Strains used in this study.

<p>Strains used in this study.</p

Asf1 and Snf2 have partially overlapping functions in cell cycle and checkpoint control.

<p>A. Rad53 activation is impaired in <i>asf1Δ snf2Δ</i> double mutants. Cells were grown in the presence or absence of 0.2 M HU for two hours. Actin is the loading control. The apparent lower expression of Rad53 in the double mutant was not reproducible – see Figure 7B. B. <i>asf1Δ snf2Δ</i> mutants fail to fully arrest in S phase in response to HU. DNA content of normally cycling cells (time 0) and cells grown in the presence of 0.2 M HU. C. Rad53 activation is delayed in <i>asf1Δ snf2Δ</i> double mutant cells. Cells from the cultures assayed in B were processed for analysis of Rad53 modification state. Actin is the loading control. D. Recovery from replication stress is compromised in <i>asf1Δ snf2Δ</i> mutants. Ten-fold serial dilutions of early log phase cells were grown in YPD or YPD + 0.2 M HU for 24 hours. Aliquots of these cultures were then diluted to 1×10⁶ cells/mL and spotted onto rich solid medium. Cells were grown at 30°C and photographs were taken after 2 and 3 days.</p

Regulation of H3K56ac is similar in wild type and <i>snf2Δ</i> cells.

<p>A. Bulk expression of H3K56ac analyzed by immunoblotting. Actin is the loading control. B, C. ChIP analysis of H3 occupancy at DDR gene promoters in response to HU (one hour). Immunoprecipitated DNA was normalised to input DNA and signal obtained in untreated wild type cells was set to 1. D, E. ChIP analysis of H3K56ac occupancy at DDR gene promoters in the indicated strains grown as in B and C. Normalization of the data is described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021633#s4" target="_blank">methods</a> section. In B-E, error bars show standard deviation between three biological replicates.</p

Asf1 and SWI/SNF contribute to survival under conditions of replication stress triggered by HU.

<p>A. <i>asf1Δ snf2Δ</i> (upper panel) and <i>asf1Δ snf5Δ</i> (lower panel) double mutants are sensitive to HU. Ten-fold serial dilutions of early log phase cells were photographed after 4 days of growth at 30°C. B. <i>asf1Δ snf2Δ</i> double mutants are slow-growing in liquid medium containing 0.2 M HU.</p

Asf1 and SWI/SNF: physical interaction and regulation of DDR gene transcription.

<p>A. Snf2 copurifies with Asf1. Asf1-TAP Snf2-HA and Snf2-HA lysates were used for tandem affinity purification. Inputs were obtained before binding to the first affinity column. Final eluates were resolved by SDS-PAGE and probed with either anti-CBP (top) or anti-HA (bottom) antibodies to detect Asf1 or Snf2, respectively. The high-contrast insert in the upper input panel has been included to more clearly show the presence of Asf1-TAP in the Asf1-TAP Snf2-HA lysate. B, C. Asf1 and SWI/SNF promote derepression of the DDR genes <i>HUG1</i> (B) and <i>RNR3</i> (C). Expression is normalised to <i>SCR1</i>. The grey shading in the right panel of B and C indicates the time period when <i>HUG1</i> and <i>RNR3</i> mRNA expression declines in the presence of HU.</p

<i>asf1Δ snf2Δ</i> mutants are sensitive to genotoxins that cause chemical modification of DNA.

<p>A. <i>asf1Δ snf2Δ</i> double mutants are sensitive to MMS. Ten-fold serial dilutions of early log phase cells were spotted onto rich medium with or without MMS and grown at 30°C. Photographs were taken after 2 and 3 days. B. <i>asf1Δ snf2Δ</i> double mutants are sensitive to UV irradiation. Ten-fold serial dilutions of cells were spotted onto rich medium and irradiated or not. Plates were incubated in the dark at 30°C and photographed as in (A).</p

Asf1 and SWI/SNF are both recruited to the promoters of DDR genes during replication stress.

<p>A. ChIP analysis of Snf2 recruitment to the promoter of <i>RNR3</i> in response to HU. In this and all other panels of Figure 2, P values obtained by statistical analysis (Student's t-test) are shown for comparisons of HU-treated and untreated cells. B. ChIP analysis of Snf5 recruitment to the promoter of <i>RNR3</i> in response to HU. In A and B, normalization takes into account SWI/SNF subunit cross-linking in the ORF of <i>POL1</i>; occupancy in untreated wild type cells is set to 1. C, D. ChIP analysis of Asf1 recruitment to the promoters of DDR genes. Immunoprecipitated DNA was normalised to input DNA and the signal obtained in untreated wild type cells was set to 1. In A-D HU treatment was for one hour. All PCR reactions were performed in triplicate to obtain the data points reported in the graphs.</p

<i>ASF1</i> shows synthetic sick interactions with components of the SWI/SNF complex.

<p>A. <i>asf1Δ snf2Δ</i> double mutants are slower-growing than either single mutant. B. <i>asf1Δ snf2Δ</i> double mutants are slightly temperature-sensitive. Ten-fold serial dilutions of early log phase cells were photographed after 3 days of growth. C. <i>asf1Δ snf5Δ</i> double mutants are slower-growing than either single mutant. Averages of three independent <i>asf1Δ snf5Δ</i> isolates are shown, with error bars (under data point icons).</p

High-Performance Chemical Isotope Labeling Liquid Chromatography–Mass Spectrometry for Profiling the Metabolomic Reprogramming Elicited by Ammonium Limitation in Yeast

Information about how yeast metabolism is rewired in response to internal and external cues can inform the development of metabolic engineering strategies for food, fuel, and chemical production in this organism. We report a new metabolomics workflow for the characterization of such metabolic rewiring. The workflow combines efficient cell lysis without using chemicals that may interfere with downstream sample analysis and differential chemical isotope labeling liquid chromatography mass spectrometry (CIL LC–MS) for in-depth yeast metabolome profiling. Using ¹²C- and ¹³C-dansylation (Dns) labeling to analyze the amine/phenol submetabolome, we detected and quantified a total of 5719 peak pairs or metabolites. Among them, 120 metabolites were positively identified using a library of 275 Dns-metabolite standards, and 2980 metabolites were putatively identified based on accurate mass matches to metabolome databases. We also applied ¹²C- and ¹³C-dimethylaminophenacyl (DmPA) labeling to profile the carboxylic acid submetabolome and detected over 2286 peak pairs, from which 33 metabolites were positively identified using a library of 188 DmPA-metabolite standards, and 1595 metabolites were putatively identified. Using this workflow for metabolomic profiling of cells challenged by ammonium limitation revealed unexpected links between ammonium assimilation and pantothenate accumulation that might be amenable to engineering for better acetyl-CoA production in yeast. We anticipate that efforts to improve other schemes of metabolic engineering will benefit from application of this workflow to multiple cell types