Interactions of the zinc-regulated factor (ZiRF1) with the mouse metallothionein Ia promoter.
A mouse cDNA clone, M96, encoding a metal-regulating-element (MRE)-binding protein, was analysed for its ability to act as a metal-regulated transcription factor. The metal depletion of a glutathione S-transferase (GST)-M96 fusion protein showed that Zn2+ ions modulate the MRE-binding activity, suggesting that the M96-encoded protein is a Zn2+-regulated factor (ZiRF1). The methylation interference assay showed the specific interactions of ZiRF1 with the MRE, MREd/c, present on the mouse metallothionein Ia promoter. Point mutations of the MREd/c nullified the metal-regulatory properties of this region. In mouse L-cell nuclear extracts, mobility-shift assays revealed a Zn2+-dependent MRE-binding complex (MBC) with DNA-recognition properties similar to those of ZiRF1. Antibodies raised against purified GST-ZiRF1 were able to specifically recognize MBC in Western-blot analyses. Competition analysis of MRE-binding proteins from mouse NIH3T3 cells with oligonucleotide matching the binding sites for SP1 and MTF1 confirmed that both the basal SP1 and the metal-regulated MBC/ZiRF1 interact with the MREd/c region. The significance of mutual interactions with the metal-responsive promoter regions of either metal-regulated or basal transcription factors is discussed.
Remondelli
P
P
Department of Pharmacology and Center for Molecular Genetics, UCSD School of Medicine, La Jolla, CA 92093-0636, USA.
Leone
A
A
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D007739:L Cells; D008668:Metallothionein / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*; D000097809:Transcription Factor MTF-1
D004247:DNA; D004268:DNA-Binding Proteins; D008668:Metallothionein; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D015032:Zinc; D000097809:Transcription Factor MTF-1