Molecular mechanisms of heavy metal homeostasis in flies and humans

Metal-responsive transcription factor-1 (MTF-1) is an evolutionarily conserved zinc finger protein that activates transcription in response to heavy metals such as Zn(II), Cd(II) and Cu(I). The DNA-binding domain of MTF-1 recognizes a specific DNA sequence termed the metal response element (MRE), located in the promoter/enhancer region of its target genes. Here we show that human MTF-1 forms homodimers in vivo via a conserved cysteine-rich cluster “CQCQCAC” located near the C-terminus that was previously shown by cell transfection experiments to be important for basal and especially metal-induced transcriptional activity. Using a mammalian two-hybrid system we find that dimerization is not further enhanced by zinc treatment, implying that dimerization via the cysteine cluster is required for metal-induced transcriptional activity but actual zinc sensing is mediated by another domain. By contrast copper, which on its own activates hMTF-1 only weakly, stabilizes the dimer by inducing intermolecular disulfide bond formation via the cysteines and can synergize with zinc to boost hMTF-1 dependent transcription. A mutant protein in which these cysteines are replaced by alanines is still able to translocate to the nucleus in response to zinc treatment and bind to DNA in a cell free system. Therefore we propose that homodimerization is important to form an interaction platform for transcriptional