Ecotopic viral integration site 1 (EVI1) regulates multiple cellular processes important for cancer and is a synergistic partner for FOS protein in invasive tumors.
ABSTRACT: 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.
Project description:The EVI1 gene encodes for a transcription factor with two zinc finger domains and is transcriptionally activated in a subset of myeloid leukemias. In leukemia, the transcriptional activation of EVI1 usually results from chromosomal rearrangements. Besides leukemia, EVI1 has also been linked to solid tumors including breast cancer, lung cancer, ovarian cancer and colon cancer. The MDS1/EVI1 gene is encoded by the same locus as EVI1. While EVI1 functions as a transcription repressor, MDS1/EVI1 acts as a transcription activator. The fusion protein encoded by the AML1/MDS1/EVI1 chimeric gene, resulting from chromosomal translocations in a subset of chronic myeloid leukemia, exhibits a similar function to EVI1. EVI1 has been shown to regulate cell proliferation, differentiation and apoptosis, whereas the functions of MDS1/EVI1 and AML1/MDS1/EVI1 remain elusive. In this review, we summarize the genetic structures, biochemical properties and biological functions of these proteins in cancer.
Project description:We studied the variations of mRNA amounts after Flag-EVI1 or Flag expression in HeLa cells. Despites EVI1 discovery in 1988, its recognized role as a dominant oncogene in myeloid leukemia and more recently in epithelial cancers, only a few target genes were known and it was not clear why EVI1 was involved in cancer progression. Here we obtained the genomic binding occupancy and expression data for EVI1 in human cells. We identified numerous EVI1 target cancer genes and genes controlling cell migration and adhesion. Moreover, we characterized a transcriptional cooperation between AP1 and EVI1 that regulated proliferation and adhesion through a feed-forward loop. Furthermore, this study provides human genome-wide mapping and downstream analyses for EVI1 that will be useful for the research community. 8 samples were collected. Each condition was done in 4 replicates, collected 24 hours after transfection (for mild expression of EVI1). Transfections with Flag-expressing vector were used as controls.
Project description:EVI1 (Ecotropic Viral Integration site I), which was originally identified as a myeloid transforming gene by means of retroviral insertional mutagenesis in mouse leukemia, encodes a nuclear DNA binding zinc finger protein. The presence of zinc fingers that are able to bind to specific sequences of DNA suggests that EVI1 is a transcriptional regulator; however, except a few, target genes of EVI1 are poorly functionally identified thus far. In this study we provide evidence that EVI1 directly induces the expression of Bcl-xL through the first set of zinc finger and thereby inhibits apoptosis. ChIP analysis showed that EVI1 binds to the Bcl-xL promoter in HT-29 cells, a colon carcinoma cell line, which expresses EVI1. The observation is also supported by the fact that EVI1 siRNA treated HT-29 cells, shows a down regulation of Bcl-xL expression and that over expression of EVI1 results in the induction of the Bcl-xL reporter construct. A set of EVI1 positive chronic myeloid leukemia (CML) samples also showed higher Bcl-xL expression with respect to EVI1 negative samples. Interestingly, co-expression of EVI1 with wild type, but not with dominant-negative form of PCAF, abolishes the effect of EVI1 on Bcl-xL, indicating that acetylation of EVI1 abrogates its ability not only to bind Bcl-xL promoter but also alleviate Bcl-xL activity. Finally we have shown that EVI1 expression regulates apoptosis in HT-29 cells, which is abrogated when HT-29 cells are transfected with EVI1 siRNA or PCAF. The result for the first time shows a direct pathway by which EVI1 can protect cells from apoptosis and also demonstrates that the pathway can be reversed when EVI1 is acetylated.
