present within the cell may determine the outcome of a TGFbeta response. We show that TGIF interacts directly with the paired amphipathic alpha-helix 2 domain of the mSin3 corepressor, and TGIF recruits mSin3 to a TGFbeta-activated Smad complex. The mSin3 interaction domain of TGIF has been shown to be essential for repression of a TGFbeta transcriptional response. Thus, TGIF represents a targeting component of the mSin3 corepressor complex.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células COS , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Histona Desacetilases , Proteínas de Homeodomínio/genética , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteína Smad2 , Transativadores/genética , Transcrição Gênica , Fator de Crescimento Transformador beta/farmacologia , Células Tumorais CultivadasRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/genética , DNA/genética , Proteínas de Homeodomínio/genética , Leucemia Mieloide/genética , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas/genética , Sequência de Aminoácidos , Animais , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia Experimental/genética , Leucemia Experimental/metabolismo , Camundongos , Dados de Sequência Molecular , Proteína Meis1 , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismoRESUMO
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.
Assuntos
Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Proteínas de Homeodomínio/fisiologia , Leucemia Mieloide/patologia , Proteínas de Neoplasias/fisiologia , Fator de Células-Tronco/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Camundongos , Camundongos Endogâmicos BALB C , Proteína Meis1 , Frações Subcelulares/metabolismoRESUMO
We have investigated the molecular changes which occur during pressure overload hypertrophy of the RV in swine. Animals were banded on the pulmonary artery so that right ventricular pressure was increased two-fold. The heart was harvested at 3, 7, 24 and 72 h after surgery. Between 7 and 72 h there was evidence of muscle damage and inflammation. Northern blot experiments showed that pressure overload induced a transient increase in the expression of the immediate early genes and in the developmentally regulated atrial natriuretic factor and skeletal muscle alpha actin genes. Consistent with the histological observations of inflammation, increases in the expression of the gene for intercellular adhesion molecule, which encodes a protein involved in the binding of leukocytes by endothelial cells and myocytes, was observed between 3 and 24 h. In addition, the expression of vascular endothelial growth factor, a growth and permeability factor specific for endothelial cells was increased at 3 and 7 h of pressure overload. An increase in the expression of urokinase plasminogen activator and its inhibitors, plasminogen activator inhibitors I and II, was also observed between 3 and 24 h. This was associated with an increase in urokinase activity in the myocardial tissue. These results indicate that hypertrophy in a large mammal such as swine induces a program of gene expression similar to that previously described in rodents and suggests that up-regulation of a variety of other genes is an early response to pressure overload.