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 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.
Sakatsume
O
O
Tsukuba Life Science Center, RIKEN (The Institute of Physical and Chemical Research), 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan. kazunari@rtc.riken.go.jp
Tsutsui
H
H
Wang
Y
Y
Gao
H
H
Tang
X
X
Yamauchi
T
T
Murata
T
T
Itakura
K
K
Yokoyama
K K
KK
D002874:Chromosome Mapping; D019175:DNA Methylation; D015342:DNA Probes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D051840:Y-Box-Binding Protein 1; D016335:Zinc Fingers
D015342:DNA Probes; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016271:Proto-Oncogene Proteins c-myc; D012097:Repressor Proteins; C102581:THZif-1 protein, human; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; C076035:c-MYC-associated zinc finger protein