The <i>hst3</i>∆ <i>hst4</i>∆ <i>sir2</i>∆ <i>fbp1</i>∆ cells harbor active glucose metabolism but cannot contribute to cell growth.

<p>(A) Pathway of central carbon metabolism in budding yeast based on information from the <i>Saccharomyces</i> genome database website (<a href="http://www.yeastgenome.org/" target="_blank">http://www.yeastgenome.org/</a>). G1P: glucose 1-phosphate, G6P: glucose 6-phosphate, PEP: phosphoenolpyruvate, PP pathway: pentose phosphate pathway, R5P: ribose 5-phosphate, and AcCoA: acetyl-CoA. (B) Comparison of glucose consumption among strains. Wild-type, <i>fbp1</i>∆, <i>hst3</i>∆ <i>hst4</i>∆ <i>sir2</i>∆ and <i>hst3</i>∆ <i>hst4</i>∆ <i>sir2</i>∆ <i>fbp1</i>∆ cells (1×10⁶ cells/ml) were released into fresh YPD medium and were cultured at 25°C. A small aliquot of medium was picked up following the time course to measure the cell number, glucose concentration, and ethanol concentration in medium (Panels A and B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194942#pone.0194942.s002" target="_blank">S2 Fig</a>). The P-value matrix contains the Mann-Whitney U-test p-value for a one-tailed test (wild-type vs. <i>hst3</i>∆ <i>hst4</i>∆ <i>sir2</i>∆ at 5 h in time course, wild-type vs. <i>hst3</i>∆ <i>hst4</i>∆ <i>sir2</i>∆ <i>fbp1</i>∆ at 24 h). P-values were calculated using ystat2008 software (Igakutosho, Japan) (*p<0.05). (C) Comparison of the ethanol productivity among yeast strains. The ethanol productivity was calculated as the concentration of ethanol released in medium per cell number at 24 h in cell cultivation (Panels A and B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194942#pone.0194942.s002" target="_blank">S2 Fig</a>). Multiple comparisons among strains (wild-type, <i>hst3</i>∆ <i>hst4</i>∆ <i>sir2</i>∆ and <i>hst3</i>∆ <i>hst4</i>∆ <i>sir2</i>∆ <i>fbp1</i>∆) were performed (non-repeated measures ANOVA with the Student-Newman-Keuls (SNK) test) (*p<0.05 and **p<0.01). Values are expressed as the means ± standard deviations. The experiments were repeated three times.</p

The <i>hst3</i>Δ <i>hst4</i>Δ <i>sir2</i>Δ <i>fbp1</i>Δ deletion increases the metabolic flux into the PP pathway.

<p>(A) Flux directions between glycolysis and the PP pathway. (B) Relative flux of the PP pathway (R5P from either G6P or G3P/S7P). (C) Relative flux of the PP pathway (E4P <i>vs</i>. P5P). (D) Relative flux of the glycolytic pathway (PEP through the PP pathway). Metabolic flux ratios between glycolysis and the PP pathway of wild-type (black: top values), <i>fbp1</i>Δ (blue: mid-upper values), <i>hst3</i>Δ <i>hst4</i>Δ <i>sir2</i>Δ (green: mid-lower values) and <i>hst3</i>Δ <i>hst4</i>Δ <i>sir2</i>Δ <i>fbp1</i>Δ (red: bottom values) cells during exponential growth on glucose. Relative flux in converging pathways (in %) as determined using FiatFlux for two nodes and one enzyme reaction in central carbon metabolism. The values are expressed as the means ± standard deviations. The minus values represent reverse flux in metabolic reaction. The values in the solid box were directly inferred from the analysis of local ¹³C patterns, whereas the other values are the calculated complements. The experiments were repeated twice. A representative experiment is shown. G6P: glucose 6-phosphate, G3P: glycerol 3-phosphate, PEP: phosphoenolpyruvate, R5P: ribose 5-phosphate, E4P: erythrose 4-phosphate, S7P: sedoheptulose 7-phosphate, ub: upper bounds, PP pathway: pentose phosphate pathway.</p

Comparison of intracellular metabolic profiles among yeast strains.

