Characterization of the Transcription Factor MTF-1 from the Japanese Pufferfish (<I>Fugu rubripes</I>) Reveals Evolutionary Conservation of Heavy Metal Stress Response

Abstract

The pufferfish Fugu rubripes was recently introduced as a new model organism for genomic studies, since it contains a full set of vertebrate genes but only 13% as much DNA as a mammal. Fugu genes tend to be smaller and densely spaced due to shortening of introns and intergenic spacers. We isolated the Fugu gene for the metal-responsive transcription factor MTF-1 (MTF1), a mediator of heavy metal regulation and oxidative stress response previously characterized in mammals. In addition, most of the cDNA sequence was also determined. The 780 amino acid MTF-1 protein of Fugu is very similar to that of mouse and human, with 90% amino acid identity in the DNA binding zinc finger domain and 57% overall identity. Expression of the pufferfish cDNA in mammalian cells shows that Fugu MTF-1 has the same DNA binding specificity as its mammalian counterpart and also induces transcription in response to zinc and cadmium. The proteincoding part of the Fugu MTF-1 gene spans 6.4 kb and consists of 11 exons. Upstream region and first exon constitute a CpG island. The distance between stop codon and polyadenylation motifs is Ͼ 2 kb, suggesting a very long 3 untranslated mRNA region, followed by another CpG island which may represent the promoter of the next gene downstream. Part of the MTF-1 genomic structure was also determined in the mouse, and some striking similarities were found: for example, the upstream adjacent gene in both species is INPP5P, encoding a phosphatase. The mouse MTF-1 promoter is also embedded in a CpG island, which however shares no sequence similarity to the one of Fugu. The Fugu CpG island is shorter than the one of the mouse and has no elevated [G+C] content; these and other data indicate that CpG islands of fish may represent a primordial stage of CpG island evolution.

Short-lived mammals (shrew, mouse) have a less robust metal-responsive transcription factor than humans and bats

“…Note that in the NES region, typical mammals are more closely related to birds and reptiles than to rodents (see also (Georgiev et al 2014 )). The MTF-1s of the animals have not been functionally tested, except those of slow worm (Georgiev et al 2014 ), and fugu (Auf der Maur et al 1999 ), both of which functioned very well in our standard reporter assay (for zebrafish, see also Cheuk ( 2008 )). The extensive alterations in the rabbit (Oryctolagus) and the two lysines replacing leucine and threonine in opossum (Monodelphis) might be worth a further investigation; both of them are relatively short-lived, rapidly multiplying species.…”

Inhibition of endogenous MTF-1 signaling in zebrafish embryos identifies novel roles for MTF-1 in development

“…Previous studies have described complete MTF-1 transcripts in two other fish species, the pufferfish ( Takifugu rubripes ) and tilapia ( Tilapia aurea×Tilapia nilotica ) [4, 31], which encode for 780 and 811 amino acid proteins, respectively. Using the Takifugu MTF-1 sequence as a guide, we were able to identify a missing exon and amplify a full zebrafish MTF-1 transcript containing an open reading frame that encodes for a 722 amino acid protein (Figure 1C).…”

“…The application of a transgenic zebrafish expressing a dominant-negative bone morphogenetic protein (Bmp) under control of a heat shock-inducible promoter [29, 30] is validation of such an approach. Previous research has identified a zebrafish MTF-1 homologue that is significantly shorter than the typical vertebrate MTF-1 and missing the cysteine-rich motif [5]; although complete MTF-1 transcripts have been described other fish species [4, 31]. Therefore in our initial effort we sought to investigate the functional diversity of MTF-1 transcripts in zebrafish, followed by an investigation of the in vivo efficacy of the dominant-negative MTF-1 by microinjection of in vitro transcribed mRNA in zebrafish embryos as a precursor to the development of a transgenic zebrafish.…”

Copper sensing function of Drosophila metal-responsive transcription factor-1 is mediated by a tetranuclear Cu(I) cluster

“…MTF-1 has been identified and at least partially characterized from human, mouse (14), pufferfish Fugu rubripes (15), zebrafish Danio rerio (16,17) and Drosophila (18). Human and mouse MTF-1 as well as the Drosophila homolog, termed dMTF-1, contain multiple functional domains, including a highly conserved zinc-finger domain that recognizes the cognate DNA sequence termed metal response element (MRE) (8).…”