THE CARBOXYL DOMAIN OF ZINC FINGERS OF THE EVI-1 MYELOID TRANSFORMINGGENE BINDS A CONSENSUS SEQUENCE OF GAAGATGAG

Inappropriate expression of the Evi-1 zinc finger gene is associated w ith myeloid leukemia and myelodysplastic syndromes in mice and humans and has been hypothesized to contribute to pathology by blocking myelo id differentiation. Evi-1 contains two domains of zinc fingers, an ami no-terminal domain of seven fingers and a carboxyl domain of three fin gers. The first domain binds a consensus sequence of GA(C/T)AAGATAAGAT AA in binding and amplication reactions or GATA repeat containing regi ons of genomic DNA. The experiments described here, establish a consen sus sequence for the carboxyl domain of zinc fingers consisting of GAA GATGAG. Unlike the first domain, the consensus sequence established fo r 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, demonstr ate that the individual fingers bind the predicted region of the conse nsus sequence. In an attempt to engineer binding of constructs contain ing the carboxyl domain, a variety of mutations were made in the middl e finger that would be predicted to change the consensus sequence in s pecific ways. Remarkably, most of the mutations were deleterious and d estroyed specific DNA binding. Although Evi-1 contains potential trans criptional activation domains, it was not able to activate gene transc ription from CAT constructs containing the consensus sequence.