Identification of Binding Sites of EVI1 in Mammalian Cells
| Autor: | Archibald S. Perkins, Dongxian Yue, Andrew J. Read, Sharon Lin, Kristin E. Yates, Pei Hui, Amanda C. Poholek, Bogdan Yatsula |
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| Rok vydání: | 2005 |
| Předmět: |
Protein Conformation
Recombinant Fusion Proteins Molecular Sequence Data Mutation Missense Biology Biochemistry Mice Proto-Oncogenes Animals Humans Electrophoretic mobility shift assay Amino Acid Sequence Binding site Molecular Biology Gene Oligonucleotide Array Sequence Analysis Binding Sites Base Sequence Wild type Herpes Simplex Virus Protein Vmw65 Zinc Fingers DNA Cell Biology DNA-binding domain Molecular biology Fusion protein MDS1 and EVI1 Complex Locus Protein DNA-Binding Proteins DNA binding site Mutagenesis Site-Directed NIH 3T3 Cells Chromatin immunoprecipitation Transcription Factors |
| Zdroj: | Journal of Biological Chemistry. 280:30712-30722 |
| ISSN: | 0021-9258 |
| Popis: | The leukemia-associated protein EVI1 possesses seven zinc fingers within an N-terminal domain (amino acids 1-250) that binds to GACAAGATA. Single amino acid missense mutants of EVI1 were developed that failed to bind DNA either in vitro, as assessed by gel shift assay, or in vivo, as shown by transactivation studies. Specifically, mutation R205N lacks high affinity binding to the GACAAGATA motif. Putative EVI1 target genes were identified by using an EVI1-(1-250)-VP16 fusion protein that acts as a transcriptional activator with the binding specificity of EVI1. Sixteen genes induced in NIH 3T3 cells by wild type EVI1-VP16 but not by mutant forms were identified. Sequence analysis revealed evolutionarily conserved GACAAGATA-like motifs within 10 kb of their transcription start sites, and by chromatin immunoprecipitation in fibroblasts, we showed occupancy of many of these sites by EVI1-VP16. To assess whether native EVI1 binds to these sites in EVI1-transformed myeloid cells, we performed chromatin immunoprecipitation in 32Dcl3 and NFS58 cells, using anti-EVI1 antisera, and we showed that the majority of these sites is bound by wild type EVI1. These putative target genes include Gadd45g, Gata2, Zfpm2/Fog2, Skil (SnoN), Klf5 (BTEB2), Dcn, and Map3k14 (Nik). In this study we demonstrated for the first time that the N-terminal DNA binding domain of EVI1 has the capacity to bind to endogenous genes. We hypothesized that these genes play a critical role in EVI1-induced transformation. |
| Databáze: | OpenAIRE |
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