<p>(A) Principal component analysis (PCA) showing the fluctuations of the intracellular metabolites of yeast strains (wild-type, <i>fbp1</i>∆, <i>hst3</i>∆ <i>hst4</i>∆ <i>sir2</i>∆ and <i>hst3</i>∆ <i>hst4</i>∆ <i>sir2</i>∆ <i>fbp1</i>∆). PCA data (PC1 and PC2) were employed from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194942#pone.0194942.s004" target="_blank">S1 Table</a>. (B) Heat map of hierarchical clustering of intracellular metabolite profiles from yeast strains. Red indicates a higher concentration of metabolites than the internal standard, while green indicates a lower concentration of metabolites than the internal standard. The relative amount of each metabolite per internal standard analyzed in CE–TOF/MS is listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194942#pone.0194942.s004" target="_blank">S1 Table</a>. Three cell samples were employed for each yeast strain. TCA cycle: tricarboxylic acid cycle, Glu: glutamine, Asn: asparagine.</p

Comparative analysis of metabolites among yeast strains.

<p>The fluctuation of metabolites was calculated using wild type as the denominator. Green indicates a decrease in the metabolite levels for wild-type cells, and red indicates an increase. Representative data are employed from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194942#pone.0194942.s006" target="_blank">S3 Table</a>. The P-value matrix contains the Welch's t-test (*<0.05, **<0.01, ***<0.001 compared with the value of wild-type cells). Leu: leucine, Phe: phenylalanine, Trp: tryptophan, Val: valine, dAMP: deoxy adenine monophosphate, ADP: adenine diphosphate, dTMP: deoxy thymidine diphosphate. UDP: uridine diphosphate, UTP: uridine triphosphate.</p

Schematic model to increase the levels of secondary metabolites in <i>hst3</i>Δ <i>hst4</i>Δ <i>sir2</i>Δ <i>fbp1</i>Δ cells.

<p>Schematic model to increase the levels of secondary metabolites in <i>hst3</i>Δ <i>hst4</i>Δ <i>sir2</i>Δ <i>fbp1</i>Δ cells.</p

Centromere Architecture Breakdown Induced by the Viral E3 Ubiquitin Ligase ICP0 Protein of Herpes Simplex Virus Type 1

<div><p>The viral E3 ubiquitin ligase ICP0 protein has the unique property to temporarily localize at interphase and mitotic centromeres early after infection of cells by the herpes simplex virus type 1 (HSV-1). As a consequence ICP0 induces the proteasomal degradation of several centromeric proteins (CENPs), namely CENP-A, the centromeric histone H3 variant, CENP-B and CENP-C. Following ICP0-induced centromere modification cells trigger a specific response to centromeres called interphase Centromere Damage Response (iCDR). The biological significance of the iCDR is unknown; so is the degree of centromere structural damage induced by ICP0. Interphase centromeres are complex structures made of proximal and distal protein layers closely associated to CENP-A-containing centromeric chromatin. Using several cell lines constitutively expressing GFP-tagged CENPs, we investigated the extent of the centromere destabilization induced by ICP0. We show that ICP0 provokes the disappearance from centromeres, and the proteasomal degradation of several CENPs from the NAC (CENP-A nucleosome associated) and CAD (CENP-A Distal) complexes. We then investigated the nucleosomal occupancy of the centromeric chromatin in ICP0-expressing cells by micrococcal nuclease (MNase) digestion analysis. ICP0 expression either following infection or in cell lines constitutively expressing ICP0 provokes significant modifications of the centromeric chromatin structure resulting in higher MNase accessibility. Finally, using human artificial chromosomes (HACs), we established that ICP0-induced iCDR could also target exogenous centromeres. These results demonstrate that, in addition to the protein complexes, ICP0 also destabilizes the centromeric chromatin resulting in the complete breakdown of the centromere architecture, which consequently induces iCDR.</p> </div

ICP0-expressing cells induce CENP-A loss from centromeres.

<p>(A) HeLa-TR, TR-ICP0, and TR-FXE cells were induced (+) or not induced (−) with tetracycline before being subjected to WB to detect ICP0 or FXE expression. The arrow indicates the ICP0 or FXE signal. Note that the level of protein for TR-FXE is 10-fold lower than that for HeLa-TR or TR-ICP0 due to the very high expression of FXE. (B) Control (not-induced) (i to iii) or tetracycline-induced (iv to ix) cells were tested by IF for: (i) the expression of ICP0 or FXE (green); and (ii) the CENP-A (red) signal at centromeres (see insets for individual ICP0, FXE or CENP-A signals). More than 99.5% of the cells are positive for the ICP0 or FXE signal. The CENP-A signal has disappeared in all the ICP0-expressing cells. Bars, 10 µm.</p

HSV-1 infection destabilizes the structure of centromeric nucleosomes in an ICP0-dependent manner.

