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Eukaryotic Cell, April 2009, p. 649-664, Vol. 8, No. 4
1535-9778/09/$08.00+0     doi:10.1128/EC.00001-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Iron Activates In Vivo DNA Binding of Schizosaccharomyces pombe Transcription Factor Fep1 through Its Amino-Terminal Region ^,

Mehdi Jbel, Alexandre Mercier, Benoit Pelletier, Jude Beaudoin, and Simon Labbé^*

Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada

Received 1 January 2009/ Accepted 9 February 2009

In Schizosaccharomyces pombe, the iron sensor Fep1 mediates the transcriptional repression of iron transport genes in response to high concentrations of iron. On the other hand, fep1⁺ expression is downregulated under conditions of iron starvation by the CCAAT-binding factor Php4. In this study, we created a fep1 php4 double mutant strain where expression of fep1⁺ was disengaged from its iron limitation-dependent repression by Php4 to examine the effects of iron on constitutively expressed functional fep1⁺-GFP and TAP-fep1⁺ alleles and their gene products. In these cells, Fep1-green fluorescent protein was invariably localized in the nucleus under both iron-limiting and iron-replete conditions. Using chromatin immunoprecipitation assays, we found that Fep1 is associated with iron-responsive promoters in vivo. Chromatin binding was iron dependent, with a loss of binding observed in the presence of low iron. Functional dissection of the protein revealed that the N-terminal 241-residue segment that includes two consensus Cys₂/Cys₂-type zinc finger motifs and a Cys-rich region is required for optimal promoter occupancy by Fep1. Within this segment, a minimal module encompassing amino acids 60 to 241 is sufficient for iron-dependent chromatin binding. Using yeast one-hybrid analysis, we showed that the replacement of the repression domain of Fep1 by fusing the activation domain of VP16 to the chromatin-binding fragment of amino acids 1 to 241 of Fep1 converts the protein from an iron-dependent repressor into an iron-dependent transcriptional activator. Thus, the repression function of Fep1 can be replaced with that of a transcriptional activation function without the loss of its iron-dependent DNA-binding activity.

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* Corresponding author. Mailing address: Département de Biochimie, Faculté de médecine, Université de Sherbrooke, 3001, 12e Ave. Nord, Sherbrooke, Quebec J1H 5N4, Canada. Phone: (819) 820-6868, ext. 15460. Fax: (819) 564-5340. E-mail: Simon.Labbe{at}USherbrooke.ca

Published ahead of print on 27 February 2009.

Supplemental material for this article may be found at http://ec.asm.org/.

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Eukaryotic Cell, April 2009, p. 649-664, Vol. 8, No. 4
1535-9778/09/$08.00+0     doi:10.1128/EC.00001-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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• Mercier, A., Labbe, S. (2009). Both Php4 Function and Subcellular Localization Are Regulated by Iron via a Multistep Mechanism Involving the Glutaredoxin Grx4 and the Exportin Crm1. J. Biol. Chem. 284: 20249-20262 [Abstract] [Full Text]