Enhancement of <i>pha-4(ts)</i> by <i>mys-1, ssl-1,</i> and <i>htz-1</i>

<div><p>(A) Feeding dsRNA to wild-type (WT) or <i>pha-4(ts)</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020161#pgen-0020161-b018" target="_blank">18</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020161#pgen-0020161-b025" target="_blank">25</a>] worms at the permissive temperature of 24 °C. WT worms generate viable progeny with <i>mys-1, ssl-1</i> or <i>htz-1</i> RNAi. In the <i>pha-4(ts)</i> background, L1 arrest increased with <i>mys-1, ssl-1,</i> or <i>htz-1</i> RNAi compared to control <i>GFP(RNAi)</i> (grey bars). Embryonic lethality remained unchanged (black bars). Effectiveness of RNAi feeding was manifest through viable, but sterile, progeny for <i>mys-1</i> and <i>ssl-1</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020161#pgen-0020161-b014" target="_blank">14</a>], as well as repeated enhancement of L1 lethality for <i>pha-4(ts),</i> performed in parallel. <i>n</i> = 100 worms/plate, three plates per column. Error bars indicate the standard deviation.</p><p>(B) Alignment of <i>C. elegans htz-1</i> (R08C7.3) with human H2A.Z, yeast Htz1, and one of the core H2A genes from yeast, Hta1. Extended acid patch region essential for H2A.Z function is indicated by the bar [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020161#pgen-0020161-b050" target="_blank">50</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020161#pgen-0020161-b060" target="_blank">60</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020161#pgen-0020161-b067" target="_blank">67</a>].</p></div

<i>htz-1</i> Influences the Onset of Late Pharyngeal Gene Expression

<div><p>(A) Onset of <i>R07B1.9::GFP</i> and <i>myo-2::</i>GFP after the comma stage for wild-type (black) or <i>htz-1(RNAi)</i> (grey) embryos. Activation of the <i>R07B1.9</i> reporter was delayed after injection of <i>htz-1</i> dsRNA (<i>p</i> < 0.0001, Student <i>t</i>-test). No RNAi: <i>n</i> = 18, <i>htz-1</i> RNAi: <i>n</i> = 12 embryos. <i>myo-2</i> activation was scored in embryos carrying either a wild-type <i>myo-2</i>::GFP reporter (WT) or a <i>myo-2::</i>GFP reporter with both PHA-4 binding sites mutated (Mut) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020161#pgen-0020161-b018" target="_blank">18</a>]. Activation of the WT reporter was delayed after injection of <i>htz-1</i> dsRNA (<i>p</i> < 0.0001, Student <i>t</i>-test), whereas onset of the Mut reporter was unchanged (<i>p</i> = 0.2068). No RNAi: <i>n</i> = 15 WT, <i>n</i> = 33 Mut Embryos; <i>htz-1</i> RNAi: <i>n</i> = 9 WT, <i>n</i> = 21 Mut embryos. Error bars indicate the standard deviation.</p><p>(B) Expression of <i>R07B1.9::GFP</i> at 90 and 180 min after the comma stage with <i>htz-1</i> RNAi or without (No RNAi). Pharyngeal <i>R07B1.9::GFP</i> is indicated by arrowheads.</p><p>(C) Expression of <i>myo-2</i>::GFP (WT) at 150, 200, and 250 min after the comma stage with <i>htz-1</i> RNAi or without (No RNAi).</p><p>(D) Expression of <i>myo-2</i>::GFP lacking PHA-4 binding sites [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020161#pgen-0020161-b018" target="_blank">18</a>] at 150, 200, 250 min after the comma stage with <i>htz-1</i> RNAi or without (No RNAi).</p></div

YFP::HTZ-1 Association Peaks at the Onset of <i>myo-2</i> Expression

<div><p>(A) Association of YFP::HTZ-1 with the <i>myo-2</i> promoter in the pharynx during the comma, 1.5-, 2-, and 3-fold stages of development (two trials: black, dark grey). YFP::HTZ-1 association in the pharynx does not peak at the 2-fold stage for a <i>myo-2</i> promoter with both PHA-4 binding sites mutated (Mut, light grey). <i>n</i> > 60 embryos imaged at each stage, for each experiment. Multiple lines were used for each trial.</p><p>(B) Pharyngeal YFP::HTZ-1 association with <i>myo-2</i> target array #1 decreased at the 2-fold stage when <i>pha-4</i> activity was reduced by RNAi<i>. n</i> > 20 embryos imaged at each stage for each of the two wild-type (WT) and two <i>pha-4(RNAi)</i> experiments.</p><p>The <i>p</i>-values for (A) and (B) indicate the significance of the WT peak at the 2-fold stage as calculated by a repeated measures analysis of variance (ANOVA).</p></div

Specificity of <i>mys-1, ssl-1,</i> and <i>htz-1</i> Synergy with <i>pha-4(ts)</i>

