Figure 1(A) Schematic representation of ChIP-Seq strategy. An antibody against GFP was used to immunoprecipitate HIS-72::GFP loaded onto chromatin. Subsequent DNA sequencing was performed onto immunoprecipitated chromatin. (B) Venn diagram of genes with H3.3-enriched promoters between wt (blue), gas-1(fc21) (red) and nuo-6(qm200) (green) mutants. (C) Heatmap of genes with H3.3-enriched promoters exclusively in nuo-6(qm200) mutants, downregulated in gas-1(fc21) vs wt, upregulated in nuo-6(qm200) vs wt and downregulated in nuo-6(qm200); his-72(tm2066); his-71(ok2289) vs nuo-6(qm200). (D) Table of genes with H3.3-enriched promoters exclusively in nuo-6(qm200) nematodes, whose gene expression was upregulated in nuo-6(qm200) vs wt, downregulated in gas-1(fc21) vs wt and downregulated in nuo-6(qm200); his-72(tm2066); his-71(ok2289) vs. nuo-6(qm200). Human orthologs and corresponding % homology are indicated. (E) qRT-PCR of cest-2.2 in wt, gas-1(fc21) and nuo-6(qm200) mutants (one-way ANOVA with Dunnet's post hoc correction, n=9-12 technical replicates pooled from 3 independent experiments). Data information: mean± SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (F) qRT-PCR of cest-2.2 mRNA expression in nuo-6(qm200) animals grown on control and cest-2.2 RNAi (unpaired t-test, n=3 biological replicates). Data information: mean± SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (G) Representative survival curves of nuo-6(qm200) mutants fed with control (C RNAi) and dsRNA against cest-2.2. Data information: , the median lifespan ± SEM is reported underneath the graphs and additional information (e.g. n numbers) (H) qRT-PCR of cest-2.2 mRNA expression in wt animals grown on control and cest-2.2 RNAi (unpaired t-test, n=3 biological replicates). Data information: mean± SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (I) Lifespan assay of wt animals grown on control and cest-2.2 RNAi. Data information: the median lifespan ± SEM is reported underneath the graphs and additional information (e.g. n numbers) (J) Representative survival curves of wt and cest-2.2 (lf) nematodes. (M) Representative survival curves of wt and cest-2.2(lf) nematodes. Data information: the median lifespan ± SEM is reported underneath the graphs and additional information (e.g. n numbers) (K) qRT-PCR of cest-2.2 mRNA expression in wt, gas-1(fc21), gas-1(fc21); cest-2.2 O/E and cest-2.2 (lf) nematodes (one-way ANOVA with Dunnett's post-hoc correction, n=3 biological replicates). Data information: , mean± SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (L) SureQuant-based quantitative mass spectrometry analysis of CEST-2.2 protein levels. Light/heavy ratios (L/H) of the two unique CEST-2.2 peptides (LSTGTIEGR and HGEDFAYAFGTNR) used for quantitation are represented as violin plots (n=3 for wt and gas-1; cest-2.2 O/E, n=2 for gas-1; the n of analyzed samples per group refers to biological replicates of pooled nematodes from a single plate; the median is indicated with a dashed line). Statistical analysis was performed using the nonparametric t-test. Data information: , middle lines represents the median, and the upper and lower lines represent the upper and lower quartiles, respectively Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (N) Lifespan assay of wt, gas-1(fc21), gas-1(fc21); cest-2.2 O/E and cest-2.2(lf); gas-1(fc21); cest-2.2 O/E mutant nematodes. Data information: the median lifespan ± SEM is reported underneath the graphs and additional information (e.g. n numbers) (O) Representative lifespan assay of wt and gas-1(fc21); cest-2.2 O/E mutant animals grown on control (solid lines) and cest-2.2 (dashed lines) RNAi bacteria from hatching. Data information: the median lifespan ± SEM is reported underneath the graphs and additional information (e.g. n numbers) (P) Quantification of body area of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes, normalized to wt (one-way ANOVA with Tukey's multiple comparison test, n=18-30 biological replicates). Data information: , middle lines represents the median, and the upper and lower lines represent the upper and lower quartiles, respectively; Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (Q) Locomotor activity (arbitrary unit) of 10-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutants (one-way ANOVA with Dunnett's post hoc correction, n=8). Data information: , middle lines represents the median, and the upper and lower lines represent the upper and lower quartiles, respectively Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (R) Number of pharyngeal pumps over 60 seconds of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes (one-way ANOVA with Tukey's multiple comparison test, n=10 biological replicates). Data information: , mean± SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (S) Total number of eggs (left panel) and number of eggs laid each day (right panel) since hatching by wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes (ordinary one-way ANOVA with Tukey's multiple comparisons test, n=8-12 biological replicates). Data information: mean± SEM points indicate mean± SEM. Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.