The carboxyl domain of zinc fingers of the Evi-1 myeloid transforming gene binds a consensus sequence of GAAGATGAG

Abstract

Inappropriate expression of the Evi-1 zinc finger gene is associated with myeloid leukemia and myelodysplastic syndromes in mice and humans and has been hypothesized to contribute to pathology by blocking myeloid differentiation. Evi-1 contains two domains of zinc fingers, an amino-terminal domain of seven fingers and a carboxyl domain of three fingers. The first domain binds a consensus sequence of GA(C/T)AAGATAAGATAA in binding and amplication reactions or GATA repeat containing regions of genomic DNA. The experiments described here, establish a consensus sequence for the carboxyl domain of zinc fingers consisting of GAAGATGAG. Unlike the first domain, the consensus sequence established for the carboxyl domain is identical to that which would be predicted by the current rules relating to C2H2 zinc fingers and DNA recognition. Substitution of sequences in finger 8 with those in finger 9, demonstrate that the individual fingers bind the predicted region of the consensus sequence. In an attempt to engineer binding of constructs containing the carboxyl domain, a variety of mutations were made in the middle finger that would be predicted to change the consensus sequence in specific ways. Remarkably, most of the mutations were deleterious and destroyed specific DNA binding. Although Evi-1 contains potential transcriptional activation domains, it was not able to activate gene transcription from CAT constructs containing the consensus sequence.