EVI1 Interferes with Myeloid Maturation via Transcriptional Repression of Cebpa, via Binding to Two Far Downstream Regulatory Elements

Abstract

One mechanism by which oncoproteins work is through perturbation of cellular maturation; understanding the mechanisms by which this occurs can lead to the development of targeted therapies. EVI1 is a zinc finger oncoprotein involved in the development of acute myeloid leukemia; previous work has shown it to interfere with the maturation of granulocytes from immature precursors. Here we investigate the mechanism by which that occurs, using an immortalized hematopoietic progenitor cell line, EML-C1, as a model system. We document that overexpression of EVI1 abrogates retinoic acid-induced maturation of EML cells into committed myeloid cells, a process that can be documented by the down-regulation of stem cell antigen-1 and acquisition of responsiveness to granulocyte-macrophage colony-stimulating factor. We show that this requires DNA binding capacity of EVI1, suggesting that downstream target genes are involved. We identify the myeloid regulator Cebpa as a target gene and identify two EVI1 binding regions within evolutionarily conserved enhancer elements at ؉35 and ؉37 kb relative to the gene. EVI1 can strongly suppress Cebpa transcription, and add-back of Cebpa into EVI1-expressing EML cells partially corrects the block in maturation. We identify the DNA sequences to which EVI1 binds at ؉35 and ؉37 kb and show that mutation of one of these releases Cebpa from EVI1-induced suppression. We observe a more complex picture in primary bone marrow cells, where EVI1 suppresses Cebpa in stem cells but not in more committed progenitors. Our data thus identify a regulatory node by which EVI1 contributes to leukemia, and this represents a possible therapeutic target for treatment of EVI1-expressing leukemia.

Expression and regulation of C/EBPα in normal myelopoiesis and in malignant transformation

“…Interestingly, CEBPA expression in these subtypes of AML is often downregulated, and the direct binding of these oncoproteins to the enhancer could explain this effect. In fact, Perkins and colleagues 94 demonstrated that Evi1 binding to the murine 137 Kb enhancer is associated with a reduction of Cebpa expression in Evi1-expressing mouse myeloid leukemia lines. Potentially, the oncoproteins may alter active chromatin states at the enhancer(s), leading to inactivation of CEBPA.…”

GATA2 mutations in myeloid malignancies: Two zinc fingers in many pies

“…In fact, AML1‐ETO represses CEBPA transcription, indicating that GATA2 mutations arise on the fertile ground of CEBPA deficiency—due to either CEBPA mutation or downregulation (Figure , right panel). Another example for this correlation is AML with 3(q21;q26) aberrations, where the overexpression of EVI1 (MECOM) represses CEBPA , thus providing the basis for acquisition of GATA2 mutations. Additionally, somatic GATA2 mutations are either lost or acquired as the disease progresses .…”

EVI1 overexpression reprograms hematopoiesis via upregulation of Spi1 transcription

“…To mimic the effects of rearrangements at 3q26 in AML, we created a tetracycline (Tet)-inducible allele of Evi1 (termed Evi1 TO ) in the mouse by inserting seven Tet operons within the first exon (Fig. 1a 15 ), allowing for the induction of all three isoforms of Evi1 8 , 9 . Evi1 TO/TO mice are viable and fertile without phenotype indicating that the allele functions normally in the uninduced state.…”

“… 22 , 23 ) and can modulate the transcription of target genes via binding in cis (e.g., refs. 15 , 24 ), we wished to determine if the myeloid skewing phenotype induced by EVI1 was dependent on its binding to DNA using two EVI1 mutants (Fig. 6a ): EVI1-R205N, which lacks DNA binding via ZF domain1 8 and EVI1-R769C, which lacks DNA binding via ZF2 25 .…”