Defining the DNA Binding Site Recognized by the Fission Yeast Zn₂Cys₆ Transcription Factor Pho7 and Its Role in Phosphate Homeostasis

Abstract

ABSTRACT Fission yeast phosphate homeostasis entails transcriptional induction of genes encoding phosphate-mobilizing proteins under conditions of phosphate starvation. Transcription factor Pho7, a member of the Zn2Cys6 family of fungal transcription regulators, is the central player in the starvation response. The DNA binding sites in the promoters of phosphate-responsive genes have not been defined, nor have any structure-function relationships been established for the Pho7 protein. Here we narrow this knowledge gap by (i) delineating an autonomous DNA-binding domain (DBD) within Pho7 that includes the Zn2Cys6 module, (ii) deploying recombinant Pho7 DBD in DNase I footprinting and electrophoretic mobility shift assays (EMSAs) to map the Pho7 recognition sites in the promoters of the phosphate-regulated pho1 and tgp1 genes to a 12-nucleotide sequence motif [5′-TCG(G/C)(A/T)xxTTxAA], (iii) independently identifying the same motif as a Pho7 recognition element via in silico analysis of available genome-wide ChIP-seq data, (iv) affirming that mutations in the two Pho7 recognition sites in the pho1 promoter efface pho1 expression in vivo, and (v) establishing that the zinc-binding cysteines and a pair of conserved arginines in the DBD are essential for Pho7 activity in vivo. IMPORTANCE Fungi respond to phosphate starvation by inducing the transcription of a set of phosphate acquisition genes that comprise a phosphate regulon. Pho7, a member of the Zn2Cys6 family of fungal transcription regulators, is the central player in the phosphate starvation response in fission yeast. The present study identifies a 12-nucleotide Pho7 DNA binding motif [5′-TCG(G/C)(A/T)xxTTxAA] in the promoters of phosphate-regulated genes, pinpoints DNA and protein features important for Pho7 binding to DNA, and correlates them with Pho7-dependent gene expression in vivo. The results highlight distinctive properties of Pho7 vis-a-vis other fungal zinc binuclear cluster transcription factors as well as the divergent cast of transcription factors deployed for phosphate homeostasis in fission yeast versus budding yeast.

Keywords

• DNA binding
• fission yeast
• phosphate homeostasis
• transcriptional regulation