Project description:The EVI1 (ecotropic viral integration site 1) gene at 3q26 codes for a transcriptional regulator with an essential role in haematopoiesis. Overexpression of EVI1 in acute myeloid leukaemia (AML) is frequently associated with 3q26 rearrangements and confers extremely poor prognosis. EVI1 mediates transcriptional regulation, signalling, and epigenetic modifications by interacting with DNA, proteins and protein complexes. To explore to what extent protein phosphorylation impacts on EVI1 functions, we analysed endogenous EVI1 protein from a high EVI1 expressing Fanconi anaemia (FA) derived AML cell line. Mass spectrometric analysis of immunoprecipitated EVI1 revealed phosphorylation at serine 196 (S196) in the sixth zinc finger of the N-terminal zinc finger domain. Mutated EVI1 with an aspartate substitution at serine 196 (S196D), which mimics serine phosphorylation of this site, exhibited reduced DNA-binding and transcriptional repression from a gene promotor selectively targeted by the N-terminal zinc finger domain. Forced expression of the S196D mutant significantly reduced EVI1 mediated transformation of Rat1 fibroblasts. While EVI1-mediated serial replating of murine haematopoietic progenitors was maintained by EVI1-S196D, this was associated with significantly higher Evi1-trancript levels compared with WT-EVI1 or EVI1-S196A, mimicking S196 non-phosphorylated EVI1. These data suggest that EVI1 function is modulated by phosphorylation of the first zinc finger domain.
Project description:DNA methylation patterns are frequently dysregulated in cancer, although little is known of the mechanisms through which specific gene sets become aberrantly methylated. The ecotropic viral integration site 1 (EVI1) locus encodes a DNA binding zinc-finger transcription factor that is aberrantly expressed in a subset of acute myeloid leukemia (AML) patients with poor outcome. We find that the promoter DNA methylation signature of EVI1 AML blast cells differs from those of normal CD34(+) bone marrow cells and other AMLs. This signature contained 294 differentially methylated genes, of which 238 (81%) were coordinately hypermethylated. An unbiased motif analysis revealed an overrepresentation of EVI1 binding sites among these aberrantly hypermethylated loci. EVI1 was capable of binding to these promoters in 2 different EVI1-expressing cell lines, whereas no binding was observed in an EVI1-negative cell line. Furthermore, EVI1 was observed to interact with DNA methyl transferases 3A and 3B. Among the EVI1 AML cases, 2 subgroups were recognized, of which 1 contained AMLs with many more methylated genes, which was associated with significantly higher levels of EVI1 than in the cases of the other subgroup. Our data point to a role for EVI1 in directing aberrant promoter DNA methylation patterning in EVI1 AMLs.
Project description:The Ecotropic viral integration site 1 (Evi1) is a zinc finger transcription factor, which is located on chromosome 3q26, over-expression in some acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Elevated Evi1 expression in AML is associated with unfavorable prognosis. Therefore, Evi1 is one of the strong candidate in molecular target therapy for the leukemia. MicroRNAs (miRNAs) are small non-coding RNAs, vital to many cell functions that negatively regulate gene expression by translation or inducing sequence-specific degradation of target mRNAs. As a novel biologics, miRNAs is a promising therapeutic target due to its low toxicity and low cost. We screened miRNAs which down-regulate Evi1. miR-133 was identified to directly bind to Evi1 to regulate it. miR-133 increases drug sensitivity specifically in Evi1 expressing leukemic cells, but not in Evi1-non-expressing cells The results suggest that miR-133 can be promising therapeutic target for the Evi1 dysregulated poor prognostic leukemia.
Project description: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-?, 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-1?. 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.
