[Negative repressor THZif-1 of protooncogene c-myc]

Abstract

A human recombinant cDNA clone that encoded 253 amino acids residues of a zinc-finger protein (THZif-1) was cloned by screening a cDNA library prepared from human promyelocytic leukemia HL60 cells with synthetic oligodeoxynucleotide probes that corresponded to the amino acid sequences of tryptic peptides derived from the DNA-binding protein specific for the nuclease-hypersensitive element (NHE) of the human c-myc gene. The predicted amino acid sequence of THZif-1 included a DNA-binding domain that contained five tandemly repeated zinc finger motifs. The three amino-terminal sets of zinc finger motifs, including the second finger, were found to be responsible for high-affinity interactions with the triple-helical conformation of NHE, as well as for high-affinity binding to the single-pyrimidine-rich strand of NHE in a sequence-specific manner. Cotransfection, trans-activation and in vitro transcription studies using the wild-type form and THZif-1 with a mutated second zinc finger motif demonstrated that the DNA-binding activity specific for H-form DNA of NHE was a prerequisite for the negative regulation of the expression of the c-myc gene.