Metal-Responsive Transcription Factor 1 (MTF-1) Activity Is Regulated by a Nonconventional Nuclear Localization Signal and a Metal-Responsive Transactivation Domain

Abstract

Metal-responsive transcription factor 1 (MTF-1) mediates both basal and heavy metal-induced transcription of metallothionein genes and also regulates other genes involved in the cell stress response and in metal homeostasis. In resting cells, MTF-1 localizes to both the cytoplasm and the nucleus but quantitatively accumulates in the nucleus upon metal load and under other stress conditions. Here we show that within the DNA-binding domain, a region spanning zinc fingers 1 to 3 (amino acids [aa] 137 to 228 in human MTF-1) harbors a nonconventional nuclear localization signal. This protein segment confers constitutive nuclear localization to a cytoplasmic marker protein. The deletion of the three zinc fingers impairs nuclear localization. The export of MTF-1 to the cytoplasm is controlled by a classical nuclear export signal (NES) embedded in the acidic activation domain. We show that this activation domain confers metal inducibility in distinct cell types when fused to a heterologous DNA-binding domain. Furthermore, the cause of a previously described stronger inducibility of human versus mouse MTF-1 could be narrowed down to a 3-aa difference in the NES; "humanizing" mouse MTF-1 at these three positions enhanced its metal inducibility to the level of human MTF-1.

The legless lizard Anguis fragilis (slow worm) has a potent metal-responsive transcription factor 1 (MTF-1)

“…Do these findings mean that metal inducibility is coupled to nuclear export function, such that MTF-1 must shuttle between nucleus and cytoplasm? This scenario is likely an oversimplification, as our previous work has shown that upon inhibition of nuclear export by the drug LMB, human MTF-1 is confined to the nucleus but still retains a large part -though not all -of its metal inducibility (Lindert et al, 2009). One also has to keep in mind that the NES motif overlaps with the acidic activation domain, thus a change in one might affect the other.…”

“…In MTF-1, the zinc fingers are involved in metal response (Bittel et al, 1998;Zhang et al, 2003) and in humans (and presumably in Anguis) -but not in rodents -the acidic activation domain can independently respond to metals (Lindert et al, 2009). Because the difference in metal response between human and mouse MTF-1 has been mapped to the nuclear export signal/ acidic activation domain (Lindert et al, 2009), we compared the activity of the NES motif of Anguis with that of its human and mouse counterparts and with selected point mutants. As shown in Figure 4, the NES domains Figure 4 In contrast to mouse MTF-1, reptile and human MTF-1 harbor a strong nuclear export (NES) function.…”

“…The NES sequences of human, mouse (Mus musculus), capybara (Hydrochoerus hydrochaeris), slow worm (Anguis fragilis), green anole (Anolis carolinensis), and pufferfish fugu (Takifugu rubripes) are aligned, with divergent amino acid positions indicated in bold red. Note that in this particular region, human MTF-1 is more closely related to reptile than to rodent (mouse, capybara) MTF-1, which is functionally significant [see Figure 4 and Lindert et al (2009)]. Other domains of functional importance are also indicated (Günther et al, 2012b).…”

“…The zinc finger region and other domains of functional importance that had been characterized in human MTF-1 are strongly conserved in the slow worm. Of note, the human nuclear export signal (NES), which is embedded in the acidic activation domain (Saydam et al, 2001;Lindert et al, 2009), is even more similar to the corresponding reptilian sequence of Anguis (and Anolis) than to the one of the house mouse (Figure 1). This region is particularly important for metal inducibility (Lindert et al, 2009).…”

“…Of note, the human nuclear export signal (NES), which is embedded in the acidic activation domain (Saydam et al, 2001;Lindert et al, 2009), is even more similar to the corresponding reptilian sequence of Anguis (and Anolis) than to the one of the house mouse (Figure 1). This region is particularly important for metal inducibility (Lindert et al, 2009). Moreover, the overall length of slow worm MTF-1 (738 aa) is similar to Anolis (754 aa) and human (753 aa) and thus clearly different from MTF-1 in the mouse (675 aa) and another rodent, the South American capybara Hydrochoerus hydrochaeris (638 aa) (Lindert et al, 2008).…”