EVI1, A Potential Regulator Of CEBPA; A Contributory Mechanism In Leukemogenesis?

Ecotropic Viral Integration Site 1 (EVI1) is a zinc finger oncoprotein implicated in a subtype of acute myeloid leukemia associated with poor prognosis. Leukemic cells overexpressing EVI1 display a block in myeloid differentiation and resistance to apoptosis, both of which are reversed with EVI1 shRNA knockdown. Previous mutagenesis studies have shown that DNA binding via the first set of zinc fingers (ZF1) is critical for leukemic transformation by EVI1. The mediators between EVI1 DNA-binding and leukemogenesis are mostly unknown. Using chromatin immunoprecipitation and sequencing (ChIP-Seq) in myeloid leukemic cells, we have recently identified an evolutionarily conserved EVI1 binding site 35 kilobases downstream of Cebpa, encoding the CCAAT enhancer-binding protein alpha, a master regulator of early granulopoiesis. In order to obtain a better understanding of the potential regulatory role of this region in leukemogenesis, we used genome database analysis to see what other transcription factors or epigenetic marks are present. ChIP-Seq data deposited in the UCSC databank indicates the presence of CTCF, GATA2 and CEBPβ, as well as TAL1, p300, and GATA1 in leukemic cells, the latter three of which are known to bind together in a complex. This data suggests that the Cebpa +35 region serves an important role in regulating hematopoietic development and Cebpa transcription. In order to characterize the binding of EVI1 ZF1 to the Cebpa +35 site, we performed methylation interference assays and oligonucleotide mutagenesis assays. EVI1 ZF1 binds within this +35kb control region to the sequence TGACAGTGACGG with 133% affinity relative to the canonical motif. In an effort to assess any biological effects linked to EVI1 binding at the Cebpa +35 motif, we conducted EVI1 overexpression studies in the EML hematopoietic progenitor cell line. While control EML cells exhibited marked upregulation of Cebpa upon induction, EVI1-transduced cells displayed a significant suppression of Cebpa transcript. To determine if this suppression requires DNA binding, we tested a mutant of EVI1 that is defective in DNA binding (EVI1R205N); this was unable to suppress Cebpa induction. To test whether EVI1 can bind to the +35 KB site, we performed ChIP-PCR, which indeed showed EVI1 occupancy at the +35 KB site. In conclusion, we have shown evidence that EVI1 suppresses Cebpa transcription in early hematopoietic progenitor cells, and that the EVI1 binding site at Cebpa +35 serves an important function in myeloid maturation. This regulatory node represents a possible therapeutic target for the treatment of acute myeloid leukemia.