Supplemental Material for Bowitch et al., 2018

<b>Figure S1</b><br><div><b><br></b></div><div>Supplemental Figure (sequence alignment) as described in Discussion</div

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

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

Synthetic Genetic Array Analysis of <i>HMT1</i>.

<p><b>A)</b> HMT1 <i>genetic interaction network.</i> Physical interactions between <i>HMT1</i> and all of its genetic interactors identified from this study using the Synthetic Genetic Array methodology. The product of the gene identified is indicated within the relevant circle. The blue line represents a previously identified physical interaction. The complete genetic interaction network was created using Cytoscape, and the physical interaction data were obtained from the <i>Saccharomyces</i> Genome Database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044656#pone.0044656-Shannon1" target="_blank">[55]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044656#pone.0044656-Cherry1" target="_blank">[56]</a>. <b>B)</b><i>GO terms for the</i> Δ<i>hmt1 SGA interaction data set reveals enrichment for components of the Rpd3(L) complex</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044656#pone.0044656-Ashburner1" target="_blank">[57]</a>. Overrepresented GO terms and corresponding adjusted p-values were determined using FuncAssociate 2.0. GO term attributes are listed based on ascending adjusted <i>p</i>-values <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044656#pone.0044656-Berriz1" target="_blank">[40]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044656#pone.0044656-Berriz2" target="_blank">[58]</a>.</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

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

Yeast strains used in this study.

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

Rpd3 occupancy at telomeric boundary regions is increased in the Hmt1 loss-of-function mutants.

<p><b>A)</b> Schematic representation of telomere VI-R and the quantitative PCR primer sets (A–E) for loci examined by ChIP in this study. <b>B)</b><i>Rpd3 occupancy across the telomeric boundary region in Hmt1 loss-of-function mutants.</i> ChIP was performed using anti-Myc antibody to immunoprecipitate myc-tagged Rpd3 from wild-type, Δ<i>hmt1,</i> and <i>hmt1(G68R)</i> cells. Bars represent the experimental signal normalized to signal from a non-transcribed intergenic region (“(–) Ctrl”). 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

Oligonucleotides used for chromatin immunoprecipitation assay in this study.

<p>Oligonucleotides used for chromatin immunoprecipitation assay in this study.</p

The <i>C. elegans</i> cGMP-Dependent Protein Kinase EGL-4 Regulates Nociceptive Behavioral Sensitivity

<div><p>Signaling levels within sensory neurons must be tightly regulated to allow cells to integrate information from multiple signaling inputs and to respond to new stimuli. Herein we report a new role for the cGMP-dependent protein kinase EGL-4 in the negative regulation of G protein-coupled nociceptive chemosensory signaling. <i>C. elegans</i> lacking EGL-4 function are hypersensitive in their behavioral response to low concentrations of the bitter tastant quinine and exhibit an elevated calcium flux in the ASH sensory neurons in response to quinine. We provide the first direct evidence for cGMP/PKG function in ASH and propose that ODR-1, GCY-27, GCY-33 and GCY-34 act in a non-cell-autonomous manner to provide cGMP for EGL-4 function in ASH. Our data suggest that activated EGL-4 dampens quinine sensitivity via phosphorylation and activation of the regulator of G protein signaling (RGS) proteins RGS-2 and RGS-3, which in turn downregulate Gα signaling and behavioral sensitivity.</p></div