BINDING OF THZIF-1, A MAZ-LIKE ZINC-FINGER PROTEIN TO THE NUCLEASE-HYPERSENSITIVE ELEMENT IN THE PROMOTER REGION OF THE C-MYC PROTOONCOGENE

A detailed analysis is reported of the binding of the zinc finger prot ein THZif-1 to the nuclease hypersensitive element (NHE) in the promot er region of the c-MYC gene using the electrophoretic mobility shift a ssay and a series of mutants of a fusion protein composed of glutathio ne S-transferase and THZif-1. The THZif-1 protein bound specifically t o the single-stranded (ss) pyrimidine-rich DNA of the NHE (ss c-myc NH E-C) with an apparent dissociation constant (K-d(app)) of 0.077 mu M. By contrast, no binding to the single-stranded purine rich DNA of the NHE (ss c-myc NHE-me(5)C) was detected. Moreover, the binding affinity of THZif-1 protein was S-fold higher for the single-stranded 5-methyl -2'-deoxycytidine derivative of NHE (ss c-myc NHE-me(5)C) than for the unmethylated NHE, in the case of the binding of THZif-1 to methylated double-stranded (ds) NHE (ds c-myc NHE-me(5)CG), no significant bindi ng to the DNA was observed. The decrease in binding: to DNA of THZif-1 was significant in the case of mutated ds c-myc NHE, in which more th an two sites of deoxycytidine residues were methylated, However, the b inding affinity of THZif-1 protein for methylated and for unmethylated triple-helical DNA of the NHE was almost identical, Moreover, the dom ain of the THZif-1 protein that made the major contribution to binding to ss c-myc NHE-C or ss c-myc NHE-me(5)C corresponded to the amino-te rminal second zinc finger motif. Taken together, the results indicate that the THZif-1 protein exhibits preferential DNA-binding activity wi th ss c-myc NHE-C, ds c-myc NHE-CG, and ts c-myc NHE but not with ss c -myc NHE-G and ds c-myc NRE-me(5)CG in vitro.