Identification of a slr-2-responsive motif. (A) Logogram of a 11-nucleotide motif identified by K-means clustering and MEME that was strongly enriched in multiple clusters of slr-2-responsive genes. (B) Seventeen examples of high-scoring genes that contain the consensus site along with their fold changes in slr-2 mutants. (C) Location of the motif within promoter regions shows a bias towards transcriptional start sites, and the motif is found in multiple copies in some genes.

The ESRE motif confers SLR-2–JMJC-1-dependent stress-induced activation. (A) A cartoon representation of the 3X-ESRE::GFP construct. (B–C) DIC (B) and GFP (C) photomicrograph of a wild-type (N2) embryo containing 3X-ESRE::GFP along with the rol-6(gf) marker that has been subjected to a 30°C heat shock. (D–E) DIC (D) and GFP (E) photomicrograph of slr-2(ku297) mutant embryo carrying the same array as depicted in (B–C) subjected to a 30°C heat shock. (F) Background-normalized quantification of fluorescence in heat-shocked, wild-type, slr-2(ku297), and jmjc-1(tm3525) embryos carrying an identical 3X-ESRE [rol-6(gf)] array subjected to a 30°C heat shock. (G–J) DIC (G), GFP (H), RFP (I), and GFP/RFP-merged (J) photomicrographs of a wild-type (N2) embryo carrying 3X-ESRE::GFP and the sur-5::RFP marker that has been subjected to a 30°C heat-shock. (K) Table showing relative frequencies of GFP expression in lines with 3X-ESRE::GFP, or a control without the 3X-ESRE, at 20 or 30°C. In (F), all images were taken using the same gain and exposure time; asterisks indicate statistical significance, P< 0.01, n=40–50. Error bars in (F) represent standard deviation (s.d.). Scale bar: 10 μm in panels (B–E) and (G–J).

SLR-2 and JMJC-1 are integral to survival under stress conditions. (A) After exposure to a 12 h lethal heat shock (37°C), both slr-2(ku297) and jmjc-1(tm3525) mutants showed reduced survival relative to wild type (N2; P<0.001, n=300). (B) slr-2 and jmjc-1 mutants displayed diminished survival under conditions of severe oxidative stress (160 μM juglone; P<0.001, n=250). (C) When exposed to brief (30 min), acute ethanol, or hypertonic stresses, slr-2 and jmjc-1 mutants showed reduced recovery after 30 min (asterisks, P<0.001, n=200).

ESRE network conservation in Drosophila. (A) When exposed to a 16 h lethal heat shock at 38°C, CG2982 mutant flies (w¹¹¹⁸P (EP) CG2982^EP1316) showed dramatically reduced survival as compared with wild-type (w¹¹¹⁸) controls (average of three biological replicates, P<0.001; n=115). (B) Logogram and locations of the ESRE motif in Drosophila genes tested in (C). (C) qRT–PCR data are shown from w¹¹¹⁸P (EP) CG2982^EP1316 and w¹¹¹⁸ control populations subjected to a 4 h, non-lethal 35°C heat shock. Note that CG2982 was upregulated after heat shock in the control strain, but its expression was nearly abolished in the strain carrying the P-element insertion. Furthermore, expression of ESRE genes was attenuated in CG2982 mutants. Light grey bars represent expression of genes in CG2982 mutants normalized to unstressed wild-type controls; grey bars, expression of genes in heat-shocked wild-type flies normalized to unstressed wild-type controls; mixed bars, gene expression in heat-shocked, CG2982 mutants normalized to either unstressed, wild-type controls (dark portion) or unstressed mutants (light portion). In (C), solid and dashed lines represent 1- and 1.5-fold change, respectively, and error bars represent s.e.m. Additional statistical information for (C) is available in Supplementary Table S1F.

Heat shock induction of ESRE genes is attenuated in slr-2 and jmjc-1 mutants. (A) 17 genes selected for qRT–PCR analysis in slr-2(ku297) and jmjc-1(tm3525) mutants on the basis of expression level and match to ESRE consensus motif. (B) After a 12 h exposure to a 30°C, non-lethal heat shock, ESRE target genes showed an upregulation in N2 and a reduction in the levels of induction in slr-2(ku297) mutants. Light grey bars represent expression of genes in unstressed slr-2(ku297) mutants normalized to unstressed wild-type (N2) controls, grey bars represent expression of genes in heat-shocked wild-type (N2) worms normalized to unstressed, wild-type controls, and mixed bars represent gene expression in heat-shocked slr-2(ku297) mutants normalized to either unstressed wild-type (N2) controls (dark portion) or unstressed slr-2(ku297) mutants (light portion). (C) After a 12 h exposure to a 30°C non-lethal heat shock, ESRE target genes showed a attenuation of upregulation in jmjc-1(tm3525) mutants. Light grey bars represent expression of genes in jmjc-1(tm3525) mutants normalized to unstressed wild-type (N2) controls, grey bars represent expression of genes in heat-shocked wild-type (N2) worms normalized to unstressed, wild-type controls, and mixed bars represent gene expression in heat-shocked jmjc-1(tm3525) mutants normalized to either unstressed wild-type controls (dark portion) or unstressed jmjc-1(tm3525) mutants (light portion). (D) After a 12 h exposure to a 30°C non-lethal heat shock, most ESRE target genes showed upregulaltion in daf-16(mu86) mutants. Light grey bars represent expression of genes daf-16(mu86) normalized to unstressed wild-type (N2) controls, grey bars represent expression of genes in heat-shocked wild-type (N2) worms normalized to unstressed, wild-type controls, the white bars represent gene expression in heat-shocked daf-16(mu86) mutants normalized to unstressed daf-16(mu86), and dark grey bars represent gene expression in heat-shocked daf-16(mu86) mutants normalized to unstressed wild-type. In (B–D), data are the means of three biological replicates in which each replicate measured in triplicate; error bars represent s.e.m. and solid and dashed lines represent 1- and 1.5-fold change, respectively. Additional statistical information for (B–D) is available in Supplementary Table S1A–C.

