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

We have identified genes encoding candidate proteins involved in iron storage (pcl(1+)), the tricarboxylic acid cycle (sdh4(+)), and iron-sulfur cluster assembly (isa(1+)) 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.