RESUMO
Ecotropic viral integration site-1 (EVI1) is an oncogenic zinc finger transcription factor whose expression is frequently up-regulated in myeloid leukemia and epithelial cancers. To better understand the mechanisms underlying EVI1-associated disease, we sought to define the EVI1 interactome in cancer cells. By using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we could confidently assign 78 proteins as EVI1-interacting partners for FLAG-tagged EVI1. Subsequently, we showed that 22 of 27 tested interacting proteins could coimmunoprecipitate with endogenous EVI1 protein, which represented an 81.5% validation rate. Additionally, by comparing the stable isotope labeling by amino acids in cell culture (SILAC) data with high-throughput yeast two hybrid results, we showed that five of these proteins interacted directly with EVI1. Functional classification of EVI1-interacting proteins revealed associations with cellular transcription machinery; modulators of transcription; components of WNT, TGF-ß, and RAS pathways; and proteins regulating DNA repair, recombination, and mitosis. We also identified EVI1 phosphorylation sites by MS analysis and showed that Ser538 and Ser858 can be phosphorylated and dephosphorylated by two EVI1 interactome proteins, casein kinase II and protein phosphatase-1α. Finally, mutations that impair EVI1 phosphorylation at these sites reduced EVI1 DNA binding through its C-terminal zinc finger domain and induced cancer cell proliferation. Collectively, these combinatorial proteomic approaches demonstrate that EVI1 interacts with large and complex networks of proteins, which integrate signals from various different signaling pathways important for oncogenesis. Comprehensive analysis of the EVI1 interactome has thus provided an important resource for dissecting the molecular mechanisms of EVI1-associated disease.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Mitose , Neoplasias/metabolismo , Proteínas Oncogênicas/metabolismo , Reparo de DNA por Recombinação , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Proteína do Locus do Complexo MDS1 e EVI1 , Neoplasias/genética , Neoplasias/patologia , Proteínas Oncogênicas/genética , Fosforilação/genética , Proto-Oncogenes/genética , Fatores de Transcrição/genéticaRESUMO
Ecotropic viral integration site 1 (EVI1) is an oncogenic dual domain zinc finger transcription factor that plays an essential role in the regulation of hematopoietic stem cell renewal, and its overexpression in myeloid leukemia and epithelial cancers is associated with poor patient survival. Despite the discovery of EVI1 in 1988 and its emerging role as a dominant oncogene in various types of cancer, few EVI1 target genes are known. This lack of knowledge has precluded a clear understanding of exactly how EVI1 contributes to cancer. Using a combination of ChIP-Seq and microarray studies in human ovarian carcinoma cells, we show that the two zinc finger domains of EVI1 bind to DNA independently and regulate different sets of target genes. Strikingly, an enriched fraction of EVI1 target genes are cancer genes or genes associated with cancer. We also show that more than 25% of EVI1-occupied genes contain linked EVI1 and activator protein (AP)1 DNA binding sites, and this finding provides evidence for a synergistic cooperative interaction between EVI1 and the AP1 family member FOS in the regulation of cell adhesion, proliferation, and colony formation. An increased number of dual EVI1/AP1 target genes are also differentially regulated in late-stage ovarian carcinomas, further confirming the importance of the functional cooperation between EVI1 and FOS. Collectively, our data indicate that EVI1 is a multipurpose transcription factor that synergizes with FOS in invasive tumors.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fatores de Transcrição/metabolismo , Sítios de Ligação , Adesão Celular , Imunoprecipitação da Cromatina , DNA/genética , DNA/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica , Células HeLa , Humanos , Proteína do Locus do Complexo MDS1 e EVI1 , Invasividade Neoplásica , Ligação Proteica , Proto-OncogenesRESUMO
The ecotropic viral integration site 1 (Evi1) oncogenic transcription factor is one of a number of alternative transcripts encoded by the Mds1 and Evi1 complex locus (Mecom). Overexpression of Evi1 has been observed in a number of myeloid disorders and is associated with poor patient survival. It is also amplified and/or overexpressed in many epithelial cancers including nasopharyngeal carcinoma, ovarian carcinoma, ependymomas, and lung and colorectal cancers. Two murine knockout models have also demonstrated Evi1's critical role in the maintenance of hematopoietic stem cell renewal with its absence resulting in the death of mutant embryos due to hematopoietic failure. Here we characterize a novel mouse model (designated Evi1(fl3)) in which Evi1 exon 3, which carries the ATG start, is flanked by loxP sites. Unexpectedly, we found that germline deletion of exon3 produces a hypomorphic allele due to the use of an alternative ATG start site located in exon 4, resulting in a minor Evi1 N-terminal truncation and a block in expression of the Mds1-Evi1 fusion transcript. Evi1(δex3/δex3) mutant embryos showed only a mild non-lethal hematopoietic phenotype and bone marrow failure was only observed in adult Vav-iCre/+, Evi1(fl3/fl3) mice in which exon 3 was specifically deleted in the hematopoietic system. Evi1(δex3/δex3) knockout pups are born in normal numbers but die during the perinatal period from congenital heart defects. Database searches identified 143 genes with similar mutant heart phenotypes as those observed in Evi1(δex3/δex3) mutant pups. Interestingly, 42 of these congenital heart defect genes contain known Evi1-binding sites, and expression of 18 of these genes are also effected by Evi1 siRNA knockdown. These results show a potential functional involvement of Evi1 target genes in heart development and indicate that Evi1 is part of a transcriptional program that regulates cardiac development in addition to the development of blood.
Assuntos
Alelos , Proteínas de Ligação a DNA/genética , Estudos de Associação Genética , Cardiopatias Congênitas/genética , Proto-Oncogenes/genética , Fatores de Transcrição/genética , Animais , Animais Recém-Nascidos , Sequência de Bases , Medula Óssea/patologia , Proteínas de Ligação a DNA/química , Modelos Animais de Doenças , Éxons , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genes Letais , Cardiopatias Congênitas/mortalidade , Cardiopatias Congênitas/patologia , Cardiopatias Congênitas/fisiopatologia , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Imunofenotipagem , Proteína do Locus do Complexo MDS1 e EVI1 , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutação , Fenótipo , Alinhamento de Sequência , Índice de Gravidade de Doença , Fatores de Transcrição/químicaRESUMO
The most common risk factor for developing hepatocellular carcinoma (HCC) is chronic infection with hepatitis B virus (HBV). To better understand the evolutionary forces driving HCC, we performed a near-saturating transposon mutagenesis screen in a mouse HBV model of HCC. This screen identified 21 candidate early stage drivers and a very large number (2,860) of candidate later stage drivers that were enriched for genes that are mutated, deregulated or functioning in signaling pathways important for human HCC, with a striking 1,199 genes being linked to cellular metabolic processes. Our study provides a comprehensive overview of the genetic landscape of HCC.