slr-2 is responsive to heat shock in a time-dependent manner. (A) GFP fluorescence of a P_slr-2::GFP transcriptional fusion reporter expression in response to a 30°C non-lethal heat shock over a 12 h period. Enhanced expression was observed by 1 h and increased steadily for at least 12 h. (B) Both slr-2 mRNA transcription (black, measured by qRT–PCR) and a P_slr-2::GFP transcriptional fusion reporter (grey, measured by fluorescence) displayed nearly identical, time-dependent increases in response to 30°C heat-shock over a 12 h period. Unstressed slr-2 controls were used for normalization for qRT—PCR. (C) Quantification of P_slr-2::GFP fluorescence intensities at 4 and 12 h after exposure to non-lethal hypertonic, ethanol, and oxidative stresses. In (B) and (C), asterisks indicate P<0.01. In (B), plus sign indicates P<0.05. Error bars in (B) and (C) represent standard error of the mean (s.e.m.). In (B) and (C), all images were taken using the same gain and exposure time (n∼50).

SLR-2–JMJC-1 overexpression and epistasis analysis. (A) qRT–PCR data for slr-2 and jmjc-1 from heat-shocked slr-2(ku297), jmjc-1(tm3525), and wild-type (N2) strains. Note that full induction of jmjc-1 requires wild-type slr-2, whereas slr-2 expression was not dependent on jmjc-1. Asterisks in (A) indicate P<0.01 as compared with non-stressed genotypic cohorts; hash sign in (A) indicates attenuation of upregulation of jmjc-1 in stressed slr-2 mutants as compared with stressed wild type (P<0.01). (B–C) Wild-type (N2), slr-2(ku297), and jmjc-1(tm3525) worms carrying either no extra-chromosomal array or arrays containing multiple copies of either slr-2 or jmjc-1 were subjected to a 12 h ethanol (induced by 4% ethanol-supplemented NGM) (B) or oxidative (induced by 160 mM juglone-supplemented NGM) (C) stress and scored for paralysis. Asterisks in (B) and (C) indicate statistical significance, P< 0.001. Each bar in (B) and (C) represents data for 150 animals. Although all three strains showed significant rescue with the extra-chromosomal array encoding jmjc-1, the slr-2 extra-chromosomal array only showed rescue or enhanced survival in slr-2 and wild-type (N2) strains, further indicating that SLR-2 functions upstream of JMJC-1. (D) Survival curves of wild type (N2), slr-2(ku297), and jmjc-1(tm3525) with and without the slr-2 high-copy extra-chromosomal array. Worms were placed at 20°C on NGM plates and scored every other day for survival. In N2 and slr-2 strains, the slr-2 extra-chromosomal array conferred a modest, but statistically significant, increase in lifespan (P<0.001).

ESRE network conservation in mammals. (A) qRT–PCR data for NO66 expression are shown from IEC-6, 3T3, LAD-II, and C4-2 cell lines subjected to a 12 h treatment with 70 mM NaCl or 200 mM ethanol. (B) qRT–PCR data for expression of ESRE-containing mammalian orthologs of C. elegans ESRE genes using 70 mM NaCl or 200 mM ethanol-stressed LAD-II cells. (C) qRT–PCR data are shown from C4-2 cells transfected with an siRNA construct targeting NO66 or a control siRNA for 48 h followed by an additional 12 h treatment with 200 mM ethanol. Light grey bars represent expression of genes in unstressed, NO66 siRNA-treated cells normalized to unstressed cells transfected with a control siRNA; grey bars, expression of genes in ethanol-stressed cells transfected with control siRNA normalized to non-stressed control siRNA; mixed bars, gene expression in ethanol-stressed NO66 siRNA-treated cells normalized to either ethanol-stressed cells transfected with a control siRNA (dark portion) or to non-stressed NO66 siRNA-treated cells (light portion). In (B) and (C), dashed lines represent 1.5-fold. In (C), solid line represents 1.0 –fold. Error bars in (A—C) represent s.e.m. In (A) and (B), all genes assayed showed significant upregulation (P<0.05) after ethanol and salt stress as compared with levels in non-stressed controls. Additional statistical information for (C) is available in Supplementary Table S1G.