RESUMO
The mammalian lymphatic vasculature is important for returning fluids from the extracellular tissue milieu back to the blood circulation. We showed previously that Prox1 dosage is important for the development of the mammalian lymphatic vasculature. The lack of Prox1 activity results in the complete absence of lymphatic endothelial cells (LECs). In Prox1 heterozygous embryos, the number of LECs is reduced because of a decrease in the progenitor pool in the cardinal vein. This reduction is caused by some progenitor cells being unable to maintain Prox1 expression. In this study, we identified Vegfr3, the cognate receptor of the lymphangiogenic growth factor Vegfc, as a dosage-dependent, direct in vivo target of Prox1. Using various mouse models, we also determined that Vegfr3 regulates Prox1 by establishing a feedback loop necessary to maintain the identity of LEC progenitors and that Vegfc-mediated activation of Vegfr3 signaling is necessary to maintain Prox1 expression in LEC progenitors. We propose that this feedback loop is the main sensing mechanism controlling the number of LEC progenitors and, as a consequence, the number of budding LECs that will form the embryonic lymphatic vasculature.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/fisiologia , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/fisiologia , Proteínas de Homeodomínio/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Contagem de Células , Embrião não Mamífero , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Vasos Linfáticos/citologia , Vasos Linfáticos/metabolismo , Camundongos , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Retinoblastoma is an aggressive childhood cancer of the developing retina that is initiated by the biallelic loss of RB1. Tumours progress very quickly following RB1 inactivation but the underlying mechanism is not known. Here we show that the retinoblastoma genome is stable, but that multiple cancer pathways can be epigenetically deregulated. To identify the mutations that cooperate with RB1 loss, we performed whole-genome sequencing of retinoblastomas. The overall mutational rate was very low; RB1 was the only known cancer gene mutated. We then evaluated the role of RB1 in genome stability and considered non-genetic mechanisms of cancer pathway deregulation. For example, the proto-oncogene SYK is upregulated in retinoblastoma and is required for tumour cell survival. Targeting SYK with a small-molecule inhibitor induced retinoblastoma tumour cell death in vitro and in vivo. Thus, retinoblastomas may develop quickly as a result of the epigenetic deregulation of key cancer pathways as a direct or indirect result of RB1 loss.
Assuntos
Epigênese Genética/genética , Genômica , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/farmacologia , Retinoblastoma/tratamento farmacológico , Retinoblastoma/genética , Aneuploidia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Instabilidade Cromossômica/genética , Regulação Neoplásica da Expressão Gênica , Genes do Retinoblastoma/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Mutação/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proto-Oncogene Mas , Retinoblastoma/patologia , Proteína do Retinoblastoma/deficiência , Proteína do Retinoblastoma/genética , Análise de Sequência de DNA , Quinase Syk , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The homeobox gene Prox1 is crucial for mammalian lymphatic vascular development. In the absence of Prox1, lymphatic endothelial cells (LECs) are not specified. The maintenance of LEC identity also requires the constant expression of Prox1. However, the mechanisms controlling the expression of this gene in LECs remain poorly understood. The SRY-related gene Sox18 is required to induce Prox1 expression in venous LEC progenitors. Although Sox18 is also expressed in embryonic arteries, these vessels do not express Prox1, nor do they give rise to LECs. This finding suggests that some venous endothelial cell-specific factor is required for the activation of Prox1. Here we demonstrate that the nuclear hormone receptor Coup-TFII is necessary for the activation of Prox1 in embryonic veins by directly binding a conserved DNA domain in the regulatory region of Prox1. In addition, we show that the direct interaction between nuclear hormone receptors and Prox1 is also necessary for the maintenance of Prox1 expression during early stages of LEC specification and differentiation.
Assuntos
Fator II de Transcrição COUP/metabolismo , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Fator II de Transcrição COUP/genética , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Células Endoteliais/citologia , Deleção de Genes , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Proteínas Supressoras de Tumor/genética , Veias/embriologiaRESUMO
Translocations interrupting the mixed lineage leukemia gene (MLL) occur in 7-10% of acute lymphoblastic leukemia (ALL) and 5-6% of acute myeloid leukemia (AML) cases. One of these translocations, t(11;15)(q23;q14), occurs rarely in both ALL and AML. The gene on chromosome 15, AF15q14, was cloned recently in a patient with AML-M4. We have identified the same gene in a de novo T-ALL patient. However, both the MLL and AF15q14 breakpoints in these patients differed: in the previously reported AML-M4, both gene breaks were within exons, while in our ALL case the MLL break is intronic and the AF15q14 break is exonic. The MLL-AF15q14 fusion described previously shares no AF15q14 residues in common with the chimera reported here. The fusion proteins also differ with respect to MLL--the previously described fusion contains 55 extra amino acids as its MLL break is in exon 11, while the chimera we report breaks in intron 9. Contrary to the originally described normal AF15q14 (5925-bp cDNA encoding a 1833-aa protein), we identify a 7542-bp cDNA and a 2342-aa AF15q14 protein. AF15q14 appears identical to an mRNA previously found to be expressed in melanoma rendered nontumorigenic by microcell-mediated introduction of normal chromosome 6, suggesting the gene may function normally to suppress cell growth and/or enhance maturation.
