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

Activation of genes by heavy metals, notably zinc, cadmium and copper, depe nds on MTF-1, a unique zinc finger transcription factor conserved from inse cts 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 promoter s. Some new candidate target genes emerged, including those encoding alpha -fetoprotein, the liver-enriched transcription factor C/EBP alpha and tear lipocalin/von Ebner's gland protein, all of which have a role in toxicity/t he cell stress response. In contrast, expression of other cell stress-assoc iated genes, such as those for superoxide dismutases, thioredoxin and heat shock proteins, do not appear to be affected by loss of MTF-1, Our experime nts have also exposed some problems with target gene searches. First, findi ng the optimal time window for detecting MTF-1 target genes in a lethal phe notype of rapid liver decay proved problematical: 12.5-day-old mouse embryo s (stage E12.5) yielded hardly any differentially expressed genes, whereas at stage 13.0 reduced expression of secretory liver proteins probably refle cted the onset of liver decay, i.e. a secondary effect. Likewise, up-regula tion 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 syn thesis of the antioxidant glutathione and which was previously suggested to be a target gene contributing to the lethal phenotype in MTF-1 knockout mi ce. gamma -GCS(hc) mRNA is reduced at the onset of liver decay but MTF-1 nu ll mutant embryos manage to maintain a very high glutathione level until sh ortly before that stage, perhaps in an attempt to compensate for low expres sion of metallothioneins, which also have a role as antioxidants.