Functional Characterization of the Iron-regulatory Transcription Factor Fep1 from Schizosaccharomyces pombe

In response to excess iron, Schizosaccharomyces pombe cells repress transcription of genes encoding components involved in iron uptake through the Fep1 transcription factor. Fep1 mediates this control by interacting with the consensus sequence 5′-(A/T)GATAA-3′, found in iron-dependent promoters. In this report, we show that Fep1 localizes to the nucleus under both iron-replete and iron-starved conditions. The Fep1 DNA binding domain (amino acids 1-241) contains two GATA-type zinc finger motifs. Although we determine that the Fep1 C-terminal zinc finger (ZF2) is essential for DNA binding, we show that the N-terminal zinc finger (ZF1) enhances DNA binding affinity ∼5-fold. Between the two zinc finger motifs of Fep1 resides an invariant amino acid sequence, denoted the Cys-rich region (amino acids 68-94), in which four highly conserved Cys residues are found. Cells harboring mutant alleles in which two or more of the conserved Cys residues were substituted by alanine exhibited elevated fio1+ mRNA levels. We determine that the dissociation constant for the resulting complex between each of the Cys mutants and the sequence 5′-(A/T)GATAA-3′ reflects a much lower affinity that correlates with failure to repress fio1+ gene expression. Deletion analysis identified two heptad repeats (amino acids 522-536) within the C-terminal region of Fep1 that are necessary and sufficient to mediate Fep1 dimerization. Moreover, mutations that impair dimerization also negatively affect transcriptional repression. Together these findings reveal several novel features of Fep1, a non-canonical GATA factor required for iron homeostasis. In response to excess iron, Schizosaccharomyces pombe cells repress transcription of genes encoding components involved in iron uptake through the Fep1 transcription factor. Fep1 mediates this control by interacting with the consensus sequence 5′-(A/T)GATAA-3′, found in iron-dependent promoters. In this report, we show that Fep1 localizes to the nucleus under both iron-replete and iron-starved conditions. The Fep1 DNA binding domain (amino acids 1-241) contains two GATA-type zinc finger motifs. Although we determine that the Fep1 C-terminal zinc finger (ZF2) is essential for DNA binding, we show that the N-terminal zinc finger (ZF1) enhances DNA binding affinity ∼5-fold. Between the two zinc finger motifs of Fep1 resides an invariant amino acid sequence, denoted the Cys-rich region (amino acids 68-94), in which four highly conserved Cys residues are found. Cells harboring mutant alleles in which two or more of the conserved Cys residues were substituted by alanine exhibited elevated fio1+ mRNA levels. We determine that the dissociation constant for the resulting complex between each of the Cys mutants and the sequence 5′-(A/T)GATAA-3′ reflects a much lower affinity that correlates with failure to repress fio1+ gene expression. Deletion analysis identified two heptad repeats (amino acids 522-536) within the C-terminal region of Fep1 that are necessary and sufficient to mediate Fep1 dimerization. Moreover, mutations that impair dimerization also negatively affect transcriptional repression. Together these findings reveal several novel features of Fep1, a non-canonical GATA factor required for iron homeostasis. The transition metal iron is both essential and, at high levels, toxic to cells (1Beutler E. Science. 2004; 306: 2051-2053Crossref PubMed Scopus (46) Google Scholar, 2Hentze M.W. Muckenthaler M.U. Andrews N.C. Cell. 2004; 117: 285-297Abstract Full Text Full Text PDF PubMed Scopus (1417) Google Scholar, 3Van Ho A. Ward D.M. Kaplan J. Annu. Rev. Microbiol. 