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SIR-2.4, but not SIR-2.1, is required for stress-induced DAF-16 nuclear localization.

<p>TJ356 animals carrying an integrated <i>daf-16::gfp</i> array were fed either vector control or <i>sir-2.4</i> RNAi bacteria for at least one generation before being subjected to heat-shock or oxidative stress. (A) Images of TJ356 animals grown on control or <i>sir-2.4</i> RNAi bacteria after 15 min heat-shock. (B) Quantification of DAF-16::GFP nuclear accumulation in response to heat-shock (35°C for 15 min.) or oxidative stress (1.5 mM H₂O₂ for 1 hr). Worms were scored for the presence or absence of GFP accumulation within the intestinal nuclei (n = 120 or greater for all treatments). An animal was scored as having nuclear GFP if one or more intestinal nuclei contained DAF-16-GFP. (C–D) Time course analysis of DAF-16::GFP nuclear accumulation in response to stress. TJ356 or EQ200 [<i>sir-2.4(n5137)</i>; <i>daf-16::gfp</i>] animals grown on either control or <i>sir-2.1</i> RNAi bacteria were subjected to (C) heat-shock (35°C) or (D) oxidative stress (1.5 mM H₂O₂). Worms were scored for GFP accumulation within the head hypodermic nuclei at day 1 of adulthood (n = 30∼50) every 5–30 min.</p

SIR-2.4 is required for optimal DAF-16–dependent gene expression.

<p>Wild-type N2 animals fed on either vector control or <i>sir-2.4</i> RNAi bacteria from the time of hatching were exposed to 10 mM H₂O₂ for 80 min. Relative mRNA levels of SOD-3, HSP-16.1, DOD-3, DOD-24, C32H11.4, and INS-7 were measured by quantitative RT-PCR and the means of three different sample sets are shown. Relative mRNA levels were normalized against ACT-1 (beta-actin). Error bars: ± STD. Statistical significance as determined by two-tailed t-test is shown in the table below; significant differences are represented in black font.</p

SIR-2.4 inhibits CBP1-mediated DAF-16 acetylation.

<p>(A) DAF-16 nuclear localization was assessed in TJ356 (expressing <i>daf-16::gfp</i> in WT background) or EQ200 (expressing <i>daf-16::gfp</i> in <i>sir-2.4(n5137)</i> background) animals. Animals (n = 90 or greater) were scored for DAF-16::GFP nuclear translocation as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002948#pgen-1002948-g004" target="_blank">Figure 4B</a>. (B) <i>cbp-1</i> KD decreases DAF-16 acetylation. DAF-16 acetylation was assessed in TJ356 worms grown on either control or <i>cbp-1</i> RNAi by acetyl-lysine immunoprecipitation followed by GFP immunoblot. (C) SIR-2.4 blocks CBP1-dependent DAF-16 acetylation <i>in vitro</i>. Purified DAF-16 was incubated with CBP in the presence of WT SIR-2.4 or the SIR-2.4 NA mutant at 37°C. DAF-16 acetylation levels were assessed as described in (B).</p

Minimal impact of SIR-2.4 on IIS-induced longevity, DAF-16 nuclear localization induced by reduced IIS, and dauer formation.

<p>(A) Lifespan analysis of wild-type (N2) animals or <i>daf-2(e1370)</i> mutants grown on vector control bacteria (black or red) or <i>sir-2.4</i> RNAi bacteria (green or blue) at 20°C. (B) DAF-16 nuclear localization was assessed in TJ356 (expressing <i>daf-16::gfp</i> in WT background) or EQ200 (expressing <i>daf-16::gfp</i> in <i>sir-2.4(n5137)</i> background) animals. Animals were fed with either vector control or <i>daf-2</i> RNAi from the time of hatching. Worms were scored for the presence or absence of GFP accumulation within the head hypodermic nuclei as day 1 adult (n = 116 or greater) under unstressed condition. <i>P</i>-values were calculated by Pearson's chi-square test. (C) <i>daf-2(e1370)</i> mutants (P₀) were fed with control or <i>sir-2.4</i> RNAi bacteria at 20°C. F₁ eggs were then moved to 22°C for 72 hours prior to being scored for dauer formation (n = 336 or greater). <i>P</i>-values were calculated by Pearson's chi-square test.</p

SIR-2.4 interacts with DAF-16 and promotes DAF-16 deacetylation and function independently of catalytic activity.

<p>(A) <i>sir-2.4</i> deletion promotes DAF-16 hyperacetylation. DAF-16 acetylation was assessed in control or <i>sir-2.4</i> KO worms by acetyl-lysine immunoprecipitation followed by GFP immunoblot. (B) SIR-2.4 and DAF-16 interact. Plasmids encoding FLAG-tagged SIR-2.4 and HA-tagged DAF-16 were transfected into 293T cells as indicated (GFP, negative control). Immunoprecipitation and immunoblotting were performed as shown. (C) Rescue of DAF-16 nuclear localization with a catalysis-defective <i>sir-2.4</i> mutant. Stable transgenic strains of <i>sir-2.4(n5137)</i> were generated expressing either wild-type SIR-2.4 or the <i>sir-2.4</i> N124A mutant. Worms were scored for GFP accumulation within the head hypodermic nuclei as day 1 adult (n = 50 or greater) after 20 min of heat-shock at 35°C. <i>P</i>-values were calculated by Pearson's chi-square test.</p

Model: SIR-2.4 promotes DAF-16 deacetylation and function during stress.

<p>See text for details.</p

SIR-2.4 promotes stress resistance.

<p>(A) Wild-type N2 or <i>sir-2.4(n5137)</i> worms grown on vector control or <i>daf-16</i> RNAi bacteria were subjected to heat-shock at 35°C and scored for viability every 1-2 hours. (B) Wild-type N2 or <i>daf-16(mu86)</i> worms grown on vector control or <i>sir-2.4</i> RNAi bacteria were treated with 1.5 mM H₂O₂ and scored for viability every 1–2 hours. Data are mean survival±SEM, in hours for (A–B). ***, <i>p</i><0.001; ns, <i>p</i>>0.05. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002948#pgen.1002948.s006" target="_blank">Table S1</a> for statistical analysis. (C) AM140 worms expressing a poly-Q tract (Q35::YFP) were seeded on the RNAi bacteria indicated and scored for poly-Q induced paralysis every other day. Data are mean time to paralysis in days±SEM. ***, <i>p</i><0.001; **, <i>p</i><0.01; ns, <i>p</i>>0.05.</p