<i>S. cerevisiae</i> forms filamentous mats.

<p><b>A</b>) Wild-type cells (PC313) were spotted 2 cm apart onto 0.3% agar media that contained (YEPD; top panel) or lacked (YEP; bottom panel) glucose. The YEPD plate was incubated for 4 days and photographed; the YEP plate for 15 days. Bar = 1 cm. <b>B</b>) Microscopic examination of perimeters of mats in 1A. Bar = 100 microns. <b>C</b>) The origin of filamentous mats. Wild type (PC538) cells were examined on synthetic medium either containing 2% glucose (SCD) or lacking glucose (SC) in 0.3% agar medium for 24 h at 30°C. A compiled Z-stack rendering of typical microcolonies are shown. Bar = 20 microns. <b>D</b>) Same strains in 1C were examined on rich medium either containing 2% glucose (YEPD) or lacking glucose (YEP) in 0.3% agar. A representative microscopic image is shown. Bar = 10 microns. E) Vegetative mats mature into filamentous mats over time as nutrients become limiting. Two mats of wild type (PC313) strain were spotted bilaterally (1.5 cm apart) on YEPD and YEP media (+0.2% galactose) containing 0.3% agar media. The number of filaments occurring along the circumference of mats was scored on a scale of 1, 2, or 3 dots at 20× magnification corresponding to 3, 6, or 9 filaments or greater, respectively. Dots were plotted on a circle representing the outline of one of the mats with right hemispheres corresponding to the side of the mat facing a second mat. Asymmetric filamentation observed in the right hemisphere of 2d, Glu can possibly result from nutritional stress compounded by nutrient depletion from adjacent mats. Filamentation was monitored and plotted after growth for 1, 2, 3, and 4 days. Quantitation of pseudohyphae was complicated at longer time points when biofilms began to variegate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032294#pone.0032294-Halme1" target="_blank">[60]</a>. Bar = 1 cm.</p

The role of Flo11 overexpression on upward growth in the plane of the Z-axis.

<p><b>A</b>) Microcolonies of wild-type cells (PC538) and cells overexpressing <i>FLO11</i> (PC2712) were examined by microscopy at 10× after 24 h incubation at 30°C. Wild type and <i>ste12</i> mats on high agar concentrations is also shown Bar = 100 microns. <b>B</b>) Contour mapping of z-stack rendering of the indicated microcolonies in panel 7A are shown. Bar = 30 microns. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032294#pone.0032294.s006" target="_blank">Supplemental Movies S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032294#pone.0032294.s007" target="_blank">S6</a>. <b>C</b>) Wild-type (PC538), <i>flo11</i>Δ (PC1029), and <i>GAL-FLO11</i> (PC2712) cells were spotted onto YEP-GAL medium (8%) agar atop nitrocellulose filters for 24 h at 30°C. Colonies were photographed in transmitted light. Bar = 1 cm. At right, separation of the <i>GAL-FLO11</i> mat from the surface using forceps. <b>D</b>) Microscopic examination of the mats in panel C. Bar = 200 microns.</p

The role of the MAPK pathway in regulating mat architecture when exposed to surfaces of different rigidities.

<p><b>A</b>) Contour maps in the Z-axis of wild type (PC538) mats incubated in media of different agar concentrations for 14d. Insets show mat morphology (left, photograph, bar, 1 cm; right, photomicrograph, bar, 200 microns) in 4% agar. The numbers in parentheses represent the average mat dry weight from two experiments with standard deviation shown. Scale bars for the X and Y-axes are in mm. <b>B</b>) Mats formed by a <i>ste12</i>Δ mutant (PC539) on different agar concentrations. Analysis is as described for panel A.</p

MAPK- and Flo11-dependent colony avoidance response.

<p><b>A</b>) Wild-type cells were spotted in three spots and examined daily. The photograph showing the embossed appearance of colonies was taken at day 3. Left three panels, Bar = 1 cm. Far right panel, micrograph of cells at the perimeter of an asymmetrically forming biofilm. Bar, 200 microns. Mat borders facing (red arrows) or not facing (blue arrows) another mat are indicated. <b>B</b>) Wild type (PC538), <i>flo</i>11Δ (PC1029), and <i>ste</i>12Δ (PC2382) cells were grown on YEPD media for 18 h. Equal concentrations of cells were spotted, 1 cm apart, on to YEPD media containing 0.3% agar. Plates were incubated for 48 h at 30°C and photographed using transmitted light. Bar = 1 cm. <b>C</b>) Bar graph of height measurements (in mm) of the mat borders facing/not facing the adjacent mats on the right in B. Contour maps in the Z-axis of mats was generated. Seven readings after the first peak in the Z-axis were averaged to plot the graph. Standard deviation between measurements were used to generate the error bars.</p

Model for the different mat responses controlled by the MAPK pathway.

