<i>DIA2</i> genetically interacts with genes encoding histone chaperones and histone H3–H4 lysine mutants with defects in nucleosome assembly.

<p>(A) Dia2 functions in parallel with known H3–H4 histone chaperones in transcriptional silencing at the silent <i>HMR</i> locus. Cells of the indicated genotype were assessed for <i>HMR</i> silencing as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002846#pgen-1002846-g001" target="_blank">Figure 1A–1B</a>. The percentage of cells expressing GFP from one of three independent experiments was reported. (B–C) Dia2 functions in parallel with known H3–H4 histone chaperones in growth and DNA damage sensitivity. (B) Genetic interactions among <i>DIA2</i>, <i>RTT106</i>, and <i>CAC1</i> were assessed by spotting a ten fold series dilution of cells of the indicated genotype onto regular growth media (YPD), media containing DMSO (0) or media with the indicated concentration of camptothecin (CPT). (C) A summary of the genetic analysis of growth and DNA damage sensitivity of histone chaperone mutations combined with deletion of <i>DIA2</i>. A ‘+’ indicates a synthetic effect in the double mutant compared to either single mutant alone. Spot assays were performed as described above using different concentrations of CPT and methyl methanesulfonate (MMS). Representative images are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002846#pgen.1002846.s002" target="_blank">Figure S2</a>. (D) The <i>dia2</i>Δ mutant is synthetic lethal with mutations at histone H4 lysine residues 5, 8 and 12 (<i>H4K5,8,12R</i>). A ten fold serial dilution of cells was spotted onto plates of the indicated media. Both copies of histone genes (<i>HHT1-HHF1</i> and <i>HHT2-HHF2</i>) were deleted. Wild type H3–H4 was expressed from a <i>URA3</i> containing plasmid, whereas the histone H4 mutant (as indicated) was expressed from a plasmid containing the <i>HIS3</i> gene. Media containing FOA was used to select for the loss of the <i>URA3</i>-containing plasmid and detect synthetic lethality.</p

Sir3 and Sir4 are mislocalized in <i>dia2</i>Δ cells and cells expressing <i>dia2</i> mutants lacking the Dia2 F-box or LRR domain.

<p>(A–C) Sir3 and Sir4 are mislocalized in <i>dia2</i>Δ and <i>dia2Δ rtt106</i>Δ cells. (A) Representative images of Sir3-GFP or Sir4-GFP foci in WT and <i>dia2</i>Δ cells. Cells expressing Sir3-GFP and GFP-Sir4 exhibited nuclear foci in WT cells, and this pattern is lost in some <i>dia2</i>Δ cells. Note that <i>dia2</i>Δ cells are larger in size than wild-type cells (images were taken under the same magnification). The scale bar represents 5 µm. Cells that exhibited Sir protein localization defects as described in the text were marked by arrows. (B–C) Quantification of the percentage of <i>dia2</i>Δ cells that had WT-like Sir3-GFP foci (B) and WT-like Sir4-GFP foci (C). (D–E) Cells expressing <i>dia2</i> mutants lacking the Dia2 F-box and LRR domains exhibit defects in the localization of Sir3 (D) and Sir4 (E). For each experiment, at least 100 cells were counted for each genotype, and the average ± s.d. percentage of cells expressing WT-like foci from at least two independent experiments was reported.</p

Sir4 binding at telomeric silent chromatin and expression of a telomeric gene are regulated during the cell cycle in a Dia2-dependent manner.

<p>(A) Schematic representation of the experiments to test Sir4 binding and gene expression at telomeric silent chromatin during the cell cycle. Briefly, WT or <i>dia2</i>Δ cells were arrested at G1 with α-factor. Samples were collected for analysis of DNA content (B), Sir4 ChIP (C) and (D) gene expression at 0, 30 and 60 minutes following released into cell cycle. (B) Cell cycle analysis of WT and <i>dia2</i>Δ cells collected in the experiment described in A. DNA was stained using propidium iodide and analyzed using flow cytometry. (C) Sir4 binding is reduced at telomere silent chromatin as cells progress through the cell cycle, whereas Sir4 levels in <i>dia2</i>Δ mutant cells are not changed during the cell cycle. At each time point, cells were collected for ChIP assay using antibodies against Sir4 and histone H3. ChIP DNA was analyzed by real time PCR using PCR primers amplifying both silent and active chromatin loci as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002846#pgen-1002846-g005" target="_blank">Figure 5D</a>. The Sir4 ChIP signal was normalized against that of H3. The data presented is the average ± s.d. of three independent experiments with the p-value indicated as determined using the student's t-test. (D) The expression of the <i>YFR057W</i> increases as the cell cycle progresses. RNA was isolated from cells collected at each time point and reverse transcribed. The expression of <i>YFR057W</i> was analyzed using quantitative real-time PCR and was normalized against the expression of <i>ACT1</i> as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002846#pgen-1002846-g001" target="_blank">Figure 1E</a>. Data is presented as the ratio of the relative expression of <i>YFR057W</i> at each indicated time point to the <i>YFR057W</i> expression at the G1 phase (0) time point for the respective cell type (thus, <i>YFR057W</i> expression at time 0 is 1 for both genotypes). The average calculated from two independent experiments is shown.</p

Effect of histone lysine mutations on the growth and DNA damage sensitivity of <i>dia2</i>Δ cells.

