RUNX1/EVI1, which blocks myeloid differentiation, inhibits CCAAT-enhancer binding protein alpha function.

The RUNX1/EVI1 chimeric transcription factor produced by t(3;21) causes leukemic transformation in hematopoietic stem cell tumors, possibly through a differentiation block of malignant myeloid progenitors. A dominant negative effect over wild-type RUNX1 has been shown to constitute one of the underlying molecular mechanisms. We introduced RUNX1/EVI1 cDNA into LG-3 cells that differentiate along the myeloid lineage upon exposure to granulocyte colony stimulating factor, and confirmed that RUNX1/EVI1 suppressed the differentiation. To further investigate the molecular mechanisms of RUNX1/EVI1-mediated differentiation block, we analyzed RUNX1/EVI1's effect on the functions of CCAAT-enhancer binding protein alpha (C/EBPalpha), a key transcriptional regulator that induces granulocytic differentiation. RUNX1/EVI1 was found to associate with C/EBPalpha. By using a reporter assay with the CEBPA promoter, we observed a dominant negative effect of RUNX1/EVI1 over C/EBPalpha-mediated transcriptional activation via the carboxyl terminal-binding protein (CtBP)-binding site in the EVI1 portion. In a gel-shift assay, RUNX1/EVI1 downregulated the DNA-binding activity of C/EBPalpha. Therefore, recruitment of histone deacetylase via CtBP and disruption of DNA binding could be likely scenarios for the RUNX1/EVI1-induced dominant repression on C/EBPalpha. Importantly, coexpression of C/EBPalpha restored the differentiation ability of the RUNX1/EVI1-expressing LG-3 cells. All of these data argue that inhibition of C/EBPalpha function may be causatively related to the leukemogenic potential of RUNX1/EVI1.