Target gene search for the metal-responsive transcription factor MTF-1

Abstract

Activation of genes by heavy metals, notably zinc, cadmium and copper, depends on MTF-1, a unique zinc finger transcription factor conserved from insects to human. Knockout of MTF-1 in the mouse results in embryonic lethality due to liver decay, while knockout of its best characterized target genes, the stress-inducible metallothionein genes I and II, is viable, suggesting additional target genes of MTF-1. Here we report on a multi-pronged search for potential target genes of MTF-1, including microarray screening, SABRE selective amplification, a computer search for MREs (DNA-binding sites of MTF-1) and transfection of reporter genes driven by candidate gene promoters. Some new candidate target genes emerged, including those encoding alpha-fetoprotein, the liver-enriched transcription factor C/EBPalpha and tear lipocalin/von Ebner's gland protein, all of which have a role in toxicity/the cell stress response. In contrast, expression of other cell stress-associated genes, such as those for superoxide dismutases, thioredoxin and heat shock proteins, do not appear to be affected by loss of MTF-1. Our experiments have also exposed some problems with target gene searches. First, finding the optimal time window for detecting MTF-1 target genes in a lethal phenotype of rapid liver decay proved problematical: 12.5-day-old mouse embryos (stage E12.5) yielded hardly any differentially expressed genes, whereas at stage 13.0 reduced expression of secretory liver proteins probably reflected the onset of liver decay, i.e. a secondary effect. Likewise, up-regulation of some proliferation-associated genes may also just reflect responses to the concomitant loss of hepatocytes. Another sobering finding concerns gamma-glutamylcysteine synthetase(hc) (gamma-GCS(hc)), which controls synthesis of the antioxidant glutathione and which was previously suggested to be a target gene contributing to the lethal phenotype in MTF-1 knockout mice. gamma-GCS(hc) mRNA is reduced at the onset of liver decay but MTF-1 null mutant embryos manage to maintain a very high glutathione level until shortly before that stage, perhaps in an attempt to compensate for low expression of metallothioneins, which also have a role as antioxidants.

Effects of non-toxic zinc exposure on human epidermal keratinocytes

“…Although the effects of zinc on intracellular signalling events are not completely known, a positive impact of Zn (II) on cell proliferation might be attributed, at least in part, to the inhibition of phosphatases, leading to augmented protein tyrosine phosphorylation 30 . The proliferation advantage by Zn (II) treatment can also be a consequence of increased calcium uptake 31 or up-regulated gene expression (e.g., NOTCH1, see Table 1) 32,33 . Interestingly, in aged skin, which is characterised by decreased epidermal renewal, the expression of MT is lower 34 .…”

A global view of the selectivity of zinc deprivation and excess on genes expressed in human THP-1 mononuclear cells

“…Nevertheless, others must exist because, whereas MTF-1 null mice display embryonic lethality (11), MT null mice are viable (33). The exact gene responsible for lethality has not been identified, but a number of candidates have been considered as database searches reveal an ever expanding list of genes with MRE sequences (10). The 104 genes in these microarray studies found responsive to zinc in a mode consistent with MTF-1-regulated genes substantially increase the number of MREcontaining candidate genes.…”

“…In contrast, the regulatory role of zinc may provide a responsive indicator. Through an interaction with a metal-responsive transcription factor, (MTF-1), zinc has been shown to regulate a number of genes (10,11). MTF-1-regulated genes include those that influence zinc trafficking, such as metallothionein (MT) and zinc transporter-1 (ZnT-1), and those associated with a variety of genes not related to zinc in an obvious way (10).…”

“…Through an interaction with a metal-responsive transcription factor, (MTF-1), zinc has been shown to regulate a number of genes (10,11). MTF-1-regulated genes include those that influence zinc trafficking, such as metallothionein (MT) and zinc transporter-1 (ZnT-1), and those associated with a variety of genes not related to zinc in an obvious way (10). The embryonic lethality of MTF-1 null mice points to the critical nature of this mode of gene regulation (11)(12).…”

Characteristics, functions, and applications of metallothionein in aquatic vertebrates

“…MTF-1 is important in the regulation of a group of genes that play a key role in cellular response to various stressors (Lichtlen et al, 2001). MREs are known in multiple copies in the metallothionein promoter region, and they appear to be variable in their response to metal-induced transcription.…”