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J. Biol. Chem., Vol. 280, Issue 26, 25146-25161, July 1, 2005

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

Benoit Pelletier, Amy Trott¶, Kevin A. Morano¶, and Simon Labbé||

From the Département de Biochimie, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada and the ^¶Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, Texas 77030-1501

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.

Received for publication, March 17, 2005 , and in revised form, May 2, 2005.

^* This work was supported in part by the Natural Sciences and Engineering Research Council of Canada Grant 238238-01 (to S. L.) and American Cancer Society Grant MBC-103134 (to K. A. M.). Infrastructure equipment essential for carrying out this investigation was obtained through Canada Foundation for Innovation Grant NOF-3754 (to S. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a studentship from the NSERC.

^|| New Investigator Scholar from the Canadian Institutes of Health Research. To whom correspondence should be addressed: Dépt. de Biochimie, Faculté de médecine, Université de Sherbrooke, 3001 12e Ave Nord, Sherbrooke (Québec) J1H 5N4, Canada. Tel.: 819-820-6868 (ext. 15460); Fax: 819-564-5340; E-mail: Simon.Labbe{at}USherbrooke.ca.

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