A Transcription Factor Cascade Involving Fep1 and the CCAAT-Binding Factor Php4 Regulates Gene Expression in Response to Iron Deficiency in the Fission Yeast Schizosaccharomyces pombe

Abstract

We have identified genes encoding candidate proteins involved in iron storage (pcl1 ؉ ), the tricarboxylic acid cycle (sdh4 ؉ ), and iron-sulfur cluster assembly (isa1 ؉ ) that are negatively regulated in response to iron deprivation. Promoter deletion and site-directed mutagenesis permitted identification of a new cis-regulatory element in the promoter region of the pcl1 ؉ gene. This cis-acting regulatory sequence containing the pentanucleotide sequence CCAAT is responsible for transcriptional repression of pcl1 ؉ under low iron supply conditions. In Schizosaccharomyces pombe, the CCAAT-binding factor is a heteromeric DNA-binding complex that contains three subunits, designated Php2, Php3, and Php5. Inactivation of the php2 ؉ locus negatively affects the transcriptional competency of pcl1 ؉ . A fourth subunit, designated Php4, is not essential for the transcriptional activation of target genes under basal and iron-replete conditions. We demonstrate that, in response to iron-limiting conditions, Php4 is required for down-regulation of pcl1 ؉ , sdh4 ؉ , and isa1 ؉ mRNA levels. In vivo RNase protection studies reveal that the expression of php4 ؉ is negatively regulated by iron and that this regulated expression requires a functional fep1 ؉ gene. The results of these studies reveal that Fep1 represses php4؉ expression in response to iron. In contrast, when iron is scarce, Fep1 becomes inactive and php4؉ is expressed to act as a regulatory subunit of the CCAAT-binding factor that is required to block pcl1 ؉ , sdh4 ؉ , and isa1 ؉ gene transcription.

Evaluating the Control of mRNA Decay in Fission Yeast

“…This type of physiological connection is nicely illustrated in the case of CTH2 from S. cerevisiae, which is an iron-regulated protein that in turn appears to regulate the stability of a group of transcripts encoding proteins involved in various aspects of iron metabolism (Puig et al, 2005). Consistent with previous observations (Mercier et al, 2006), zfs1 deficiency does not appear to affect iron metabolism (Cuthbertson et al, 2007). Our hope is that not only will the zfs1 protein be found to coordinate the stability of a similar collection of transcripts involved in an important physiological process in S. pombe, but that we can use this system to gain insights into the biochemical function of the whole class of CCCH tandem zinc finger proteins.…”

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

“…As part of the coordinated response to iron deprivation, S. pombe expresses the Php4 subunit of the heteromeric CCAATbinding complex (18). Php4 associates with the Php2/Php3/ Php5 heterotrimer to reprogram the cell for iron use.…”

“…Protein Extraction and Immunoblotting-Total cell lysates were prepared as described previously (18). Cell lysates were quantitated using the Bradford assay (47), and equal amounts of each sample were subjected to electrophoresis on 9% SDS-polyacrylamide gels.…”

“…we have previously shown (18,19) inactivation of Php4 in response to iron is regulated at two distinct levels. First, in the presence of iron, Fep1 associates with GATA elements in the php4 ϩ promoter to repress transcription, ensuring the extinction of most php4 ϩ transcripts.…”

“…php4 ϩ encodes a subunit of the CCAAT-binding protein complex, which includes three other subunits, denoted Php2, Php3, and Php5 (17,18). Genes encoding Php2, Php3, and Php5 are constitutively expressed, whereas transcript levels of php4 ϩ are induced under conditions of iron starvation and repressed under iron-replete conditions (18). Php4 is responsible for the transcriptional repression capability of the Php complex and is not required for the DNA binding activity of the complex.…”