Assuntos
Proteínas de Transporte , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 15/genética , Leucemia Mielomonocítica Aguda/genética , Leucemia-Linfoma de Células T do Adulto/genética , Melanoma/genética , Proteínas de Fusão Oncogênica/genética , Proteínas/genética , Translocação Genética/genética , Sequência de Aminoácidos , Quebra Cromossômica , Cromossomos Humanos Par 11/ultraestrutura , Cromossomos Humanos Par 15/ultraestrutura , Cromossomos Humanos Par 6/genética , Teste de Complementação Genética , Hematopoese/genética , Humanos , Íntrons/genética , Melanoma/patologia , Proteínas Associadas aos Microtúbulos , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide , Proteínas/fisiologia , RNA Mensageiro/genética , Células Tumorais CultivadasRESUMO
Myelodysplasia/acute myeloid leukemia (MDS/AML) is characterized by a t(3;5)(q25.1;q34) chromosomal translocation that forms a fusion gene between nucleophosmin (NPM) and MDS/myeloid leukemia factor 1 (MLF1). We identified a novel protein, MLF1-interacting protein (MLF1IP), that specifically associates with MLF1 by yeast two-hybrid analysis and in pulldown assays, and colocalizes with it in both the nuclei and cytoplasm of cells. The MLF1IP gene locus is at chromosome 4q35.1 and is composed of 14 exons spanning 75.8 kb of genomic DNA. The MLF1IP cDNA encodes a 46-kDa protein that contains two bipartite and two classical nuclear localization signals, two nuclear receptor-binding motifs (LXXLL), two leucine zippers, two PEST residues and several potential phosphorylation sites. MLF1IP transcripts are expressed in a variety of tissues (e.g. fetal liver, bone marrow, thymus and testis). MLF1IP appears to be a lineage-specific gene whose expression is confined exclusively to the CFU-E erythroid precursor cells, but not in mature erythrocytes. These observations, together with previous data demonstrating a role for MLF1 in suppressing red cell maturation, suggest a possible role for MLF1IP and MLF1 deregulation in the genesis of erythroleukemias.
Assuntos
Proteínas Nucleares/genética , Proteínas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Proteínas de Ciclo Celular , DNA Complementar , Proteínas de Ligação a DNA , Imunofluorescência , Células-Tronco Hematopoéticas/metabolismo , Histonas , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Técnicas do Sistema de Duplo-HíbridoRESUMO
Avian influenza viruses have adapted to human hosts, causing pandemics in humans. The key host-specific amino acid mutations required for an avian influenza virus to function in humans are unknown. Through multiple-sequence alignment and statistical testing of each aligned amino acid, we identified markers that discriminate human influenza viruses from avian influenza viruses. We applied strict thresholds to select only markers which are highly preserved in human influenza virus isolates over time. We found that a subset of these persistent host markers exist in all human pandemic influenza virus sequences from 1918, 1957, and 1968, while others are acquired as the virus becomes a seasonal influenza virus. We also show that human H5N1 influenza viruses are significantly more likely to contain the amino acid predominant in human strains for a few persistent host markers than avian H5N1 influenza viruses. This sporadic enrichment of amino acids present in human-hosted viruses may indicate that some H5N1 viruses have made modest adaptations to their new hosts in the recent past. The markers reported here should be useful in monitoring potential pandemic influenza viruses.
Assuntos
Aves/virologia , Surtos de Doenças , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/virologia , Influenza Humana/virologia , Substituição de Aminoácidos/genética , Animais , Evolução Molecular , Marcadores Genéticos , Humanos , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/epidemiologia , Influenza Humana/epidemiologia , Mutagênese , Estações do Ano , Alinhamento de Sequência , Análise de Sequência de Proteína , Análise de Sequência de RNA , Proteínas Virais/genéticaRESUMO
The spread of H5N1 avian influenza viruses (AIVs) from China to Europe has raised global concern about their potential to infect humans and cause a pandemic. In spite of their substantial threat to human health, remarkably little AIV whole-genome information is available. We report here a preliminary analysis of the first large-scale sequencing of AIVs, including 2196 AIV genes and 169 complete genomes. We combine this new information with public AIV data to identify new gene alleles, persistent genotypes, compensatory mutations, and a potential virulence determinant.
Assuntos
Genes Virais , Virus da Influenza A Subtipo H5N1/genética , Vírus da Influenza A/genética , Proteínas não Estruturais Virais/química , Fatores de Virulência/química , Animais , Aves/virologia , Biologia Computacional , Genoma Viral , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H2N2/genética , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N8/genética , Virus da Influenza A Subtipo H5N1/química , Virus da Influenza A Subtipo H5N1/patogenicidade , Vírus da Influenza A Subtipo H5N2/genética , Vírus da Influenza A Subtipo H7N7/genética , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A/química , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/patogenicidade , Influenza Aviária/virologia , Influenza Humana/virologia , Dados de Sequência Molecular , Mutação , Filogenia , RNA Viral/genética , Vírus Reordenados/genética , Análise de Sequência de DNA , Proteínas não Estruturais Virais/genética , Proteínas Virais/química , Proteínas Virais/genética , Fatores de Virulência/genéticaRESUMO
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with glycolytic and non-glycolytic functions, including pro-apoptotic activity. GAPDH accumulates in the nucleus after cells are treated with genotoxic drugs, and it is present in a protein complex that binds DNA modified by thioguanine incorporation. We identified a novel CRM1-dependent nuclear export signal (NES) comprising 13 amino acids (KKVVKQASEGPLK) in the C-terminal domain of GAPDH, truncation or mutation of which abrogated CRM1 binding and caused nuclear accumulation of GAPDH. Alanine scanning of the sequence encompassing the putative NES demonstrated at least two regions important for nuclear export. Site mutagenesis of Lys259 did not affect oligomerization but impaired nuclear efflux of GAPDH, indicating that this amino acid residue is essential for proper functioning of this NES. This novel NES does not contain multiple leucine residues unlike other CRM1-interacting NES, is conserved in GAPDH from multiple species, and has sequence similarities to the export signal found in feline immunodeficiency virus Rev protein. Similar sequences (KKVV*7-13PLK) were found in two other human proteins, U5 small nuclear ribonucleoprotein, and transcription factor BT3.