<p>The indicated worm strains were fed dsRNA for GFP (negative control) or <i>mys-1, ssl-1,</i> or <i>htz-1</i> at 20 °C (or 24 °C for WT and <i>pha-4(ts)</i>)<i>.</i> Lethal embryos (black bars) or lethal L1 progeny (grey bars) were scored for each strain. Effectiveness of RNAi feeding was manifest through viable, but sterile, progeny for <i>mys-1</i> and <i>ssl-1</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020161#pgen-0020161-b014" target="_blank">14</a>], as well as repeated enhancement of L1 lethality for <i>pha-4(ts). n</i> =1 00 worms/plate, three plates per column. Error bars indicate the standard deviation.</p

<i>htz-1</i> Influences the Onset of Early PHA-4–Dependent Expression

<div><p>(A) Onset of <i>3XPRE::GFP</i> after the two-cell stage for wild-type (black) or <i>htz-1(RNAi)</i> (grey) embryos<i>. 3XPRE::GFP</i> is a reporter construct with three copies of a high-affinity PHA-4 response element upstream of the <i>Δpes-10</i> promoter that reproducibly activates early pharyngeal expression [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020161#pgen-0020161-b019" target="_blank">19</a>]. Activation of the <i>3XPRE::GFP</i> reporter is delayed after injection of <i>htz-1</i> dsRNA (<i>p</i> = 0.0289, Student <i>t</i>-test). No RNAi: <i>n</i> = 23, <i>htz-1</i> RNAi: <i>n</i> = 9 embryos. Error bars indicate the standard deviation.</p><p>(B) Expression of <i>3XPRE::GFP</i> at 150 and 200 min after the two-cell stage with <i>htz-1</i> RNAi or without (No RNAi).</p></div

Summary and Model: HTZ-1 Synergizes with PHA-4 to Establish the Foregut

<div><p>(A) Summary of the data presented in this paper and in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020161#pgen-0020161-b018" target="_blank">18</a>]. <i>myo-2</i> expression initiates at the 2-fold stage, and onset at this stage requires <i>pha-4</i> and <i>htz-1</i> because mutation of PHA-4 binding sites (Mut) or <i>htz-1</i> RNAi lead to a delay in <i>myo-2</i> activation. HTZ-1 association with the <i>myo-2</i> promoter peaks at the onset of <i>myo-2</i> transcription, and this association requires <i>pha-4</i>.</p><p>(B) Model to explain how HTZ-1 synergizes with PHA-4. PHA-4 association with the <i>myo-2</i> promoter leads to exchange of H2A-containing nucleosomes for one or more nucleosomes carrying HTZ-1/H2A.Z at the 2-fold stage. Based on data from other organisms [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020161#pgen-0020161-b001" target="_blank">1</a>], we propose MYS-1 and/or SSL-1 function in a complex that performs the exchange reaction. HTZ-1–containing nucleosomes promote transcriptional activation by the 2-fold stage.</p></div

<i>mys-1, ssl-1,</i> and <i>htz-1</i> Enhance Pharyngeal Defects of <i>pha-4(ts)</i>

<div><p>(A–C) Feeding dsRNA to <i>pha-4(ts)</i> worms at the permissive temperature of 24 °C.</p><p>(A) A <i>pha-4(ts); GFP(RNAi)</i> L1 with a wild-type pharynx (arrowheads).</p><p>(B) A <i>pha-4(ts); ssl-1(RNAi)</i> L1 with an unattached pharynx (arrowheads).</p><p>(C) Quantitation of <i>pha-4(ts)</i> animals exhibiting a normal pharynx (WT), an unattached or incomplete pharynx (Pun), or no detectable pharynx. <i>htz-1, ssl-1,</i> or <i>mys-1</i> RNAi significantly increased the number of Pun <i>pha-4(ts)</i> animals (<i>htz-1: p</i>=0.0290; <i>ssl-1</i> and <i>mys-1: p</i> < 0.0001, Fisher exact test).</p><p>(D–F) Feeding dsRNA to <i>pha-4(ts)</i> animals at the intermediate temperature of 20 °C.</p><p>(D) A <i>pha-4(ts); GFP(RNAi)</i> L1 at the intermediate temperature of 20 °C with a morphologically wild-type pharynx (arrowheads).</p><p>(E) A <i>pha-4(ts); htz-1(RNAi)</i> worm at 20 °C missing a detectable pharynx.</p><p>(F) Quantitation of <i>pha-4(ts)</i> animals exhibiting a normal pharynx (WT), an unattached or incomplete pharynx (Pun), or no detectable pharynx. <i>htz-1, ssl-1,</i> or <i>mys-1</i> RNAi significantly increased the number of worms with no detectable pharynx (<i>htz-1</i> and <i>ssl-1: p</i> < 0.0001; <i>mys-1: p</i> = 0.0015; Fisher exact test). WT worms with reduced <i>htz-1, ssl-1,</i> or <i>mys-1</i> activity had a wild-type pharynx at 24 °C and 20 °C (unpublished data). RNAi was conducted by feeding dsRNA [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020161#pgen-0020161-b029" target="_blank">29</a>].</p></div