Abstract
A detailed analysis is reported of the binding of the zinc finger protein THZif-1 to the nuclease-hypersensitive element (NHE) in the promoter region of the c-MYC gene using the electrophoretic mobility shift assay and a series of mutants of a fusion protein composed of glutathione S-transferase and THZif-1. The THZif-1 protein bound specifically to the single-stranded (ss) pyrimidine-rich DNA of the NHE (ss c-myc NHE-C) with an apparent dissociation constant (Kd (app)) of 0.077 microM. By contrast, no binding to the single-stranded purine-rich DNA of the NHE (ss c-myc NHE-G) was detected. Moreover, the binding affinity of THZif-1 protein was 2-fold higher for the single-stranded 5-methyl-2'-deoxycytidine derivative of NHE (ss c-myc NHE-me5C) than for the unmethylated NHE. In the case of the binding of THZif-1 to methylated double-stranded (ds) NHE (ds c-myc NHE-me5CG), no significant binding 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 than two sites of deoxycytidine residues were methylated. However, the binding affinity of THZif-1 protein for methylated and for unmethylated triple-helical DNA of the NHE was almost identical. Moreover, the domain of the THZif-1 protein that made the major contribution to binding to ss c-myc NHE-C or ss c-myc NHE-me5C corresponded to the amino-terminal second zinc finger motif. Taken together, the results indicate that the THZif-1 protein exhibits preferential DNA-binding activity with 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 NHE-me5CG in vitro.
Highlights
Recent studies suggest that the c-Myc oncoprotein functions, in part, as a sequence-specific transcription factor
We showed previously that synthesis of the transcription factor THZif-1, a MAZ-like zinc finger protein, can be induced in the nuclei of HL60 cells that have been transformed with antisense c-MYC, and that it serves as a repressor of the endogenous transcription of the c-MYC protooncogene via binding to the nuclease-hypersensitive element (NHE) element [57,58,59,60]
Regulation of Transcription of the c-MYC Gene by THZif1—We isolated a cDNA clone for a protein that binds to CACCTCC repeated elements, designated THZif-1, which is a member of the ZF87/Maz (Pur-1) family [53,54,55]
Summary
Recent studies suggest that the c-Myc oncoprotein functions, in part, as a sequence-specific transcription factor. This protein is induced in a variety of cellular processes, which include regulation of progression of the cell cycle, proliferation, differentiation, and programmed cell death (apoptosis) [1,2,3,4]. Inactivation of c-myc and N-myc results in death of embryos by mid-gestation (6 –9), a phenomenon that demonstrates that each is required for normal development, the survival of the mutant embryos to such a late stage of embryogenesis suggests that members of the c-myc family of genes might have overlapping functions at early, but not later, stages of development. Several cis- and trans-acting components that regulate the initiation of transcription of the c-MYC gene have been described [1,2,3,4,5]
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