Iron activates in vivo DNA binding of Schizosaccharomyces pombe transcription factor Fep1 through its amino-terminal region.
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 fep1Delta php4Delta 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 Cys(2)/Cys(2)-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.
Jbel
Mehdi
M
Département de Biochimie, Faculté de médecine, Université de Sherbrooke, Sherbrooke, Quebec, Canada.
Mercier
Alexandre
A
Pelletier
Benoit
B
Beaudoin
Jude
J
Labbé
Simon
S
D020816:Amino Acid Motifs; D023081:CCAAT-Binding Factor / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050980:GATA Transcription Factors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D007501:Iron / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D021381:Protein Transport; D012568:Schizosaccharomyces / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation
D023081:CCAAT-Binding Factor; C459859:Fep1 protein, S pombe; D050980:GATA Transcription Factors; C519990:Php4 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; D007501:Iron