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

Abstract

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 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.

Cells Lacking Pfh1, a Fission Yeast Homolog of Mammalian Frataxin Protein, Display Constitutive Activation of the Iron Starvation Response

“…Downstream of Grx4, the inactivation of Fep1 and activation of Php4 transcriptional repressors mediate the cellular response to iron deficiency (44). Briefly, when iron is experimentally depleted by the use of chelators, Fep1 is released from promoters of genes involved in iron uptake (45), whereas Php4 accumulates at the nucleus and represses transcription of genes coding for iron storage or iron-consuming proteins (46). Importantly enough, Grx4 is the real sensor of iron deprivation, probably through its ISC.…”

“…Thus, upon low iron conditions Fep1 is released from promoters of genes involved in iron uptake, such as fio1 or str3 (Fig. 4, A and B) (45). As shown in Fig.…”

Both Php4 Function and Subcellular Localization Are Regulated by Iron via a Multistep Mechanism Involving the Glutaredoxin Grx4 and the Exportin Crm1

“…Cell lysates were quantitated using the Bradford assay (47), and equal amounts of each sample were subjected to electrophoresis on 9% SDS-polyacrylamide gels. After electrophoresis, protein samples were electroblotted as described previously (9). The following primary antisera were used for immunodetection: monoclonal anti-GFP antibody (B-2; Santa Cruz Biotechnology) and monoclonal anti-PCNA antibody (PC10; Sigma).…”

“…fip1 ϩ , fio1 ϩ , frp1 ϩ , and str1 ϩ /2 ϩ /3 ϩ ) and shuts down their expression to avoid deleterious consequences of iron overload (6,7). When iron levels are low, Fep1 is unable to bind DNA, resulting in the transcriptional induction of genes involved in iron acquisition (6,8,9). Analogous to S. pombe, other fungi use Fep1 orthologs to repress transcription of target genes in response to high iron.…”

Simon Labbé’s work on iron and copper homeostasis

“…Labbé and his team took advantage of the Schiz. pombe model to identify the GATA-type transcription factor Fep1 [1][2][3] . The identification of Fep1 was significant because it revealed that iron-regulated gene expression in fungi other than S. cerevisiae is mediated by a group of non-canonical GATA-type transcription factors.…”