<p>(A) Micrococcal nuclease (MNase) digestion patterns of the chromatin from HeLa cells subjected to: mock infection; infection with the ICP0-null HSV-1 mutant virus (dl1403); and infection with HSV-1 wt (17syn+). Left panel: MNase digestion pattern of total DNA stained with ethidium bromide. Right panel: Southern blot hybridization of the gel shown in left panel using an alphoid DNA probe. M = 1 Kb Plus DNA ladder (in base pair, Invitrogen). (B) Plot of the digestion profiles after 30 min and 50 min of MNase digestion (curves corresponding to the colored frames in A). Red curve: HSV-1 wt infection; black curve, HSV-1 dl1403 infected cells; green curve: non-infected cells (C) Quantification of mono- and di-nucleosomes over time of MNase digestion. Red curve: HSV-1 wt infection; black curve, HSV-1 dl1403 infected cells; green curve: non-infected cells (D) Rate of chromatin digestion. The ratios of LMW-normalized mono- over di-nucleosomes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044227#pone.0044227.s001" target="_blank">Figure S1</a>) give the rate of digestion of total chromatin (left panels) or centromeric chromatin (right panels). The graphs represent the mean values of the ratios of mono- over di-nucleosomes ± SD for at least three independent experiments. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044227#s2" target="_blank">Results</a> were considered as significant for a p value≤0.05 (Student's <i>t</i> test).</p

ICP0 alone destabilizes the centromere nucleosome structure.

<p>(A) Micrococcal nuclease (MNase) digestion patterns of the chromatin of three inducible cell lines: Control (no protein expressed) (HeLa-TR); expressing the ICP0 RING finger mutant FXE (TR-FXE); and expressing ICP0 (TR-ICP0). All cells were treated with tetracycline (1 µg/ml) for 19 h before the analysis. Left panel: MNase digestion patterns of total DNA. Right panel: Southern blot hybridization of the gel shown on the left using an alphoid DNA probe. M = 1 Kb Plus DNA ladder (in base pair, Invitrogen). (B) Plot of the digestion profiles after 30 min and 50 min of MNase digestion (curves corresponding to the colored frames in A). Red curve: HSV-1 wt infection; black curve, HSV-1 dl1403 infected cells; green curve: non-infected cells. (C) Quantification of mono- and di-nucleosomes during MNase digestion. Red curve: HSV-1 wt infection; black curve, HSV-1 dl1403 infected cells; green curve: non-infected cells. (D) Rate of chromatin digestion. The ratios of LMW-normalized mono- over di-nucleosomes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044227#pone.0044227.s004" target="_blank">Figure S4</a>) give the rate of digestion of total chromatin (left panels) or centromeric chromatin (right panels). The graphs represent the mean values of the ratios of mono- over di-nucleosomes ± SD for at least three independent experiments. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044227#s2" target="_blank">Results</a> were considered as significant for a p value≤0.05 (Student's <i>t</i> test).</p

ICP0 triggers iCDR on human artificial chromosomes.

<p>(A) Upper panel, Formation and schematic structure of a human artificial chromosome (HAC). Lower panel, DNA-FISH showing the co-detection of HAC and alphoid DNA; the position of the probes for detection of HAC and alphoid DNA sequences are indicated above the images. Bar, 10 µm. (B) Immuno-DNA FISH showing the co-localization of ICP0 and endogenous centromeres in the interphase, different stages of mitosis, and on a mitotic chromosome (i to v). HT1080 W0210-R8 cells that contain a HAC were infected for 3 h with HSV-1 wt, and ICP0 and the HAC were detected (vi). Bars, 10 µm. (C) Immuno-DNA FISH showing the presence of CENP-A, -B, -C, and -I on the HAC in interphase cells. Images i, ii, and iii were obtained using a wide-field microscope, while image iv was acquired using a confocal microscope. The nucleus is outlined by a dashed line in iv. Bars, 10 µm. (D) Immuno-DNA FISH showing the disappearance of CENPs proteins from the HAC in HT1080 W0210-R8 cells infected for 5 h with the HSV-1 wt virus. DNA (i) or ICP0 (ii to iv) is visualized in blue. Arrowheads indicate HACs with CENPs co-localized (in ICP0- cells), while the arrows indicate HACs with no CENP signals (in ICP0+ cells). Bars, 10 µm. (E and F) Immuno-DNA FISH detection of coilin (E), fibrillarin (F), and HAC in HT1080 W0210-R8 cells infected with HSV-1 wt for 4 h. Coilin and fibrillarin are shown in control (not expressing ICP0) cells together with the HAC. Multiple spots of coilin and fibrillarin in ICP0-expressing cells are representative of the iCDR on endogenous centromeres in the ICP0-expressing cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044227#pone.0044227-Morency2" target="_blank">[47]</a>). Arrows indicate coilin or fibrillarin colocalizing with the HAC. Dotted lines in control images delimit the nucleus edge. Bars, 10 µm.</p