2002; 56: 237-261Crossref PubMed Scopus (181) Google Scholar, 4Wessling-Resnick M. Crit. Rev. Biochem. Mol. PubMed Scopus Google The of iron is to between two and of iron a for several that DNA the acid and N.C. PubMed Scopus Google Scholar, E. PubMed Scopus Google In iron by with or to a that is for of and of DNA or Biochem. J. PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Cells to that sufficient and of iron are in the at M.W. Muckenthaler M.U. Andrews N.C. Cell. 2004; 117: 285-297Abstract Full Text Full Text PDF PubMed Scopus (1417) Google Scholar, E. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, E. A. 2002; PubMed Scopus Google Scholar, J. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Biochem. 2002; PubMed Scopus Google Scholar, Kaplan J. J. Full Text Full Text PDF PubMed Scopus Google Scholar, Annu. Rev. PubMed Scopus Google In the Schizosaccharomyces two identified for iron M. 2004; PubMed Scopus Google Scholar, Kaplan J. J. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Mol. Cell. PubMed Scopus Google The of the and of a M. 2004; PubMed Scopus Google this and the Although the the pombe also of the by J. PubMed Scopus Google and genes identified are involved in uptake of iron J. PubMed Scopus Google these for is for and to a J. PubMed Scopus Google Although in the of iron to In a for iron uptake of iron to by the A. Mol. Cell. PubMed Scopus Google to iron is the by a complex of the and the Kaplan J. J. Full Text Full Text PDF PubMed Scopus Google this iron to which is the by the high affinity of iron and mRNA are under iron-replete of these genes is Kaplan J. J. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Mol. Cell. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Full Text Full Text PDF PubMed Scopus Google The necessary for of the and genes to a GATA-type sequence 5′-(A/T)GATAA-3′ J. PubMed Scopus Google Scholar, J. J. 2002; Full Text Full Text PDF PubMed Scopus Google the for of these genes in the of iron to Fep1, a of the GATA factor J. PubMed Scopus Google Scholar, J. J. 2002; Full Text Full Text PDF PubMed Scopus Google pombe cells in which elevated of of the fio1+ gene J. J. 2002; Full Text Full Text PDF PubMed Scopus Google mutant cells of the J. J. 2002; Full Text Full Text PDF PubMed Scopus Google Moreover, the is to an that acids in the of excess iron J. J. 2002; Full Text Full Text PDF PubMed Scopus Google We identified two and which for transcriptional J. 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Science. 2004; 306: 2051-2053Crossref PubMed Scopus (46) Google Scholar, 2Hentze M.W. Muckenthaler M.U. Andrews N.C. Cell. 2004; 117: 285-297Abstract Full Text Full Text PDF PubMed Scopus (1417) Google In several genes encoding components of the and iron are by the iron-dependent Fep1 transcription factor in response to high iron J. PubMed Scopus Google Scholar, J. J. 2002; Full Text Full Text PDF PubMed Scopus Google Fep1 is a of the GATA factor that to DNA 5′-(A/T)GATAA-3′ J. PubMed Scopus Google Scholar, J. J. 2002; Full Text Full Text PDF PubMed Scopus Google with and to Fep1 found in A. and in Microbiol. PubMed Scopus Google these are GATA transcription by the of two consensus zinc and a highly conserved amino acid sequence with four Cys between the two zinc Microbiol. PubMed Scopus Google Scholar, Microbiol. PubMed Scopus Google the sequence in these also C-terminal region in which a domain is that dimerization 2002; PubMed Scopus Google Scholar, J. 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PubMed Scopus Google We the of these Cys residues by the two and the two and or Cells these mutants exhibited of the fio1+ in the of iron and a of the and the the Cys mutants the to fio1+ we for each of the Cys mutants the dissociation constant of the complex in an analysis We found that Fep1 Fep1 and Fep1 much lower binding for GATA in with a of and an of and in binding affinity with the for the the of the Cys mutant reflects a much lower affinity that correlates with failure to repress fio1+ gene expression. to that of conserved Cys residues between the two zinc of to gene 2002; PubMed Scopus Google We the In by of the and iron of gene that involved in of iron PubMed Scopus Google Scholar, Microbiol. 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