Structural Analysis of the CDK-Cyclin Complex of Pho85-Pho80 and Genome-Wide Characterization of the Phosphate Starvation Response in Schizosaccharomyces pombe

Abstract

Inorganic phosphate is an essential nutrient required by all organisms for optimal growth. During phosphate starvation, Saccharomyces cerevisiae induces a set of genes responsible for the regulation of inorganic phosphate acquisition. The phosphate-responsive signaling (PHO) pathway controls this response, with the CDK-cyclin complex Pho85-Pho80 playing a prominent role. Here we report the X-ray structure of the Pho85-Pho80 complex, identifying the unique structural features that distinguish it from other cell cycle associated CDK-cyclin complexes. The structure reveals a specific salt bridge between a Pho85 arginine and a Pho80 aspartate that maintains a Pho80 loop confirmation important for substrate recognition and makes phosphorylation of the Pho85 activation loop dispensible. We show that a cluster of residues distal to the kinase active site are involved in a high affinity interaction between the Pho80 cyclin and the transcription factor substrate (Pho4). The structure also reveals a separate high affinity binding site for the CDK inhibitor (Pho81). The fission yeast, Schizosaccharomyces pombe, regulates expression of the secreted acid phosphatase \((pho1^+)\) via a non-orthologous PHO pathway. The genes induced by phosphate limitation and the molecular mechanism by which the genetically identified positive \((pho7^+)\) and negative \((csk1^+)\) regulators function are not known. Here we use a combination of molecular biology, expression microarrays, chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq), and global transcriptome sequencing (RNA-Seq) to characterize the role of \(pho7^+\) and \(csk1^+\) in the PHO response. We show that there is a fast and slow response to phosphate starvation, each with defined regulatory roles. We use ChIP-Seq to identify members of the Pho7 regulon and characterize Pho7 binding dynamics in response to phosphate-limitation and Csk1 activity. We identify a conserved PHO response for the PHO5 \((pho1^+)\), PHO84 \((spbc8e4.01c^+)\), and GIT1 \((spbc1271.09^+)\) orthologs. We show that activation of \(pho1^+\) requires Pho7 binding to a UAS in the \(pho1^+\) promoter and that a URS is necessary for Csk1 repression. We find that Pho7-dependent activation is not limited to phosphate-starvation, as additional environmental stress response pathways require \(pho7^+\) for maximal induction. Using RNA-Seq we show that Pho7 is also involved in regulating non-coding transcription and is a bi-functional transcription factor.