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Genome-Wide Characterization of the Phosphate Starvation Response in Schizosaccharomyces pombe

Ian Carter-O’Connell12, Michael T Peel4, Dennis D Wykoff4* and Erin K O’Shea123*

Author Affiliations

1 Howard Hughes Medical Institute, Faculty of Arts and Sciences, Center for Systems Biology, Northwest Labs, Harvard University, 52 Oxford Street, Cambridge, MA, 02138, USA

2 Department of Molecular and Cellular Biology, Harvard University, Faculty of Arts and Sciences, Center for Systems Biology, Northwest Labs, 52 Oxford Street, Cambridge, MA, 02138, USA

3 Department of Chemistry and Chemical Biology, Harvard University, Faculty of Arts and Sciences, Center for Systems Biology, Northwest Labs, 52 Oxford Street, Cambridge, MA, 02138, USA

4 Department of Biology, Villanova University, 800 Lancaster Ave, Villanova, PA, 19085, USA

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BMC Genomics 2012, 13:697  doi:10.1186/1471-2164-13-697

Published: 12 December 2012

Additional files

Additional file 1:

Figure S1. Temporal Dynamics of the Phosphate Starvation Response in S. pombe. (A) Line plot depicting the induction profile for genes displaying a fast (red) or slow (blue) response to phosphate starvation as measured by microarray analysis. Thresholds used to delineate the response time are described in the text. Induction was normalized to the initial sample pre-starvation (t=0). (B) Average expression values for genes in the fast (■) and slow (O) response are shown ± SD.

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Additional file 2:

Table S1. Microarray Results for Genes Identified in the Pi Starvation Time Course. Genes induced following two hours (fast response, bold) or four hours (slow response) of Pi depletion were tabulated and their normalized expression levels (to t=0 min) are shown. Induction thresholds for the fast response were set at ≥ 2σ + median log2 fold change for each time point (1.00 log2 fold change at 120 minutes, 1.24 log2 fold change at 240 minutes). Genes above threshold at both 120 minutes and 240 minutes post-starvation were classified as the rapid response. Genes above only the 240-minute threshold were classified as the slow response.

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Additional file 3:

Table S2. Microarray Results for Genes Regulated by Pi Starvation, pho7+, and/or csk1+. Genes induced following Pi starvation, in pho7+ versus pho7Δ cells during Pi starvation, or in csk1Δ versus csk1+ cells in non-stressed (high-Pi) conditions were tabulated and analyzed for orthologs in the S. cerevisiae PHO pathway. Induction thresholds were set at ≥ 1.8 fold-change with a p-value ≤ 0.10. Values shown are the average of two independent replicates.

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Additional file 4:

Figure S2. Global Pho7-TAP Enrichment During Pi-Starvation. Cells containing the tagged variant of Pho7 (Pho7-TAP) were grown in either high-Pi (blue) or starvation (red) media for 120 minutes prior to cross-linking and ChIP-Seq processing. Shown are the chromosomal enrichment profiles for Pho7 for the S. pombe genome. Reads were normalized to total counts for each chromosome.

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Additional file 5:

Table S3. Peak List for Pho7-TAP ChIP-Seq Data Set. Pho7-TAP peaks detected in both high-Pi (Hi) or no-Pi (No) conditions with maximum height ≥ 2x genome average are given. Peak detection was performed against a mock sample lacking a TAP epitope as described in materials and methods.

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Additional file 6:

Figure S3. Pho7-TAP Protein Levels Remain Constant During Pi Starvation in csk1+ and csk1Δ Backgrounds. csk1+ or csk1Δ cells containing the tagged variant of Pho7 (Pho7-TAP) were grown in high-Pi (+Pi) media followed by Pi starvation for 60 or 120 minutes (-Pi). Western blot analysis reveals similar levels of the Pho7-TAP protein in all conditions as detected by rabbit IgG. The strain lacking the TAP tag is shown (Pho7) as a negative control. The Western analysis was completed in duplicate, shown is a representative blot.

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Additional file 7:

Figure S4. Global Pho7-TAP Enrichment in the Absence of Csk1. csk1+ (blue) or csk1Δ (cyan) cells containing the tagged variant of Pho7 (Pho7-TAP) were grown in high-Pi media for 120 minutes prior to cross-linking and ChIP-Seq processing. Shown are the chromosomal enrichment profiles for Pho7 for the S. pombe genome. Reads were normalized to total counts for each chromosome.

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Additional file 8:

Figure S5. ChIP-Seq Binding Profiles for Pho7-TAP at Non-PHO Promoters. Shown are ChIP-Seq profiles for the genes identified in Figure 6. As previously described, wild-type cells containing Pho7-TAP were grown in either high-Pi (blue) or no-Pi (red) conditions and ChIP-Seq libraries were prepared from purified DNA. For comparison, the ChIP-Seq signal from mock (black) cells grown in no-Pi and csk1Δ (cyan) cells incubated in high-Pi is included. The gene product of interest is plotted based on transcript direction with the plus (+) strand above and the minus (-) strand below. Reads were normalized as described in the text and the location within the genome is plotted on the x-axis.

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Additional file 9:

Table S4. Gene Ontology Enrichment for Pho7 Dependent Genes Identified via Microarray and ChIP-Seq Analysis. Genes possessing a pho7+-dependency independent of Pi availability were cross-referenced with the Pho7-TAP peak list to identify pho7+-regulated genes with promoters enriched by Pho7-TAP. Thresholds for induction and peak enrichment are described in materials and methods. The gene set was processed through the AmiGO toolkit [34,52-55] and GO terms containing at least 3 genes with p-values ≤ 0.01 are displayed.

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Additional file 10:

Table S5. Microarray Results for pho7+-Dependent Genes Regulated by Phosphate, Iron, Copper, Osmotic, and/or Carbon Switching Stress. pho7+ genes passing the thresholds (described in materials and methods) for each stress condition assayed were tabulated with the corresponding fold-change (Log2) and p-values (based on at least two independent biological replicates). Genes regulated in multiple stress conditions by pho7+ are indicated in bold. All comparisons between stressed and non-stressed conditions (e.g., [-Pi/+Pi]) were done in a pho7+ background. Genes passing the thresholds for each array condition have fold-change (Log2) values indicated in bold. +Pi: 10 mM H2KPO4, -Pi: 0 mM H2KPO4, +Fe: 100 uM Fe(III)Cl3, -Fe: 250 uM DIP, +Cu: 100 uM Cu(II)SO4, -Cu: 100 uM BCS, 1.2M: 1.2M NaCl, 0.1M: 0.1M NaCl, G: 2% glucose, GE: 2% glycerol, 1% ethanol.

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Additional file 11:

Table S6. All primers utilized in this manuscript are listed with their primer ID, nucleotide sequence, and experimental purpose.

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