Identification of Binding Sites of EVI1 in Mammalian Cells

Abstract

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 GACAA-GATA-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.

Ecotopic viral integration site 1 (EVI1) regulates multiple cellular processes important for cancer and is a synergistic partner for FOS protein in invasive tumors

“…S1B). For instance, Flag-EVI1 occupied the GATA2 (12,20) 1S promoter similar to a previous report (21). We also found EVI1 binding sites at the FOS and JUN promoters (Fig.…”

“…Previous studies have shown that the EVI1 N-and C-terminal ZNF domains recognize a GATA-like motif (12,14,20) and an ETS-like motif (15), respectively. Using de novo motif search, we identified GATAGA and ETS-like DNA binding motifs for EVI1 that represented its genome-wide occupancy ( Fig.…”

“…Despite its discovery in 1988 (16,17), very few EVI1 target genes have been identified, and most of these loci are known to be bound by EVI1 N-terminal ZNF domain (18)(19)(20). This lack of knowledge regarding the genes transcriptionally regulated by EVI1 has precluded a complete understanding of EVI1's role in development and cancer.…”

Pbx1 is a downstream target of Evi-1 in hematopoietic stem/progenitors and leukemic cells

“…Genes expressed in the para-aortic splanchnopleural (P-Sp) region of mouse embryos (Gata1, Gata2, and Angpt1), genes involved in the HSC regulation (Gata2, Angpt1, Mpl, Jag2, Pbx1, and Setbp1), and genes related to platelet formation (Gata1, Mpl, Itga2b, and Itgb3) were identified. Another group also provided set of genes as potential targets for Evi-1 regulation using an inducible EVI1-VP16 chimera, including Gadd45g, Gata2, and Klf5 (Yatsula et al, 2005). Nevertheless, whether those are truly regulated by Evi-1 has yet to be determined.…”

Ethanol-responsive genes: identification of transcription factors and their role in metabolomics

“…Interestingly, growth arrest DNA-damage-inducible 45 gamma has been identified as a target gene of proto-oncogene EVI1. 52 Since a majority of the ER genes (e.g. v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian) (Erbb3), cyclin-dependent kinase inhibitor 1A (p21) (Cdkn1a), v-maf musculoaponeurotic fibrosarcoma oncogene family, protein F (avian) (Maff) and B-cell translocation gene 3 (Btg3)) contain the CRM model Ccr_1 with a statistically significant score, this makes EVI1 the most prominent candidate TF as a regulator of these genes involved in cell cycle regulation (Figure 1b).…”