Abstract
The metal-regulatory transcription factor 1 (MTF-1) is a key regulator of heavy metal-induced transcription of metallothionein I and II and other genes in mammals and other metazoans. Transcriptional activation of genes by MTF-1 is mediated through binding to metal-responsive elements of consensus TGCRCNC in the target gene promoters. In an attempt to further clarify the mechanisms by which certain external signals activate MTF-1 and in turn modulate gene transcription, we show here that human MTF-1 has a dual nuclear and cytoplasmic localization in response to diverse stress stimuli. MTF-1 contains a consensus nuclear localization signal located just N-terminal to the first zinc finger that contributes to but is not essential for nuclear import. MTF-1 also harbors a leucine-rich, nuclear export signal. Under resting conditions, the nuclear export signal is required for cytoplasmic localization of MTF-1 as indicated by mutational analysis and transfer to the heterologous green fluorescent protein. Export from the nucleus was inhibited by leptomycin B, suggesting the involvement of the nuclear export protein CRM1. Our results further show that in addition to the heavy metals zinc and cadmium, heat shock, hydrogen peroxide, low extracellular pH (pH 6.0), inhibition of protein synthesis by cycloheximide, and serum induce nuclear accumulation of MTF-1. However, heavy metals alone (and not the other stress conditions) induce a significant transcriptional response via metal-responsive element promoter sequences, implying that nuclear import of MTF-1 is necessary but not sufficient for transcriptional activation. Possible roles for nuclear import under non-metal stress conditions are discussed.
Highlights
Gene regulation in response to cellular stress is mediated through a variety of signaling pathways
The cells were treated with substances that induce nuclear translocation of other transcription factors: tumor necrosis factor ␣ (50 ng/ml, 1 h) and 12-O-tetradecanoylphorbol-13-acetate (100 ng/ml, 1 h), which activate nuclear factor B by inducing the nuclear translocation of RelA [29], and sorbitol (400 mM, 1 h) which is known to stimulate the nuclear translocation of signal transduceractivators of transcription in mammalian cells [30] and HOG1 mitogen-activated protein kinase in yeast [31]; these treatments did not induce any obvious change in the subcellular localization of metal-regulatory transcription factor 1 (MTF-1)
This indicates that the nuclear translocation of MTF-1 in response to stress conditions is a specific property of this transcription factor
Summary
Gene regulation in response to cellular stress is mediated through a variety of signaling pathways. The mammalian metal-regulatory transcription factor 1 (MTF-1) is a zinc finger transcription factor that activates the transcription of target genes in response to heavy metal exposure via binding to MREs [6]. These sequence motifs of consensus TGCRCNC are present in the promoter regions of MT-I, MT-II, and a number of other genes, many of which are involved in stress [7,8,9]. Several transcription factors involved in stress-regulated rapid events, including p53, nuclear factor B, and nuclear factor of activated T cells, localize differentially between the cytoplasm and the nucleus in response to DNA damage, growth signals, or environmental stimuli [12, 13]. We demonstrate that the nuclear accumulation of MTF-1 per se does not result in transcriptional activation via MRE sequences, indicating that nuclear translocation and transcriptional activation functions can be separated
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