The Zinc-Sensing Mechanism of Mouse MTF-1 Involves Linker Peptides between the Zinc Fingers

Mouse metal response element-binding transcription factor-1 (MTF-1) regulates the transcription of genes in response to a variety of stimuli, including exposure to zinc or cadmium, hypoxia, and oxidative stress. Each of these stresses may increase labile cellular zinc, leading to nuclear translocation, DNA binding, and transcriptional activation of metallothionein genes (MT genes) by MTF-1. Several lines of evidence suggest that the highly conserved six-zinc finger DNA-binding domain of MTF-1 also functions as a zinc-sensing domain. In this study, we investigated the potential role of the peptide linkers connecting the four N-terminal zinc fingers of MTF-1 in their zinc-sensing function. Each of these three linkers is unique, completely conserved among all known vertebrate MTF-1 orthologs, and different from the canonical Cys2His2 zinc finger TGEKP linker sequence. Replacing the RGEYT linker between zinc fingers 1 and 2 with TGEKP abolished the zinc-sensing function of MTF-1, resulting in constitutive DNA binding, nuclear translocation, and transcriptional activation of the MT-I gene. In contrast, swapping the TKEKP linker between fingers 2 and 3 with TGEKP had little effect on the metal-sensing functions of MTF-1, whereas swapping the canonical linker for the shorter TGKT linker between fingers 3 and 4 rendered MTF-1 less sensitive to zinc-dependent activation both in vivo and in vitro. These observations suggest a mechanism by which physiological concentrations of accessible cellular zinc affect MTF-1 activity. Zinc may modulate highly specific, linker-mediated zinc finger interactions in MTF-1, thus affecting its zinc- and DNA-binding activities, resulting in translocation to the nucleus and binding to the MT-I gene promoter.