<p>Schematic of a mat expanding under nutrient-limiting conditions is shown. Different responses regulated by the MAPK pathway may include: 1) mat expansion in the plane of the XY-axis (surface growth, through Flo11 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032294#pone.0032294-Reynolds1" target="_blank">[23]</a>), 2) cell differentiation that causes invasive growth in the Z-axis (downward growth, Flo11 and differentiation), and 3) upward growth in the plane of the Z-axis in response to surface rigidity and nutrient-limiting conditions (Flo11 and differentiation). This upward growth may represent a type of chemorepulsion. An extracellular matrix (ECM), which may contain shed Flo11 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032294#pone.0032294-Karunanithi1" target="_blank">[48]</a> as well as other proteins is depicted.</p

Genetic interactions between <i>HMT1</i> and genes encoding Rpd3L complex components.

<p><b>A)</b> Spot assay of 10-fold serially diluted haploid cells with single Rpd3 complex component deletion (+Kan) or double mutant selection (+Kan+Nat) resulting from mating with either <i>HMT1</i> or Δ<i>hmt1</i> query strains. <b>B)</b> Growth of haploid, double-drug resistant strains produced from matings between strains deleted for genes encoding components of the Rpd3L complex, or components specific for Rpd3S complex (Δ<i>eaf3</i> and Δ<i>rco1</i>), with either <i>HMT1</i> or Δ<i>hmt1</i> query strains. <b>C)</b> Tabulated results of growth differences for Rpd3 complex components from genome-wide SGA screen.</p

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

Hmt1 loss-of-function mutants display various changes in the occupancy of acetyl-K5 and -K16 of histone H4 at telomeric boundary regions.

<p>Directed ChIPs using anti-acetyl-H4K5 antibody <b>(part A)</b>, or anti-acetyl-H4K16 antibody <b>(part B)</b> in wild-type, Δ<i>hmt1,</i> and <i>hmt1(G68R)</i> cells. Primer sets used for this analysis were the same as those used in Fig. 3. Bars represent the experimental signals normalized to signal for the highly transcribed control, <i>ACT1.</i> Error bars represent standard deviation of three biological samples (n = 3) per genotype, and asterisks denote <i>p-</i>value of 0.05 by Student’s t-Test.</p

Epistatic analysis of silencing in Hmt1 and Rpd3 mutants.

<p><b>A)</b> Telomeric Silencing Assay comparing Δhmt1/Δrpd3 to either Δhmt1 or Δrpd3 single mutant. <b>B)</b> Sir2 occupancy across the telomeric boundary region in Δhmt1, Δrpd3, or Δhmt1/Δrpd3 mutants. ChIP was performed using anti-Sir2 antibody to immunoprecipitate Sir2 from Δhmt1, Δrpd3, and Δhmt1/Δrpd3 cells. Primer sets are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044656#pone-0044656-g003" target="_blank">Fig. 3</a>. Bars represent the experimental signal normalized to the GAL1 ORF. Error bars represent standard deviation of three biological samples (n = 3) per genotype, and asterisks denote p-value of 0.05 by Student’s t-Test.</p

Recruitment of Rpd3 to the Telomere Depends on the Protein Arginine Methyltransferase Hmt1

<div><p>In the yeast <em>Saccharomyces cerevisiae</em>, the establishment and maintenance of silent chromatin at the telomere requires a delicate balance between opposing activities of histone modifying enzymes. Previously, we demonstrated that the protein arginine methyltransferase Hmt1 plays a role in the formation of yeast silent chromatin. To better understand the nature of the Hmt1 interactions that contribute to this phenomenon, we carried out a systematic reverse genetic screen using a null allele of <em>HMT1</em> and the synthetic genetic array (SGA) methodology. This screen revealed interactions between <em>HMT1</em> and genes encoding components of the histone deacetylase complex Rpd3L (large). A double mutant carrying both <em>RPD3</em> and <em>HMT1</em> deletions display increased telomeric silencing and Sir2 occupancy at the telomeric boundary regions, when comparing to a single mutant carrying Hmt1-deletion only. However, the dual <em>rpd3/hmt1</em>-null mutant behaves like the <em>rpd3</em>-null single mutant with respect to silencing behavior, indicating that <em>RPD3</em> is epistatic to <em>HMT1</em>. Mutants lacking either Hmt1 or its catalytic activity display an increase in the recruitment of histone deacetylase Rpd3 to the telomeric boundary regions. Moreover, in such loss-of-function mutants the levels of acetylated H4K5, which is a substrate of Rpd3, are altered at the telomeric boundary regions. In contrast, the level of acetylated H4K16, a target of the histone deacetylase Sir2, was increased in these regions. Interestingly, mutants lacking either Rpd3 or Sir2 display various levels of reduction in dimethylated H4R3 at these telomeric boundary regions. Together, these data provide insight into the mechanism whereby Hmt1 promotes the proper establishment and maintenance of silent chromatin at the telomeres.</p> </div