Project description:Amplification of 3q26.2, found in many cancer lineages, is a frequent and early event in ovarian cancer. We previously defined the most frequent region of copy number increase at 3q26.2 to EVI1 (ecotropic viral integration site-1) and MDS1 (myelodysplastic syndrome 1) (aka MECOM), an observation recently confirmed by the cancer genome atlas (TCGA). MECOM is increased at the DNA, RNA, and protein level and likely contributes to patient outcome. Herein, we report that EVI1 is aberrantly spliced, generating multiple variants including a Del(190-515) variant (equivalent to previously reported) expressed in >90% of advanced stage serous epithelial ovarian cancers. Although EVI1(Del190-515) lacks ?70% of exon 7, it binds CtBP1 as well as SMAD3, important mediators of TGF? signaling, similar to wild type EVI1. This contrasts with EVI1 1-268 which failed to interact with CtBP1. Interestingly, the EVI1(Del190-515) splice variant preferentially localizes to PML nuclear bodies compared to wild type and EVI1(Del427-515). While wild type EVI1 efficiently repressed TGF?-mediated AP-1 (activator protein-1) and plasminogen activator inhibitor-1 (PAI-1) promoters, EVI1(Del190-515) elicited a slight increase in both promoter activities. Expression of EVI1 and EVI1(Del427-515) (but not EVI1(Del190-515)) in OVCAR8 ovarian cancer cells increased cyclin E1 LMW expression and cell cycle progression. Furthermore, knockdown of specific EVI1 splice variants (both MDS1/EVI1 and EVI1(Del190-515)) markedly increased claudin-1 mRNA and protein expression in HEY ovarian and MDA-MB-231 breast cancer cells. Changes in claudin-1 were associated with alterations in specific epithelial-mesenchymal transition markers concurrent with reduced migratory potential. Collectively, EVI1 is frequently aberrantly spliced in ovarian cancer with specific forms eliciting altered functions which could potentially contribute to ovarian cancer pathophysiology.
Project description:We studied the variations of mRNA amounts after Evi1 knockdown or Flag-Evi1 overexpression in SKOV-3 cells. Despites Evi1 discovery in 1988, its recognized role as a dominant oncogene in myeloid leukemia and more recently in epithelial cancers, only a few target genes were known and it was not clear why Evi1 was involved in cancer progression. Here we obtained the genomic binding occupancy and expression data for Evi1 in human ovarian carcinoma cells. We identified numerous Evi1 target cancer genes and genes controlling cell migration and adhesion. Moreover, we characterized a transcriptional cooperation between AP1 and Evi1 that regulated proliferation and adhesion through a feed-forward loop. Furthermore, this study provides human genome-wide mapping and downstream analyses for Evi1 that will be useful for the research community. Overall design: 16 samples were collected. Each condition was done in 4 replicates, collected 65 hours after transfection. Transfections with control siRNA or Flag-expressing vector were used as controls.
Project description:The zinc finger protein EVI1 is causally associated with acute myeloid leukemogenesis, and inhibition of its function with a small molecule therapeutic may provide effective therapy for EVI1-expressing leukemias. In this paper we describe the development of a pyrrole-imidazole polyamide to specifically block EVI1 binding to DNA. We first identify essential domains for leukemogenesis through structure-function studies on both EVI1 and the t(3;21)(q26;q22)-derived RUNX1-MDS1-EVI1 (RME) protein, which revealed that DNA binding to the cognate motif GACAAGATA via the first of two zinc finger domains (ZF1, encompassing fingers 1-7) is essential transforming activity. To inhibit DNA binding via ZF1, we synthesized a pyrrole-imidazole polyamide 1, designed to bind to a subsite within the GACAAGATA motif and thereby block EVI1 binding. DNase I footprinting and electromobility shift assays revealed a specific and high affinity interaction between polyamide 1 and the GACAAGATA motif. In an in vivo CAT reporter assay using NIH-3T3-derived cell line with a chromosome-embedded tet-inducible EVI1-VP16 as well as an EVI1-responsive reporter, polyamide 1 completely blocked EVI1-responsive reporter activity. Growth of a leukemic cell line bearing overexpressed EVI1 was also inhibited by treatment with polyamide 1, while a control cell line lacking EVI1 was not. Finally, colony formation by RME was attenuated by polyamide 1 in a serial replating assay. These studies provide evidence that a cell permeable small molecule may effectively block the activity of a leukemogenic transcription factor and provide a valuable tool to dissect critical functions of EVI1 in leukemogenesis.