<p>The <i>dia2</i>Δ mutant, each histone mutant and their corresponding double mutant, containing both the <i>dia2</i>Δ mutation and the indicated histone mutation, were assayed for cell growth and sensitivity towards the DNA damaging agent camptothecin (CPT). A ‘−’ represents no effect on growth or DNA damage sensitivity over the individual single mutants. A ‘+’ represents a synthetic effect in growth on regular media (YPD) or DNA damage sensitivity (growth on medium containing CPT). More+s indicate a more dramatic synthetic effect in comparison with other strains tested. Note that the <i>dia2Δ H4K5,8,12R</i> mutant is synthetic lethal, and therefore, DNA damage sensitivity could not be assessed (n/a).</p

Dia2's F-box and LRR domains are indispensable for transcriptional silencing.

<p>(A) A schematic drawing of the domain structure of Dia2. Dia2 contains a tetratricopeptide repeat (TPR) region, nuclear localization signal (NLS), F-box and leucine rich repeat (LRR) domain. (B) The Dia2 F-box and LRR domains are important for silencing at the <i>HMR</i> locus. The <i>dia2</i>Δ mutant cells containing the <i>HMR::GFP</i> reporter were transformed with empty vector or a plasmid expressing WT or different <i>dia2</i> mutants, and the percentage of cells expressing GFP was determined as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002846#pgen-1002846-g001" target="_blank">Figure 1B</a>. Note that the percentage of cells expressing GFP in <i>dia2</i>Δ cells was higher when using selective media compared to cells growing in YPD. (C) The Dia2 F-box and LRR domains are important for telomere silencing. The <i>dia2</i>Δ mutants containing the <i>VIIL-URA3</i> reporter were transformed with empty vector or a plasmid expressing the indicated <i>dia2</i> mutant and spotted onto selective media (-HIS) and media containing FOA (-HIS, FOA) to assay silencing. (D–E) The Dia2 LRR domain is important for <i>HMR</i> silencing (D) and telomere silencing (E). Assays were performed as described in B and C.</p

A Cell-Free Fluorometric High-Throughput Screen for Inhibitors of Rtt109-Catalyzed Histone Acetylation

<div><p>The lysine acetyltransferase (KAT) Rtt109 forms a complex with Vps75 and catalyzes the acetylation of histone H3 lysine 56 (H3K56ac) in the Asf1-H3-H4 complex. Rtt109 and H3K56ac are vital for replication-coupled nucleosome assembly and genotoxic resistance in yeast and pathogenic fungal species such as <i>Candida albicans</i>. Remarkably, sequence homologs of Rtt109 are absent in humans. Therefore, inhibitors of Rtt109 are hypothesized as potential and minimally toxic antifungal agents. Herein, we report the development and optimization of a cell-free fluorometric high-throughput screen (HTS) for small-molecule inhibitors of Rtt109-catalyzed histone acetylation. The KAT component of the assay consists of the yeast Rtt109-Vps75 complex, while the histone substrate complex consists of full-length <i>Drosophila</i> histone H3-H4 bound to yeast Asf1. Duplicated assay runs of the LOPAC demonstrated day-to-day and plate-to-plate reproducibility. Approximately 225,000 compounds were assayed in a 384-well plate format with an average Z' factor of 0.71. Based on a 3σ cut-off criterion, 1,587 actives (0.7%) were identified in the primary screen. The assay method is capable of identifying previously reported KAT inhibitors such as garcinol. We also observed several prominent active classes of pan-assay interference compounds such as Mannich bases, catechols and p-hydroxyarylsulfonamides. The majority of the primary active compounds showed assay signal interference, though most assay artifacts can be efficiently removed by a series of straightforward counter-screens and orthogonal assays. Post-HTS triage demonstrated a comparatively small number of confirmed actives with IC₅₀ values in the low micromolar range. This assay, which utilizes five label-free proteins involved in H3K56 acetylation <i>in vivo</i>, can in principle identify compounds that inhibit Rtt109-catalyzed H3K56 acetylation via different mechanisms. Compounds discovered via this assay or adaptations thereof could serve as chemical probes or leads for a new class of antifungals targeting an epigenetic enzyme.</p></div

Noteworthy PAINS substructures in the primary Rtt109-Vps75 HTS.

<p><i>Italics</i> denote the original names of published PAINS substructures <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078877#pone.0078877-Baell1" target="_blank">[51]</a>. For individual substructures, the ratios denote the number of primary active compounds divided by the number of compounds tested for each HTS production run.</p

Assay design and optimization.

<p>(A) Titration matrix of CoA and CPM in buffer-only conditions to determine the optimal assay levels of acetyl-CoA and CPM. (B) Titration matrix of CPM and acetyl-CoA in buffer-only conditions to verify acetyl-CoA and CPM do not form fluorescent adducts under HTS conditions. (C) Time-course study of CoA titrations with 20 µM CPM in buffer-only conditions to determine the optimal time for the reaction involving CoA and CPM. (D) HTS plate template. Arrows denote chosen HTS conditions.</p

Assay validation using the LOPAC ± detergent.

<p>(A) Z' factors for replicate LOPAC experiments ± detergent. (B–C) Comparison of duplicate runs of the LOPAC ± detergent. Each point represents the activity of a discrete compound from the LOPAC. (D) Comparison of the LOPAC results ± detergent. Percent inhibitions represent the means of the replicate LOPAC experiments. Trend line (solid, red), ideal correlation line (dashed, blue). (E) Percent inhibition distribution of the averaged LOPAC results ± detergent, binned in 5% intervals.</p

Fluorometric Rtt109-Vps75 HTS schematic.

<p>The Rtt109-Vps75 KAT complex catalyzes the transfer of an acetyl group from acetyl-CoA to specific histone lysine residues within the Asf1-dH3-H4 substrate complex. The resulting CoA contains a free thiol group (-SH), which can react with the sulfhydryl-sensitive probe CPM to form a fluorescent adduct, thereby permitting the quantification of free CoA as a measure of KAT activity.</p