Defining the DNA Binding Site Recognized by the Fission Yeast Zn<sub>2</sub>Cys<sub>6</sub> Transcription Factor Pho7 and Its Role in Phosphate Homeostasis

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 starvati...

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Autores principales:	Beate Schwer, Ana M. Sanchez, Angad Garg, Debashree Chatterjee, Stewart Shuman
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Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	DNA binding fission yeast phosphate homeostasis transcriptional regulation Microbiology QR1-502
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spelling	oai:doaj.org-article:2dddd7a272da41ddb0b48a57202e0fcb2021-11-15T15:51:43ZDefining the DNA Binding Site Recognized by the Fission Yeast Zn<sub>2</sub>Cys<sub>6</sub> Transcription Factor Pho7 and Its Role in Phosphate Homeostasis10.1128/mBio.01218-172150-7511https://doaj.org/article/2dddd7a272da41ddb0b48a57202e0fcb2017-09-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01218-17https://doaj.org/toc/2150-7511ABSTRACT 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.Beate SchwerAna M. SanchezAngad GargDebashree ChatterjeeStewart ShumanAmerican Society for MicrobiologyarticleDNA bindingfission yeastphosphate homeostasistranscriptional regulationMicrobiologyQR1-502ENmBio, Vol 8, Iss 4 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	DNA binding fission yeast phosphate homeostasis transcriptional regulation Microbiology QR1-502
spellingShingle	DNA binding fission yeast phosphate homeostasis transcriptional regulation Microbiology QR1-502 Beate Schwer Ana M. Sanchez Angad Garg Debashree Chatterjee Stewart Shuman Defining the DNA Binding Site Recognized by the Fission Yeast Zn<sub>2</sub>Cys<sub>6</sub> Transcription Factor Pho7 and Its Role in Phosphate Homeostasis
description	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.
format	article
author	Beate Schwer Ana M. Sanchez Angad Garg Debashree Chatterjee Stewart Shuman
author_facet	Beate Schwer Ana M. Sanchez Angad Garg Debashree Chatterjee Stewart Shuman
author_sort	Beate Schwer
title	Defining the DNA Binding Site Recognized by the Fission Yeast Zn<sub>2</sub>Cys<sub>6</sub> Transcription Factor Pho7 and Its Role in Phosphate Homeostasis
title_short	Defining the DNA Binding Site Recognized by the Fission Yeast Zn<sub>2</sub>Cys<sub>6</sub> Transcription Factor Pho7 and Its Role in Phosphate Homeostasis
title_full	Defining the DNA Binding Site Recognized by the Fission Yeast Zn<sub>2</sub>Cys<sub>6</sub> Transcription Factor Pho7 and Its Role in Phosphate Homeostasis
title_fullStr	Defining the DNA Binding Site Recognized by the Fission Yeast Zn<sub>2</sub>Cys<sub>6</sub> Transcription Factor Pho7 and Its Role in Phosphate Homeostasis
title_full_unstemmed	Defining the DNA Binding Site Recognized by the Fission Yeast Zn<sub>2</sub>Cys<sub>6</sub> Transcription Factor Pho7 and Its Role in Phosphate Homeostasis
title_sort	defining the dna binding site recognized by the fission yeast zn<sub>2</sub>cys<sub>6</sub> transcription factor pho7 and its role in phosphate homeostasis
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/2dddd7a272da41ddb0b48a57202e0fcb
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Defining the DNA Binding Site Recognized by the Fission Yeast Zn<sub>2</sub>Cys<sub>6</sub> Transcription Factor Pho7 and Its Role in Phosphate Homeostasis

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