Evi-1 transforming and repressor activities are mediated by CtBP Co-repressor proteins

Ectopic production of the Evil transcriptional repressor zinc finger protei n is seen in 4-6% of human acute myeloid leukemias. Overexpression also tra nsforms Rat1 fibroblasts by an unknown mechanism, which is likely to be rel ated to its role in leukemia and which depends upon its repressor activity. We show here that mutant murine Evi-1 proteins, lacking either the N-termi nal zinc finger DNA binding domain or both DNA binding zinc finger clusters , function as dominant negative mutants by reverting the transformed phenot ype of Evi-1 transformed Rat1 fibroblasts, The dominant negative activity o f the non-DNA binding mutants suggests sequestration of transformation-spec ific cofactors and that recruitment of these cellular factors might mediate Evi-1 transforming activity. C-terminal binding protein (CtBP) co-represso r family proteins bind PLDLS-like motifs, We show that the murine Evi-1 rep ressor domain has two such sites, PFDLT (site a, amino acids 553-659) and P LDLS (site b, amino acids 584-599), which independently can bind CtBP famil y co-repressor proteins, with site b binding with higher affinity than site a. Functional analysis of specific CtBP binding mutants show site b is abs olutely required to mediate both transformation of Rat1 fibroblasts and tra nscriptional repressor activity. This is the first demonstration that the b iological activity of a mammalian cellular transcriptional repressor protei n is mediated by CtBPs. Furthermore, it suggests that CtBP proteins are inv olved in the development of some acute leukemias and that blocking their ab ility to specifically interact with EVI1 might provide a target for the dev elopment of pharmacological therapeutic agents.