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.

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