1. Volume 22, Issue 10, Pages 2716-2729 (March 2018)
Muscle-Specific Histone H3K36 Dimethyltransferase SET-18 Shortens Lifespan of Caenorhabditis elegans by Repressing daf-16a Expression 
Liangping Su, Hongyuan Li, Cheng Huang, Tingting Zhao, Yongjun Zhang, Xueqing Ba, Zhongwei Li, Yu Zhang, Baiqu Huang, Jun Lu, Yanmei Zhao, Xiaoxue Li 
Cell Reports 
Volume 22, Issue 10, Pages (March 2018)
DOI: /j.celrep
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2. Cell Reports 2018 22, 2716-2729DOI: (10.1016/j.celrep.2018.02.029)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1
Muscle-Specific Expression of SET-18 is Increased in Aging Worms
(A) The fold changes in mRNA expression levels of genes encoding known histone methyltransferases and proteins containing SET domain in old-aged worms. The changes in mRNA levels of selected genes in whole-worm extract of old worms (day 11) compared with those of young ones (day 3) were analyzed by RT-qPCR. Error bars represented the SEM. act-1 was used as an internal control. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p <
(B and C) The lifespan of wild-type worms (N2) was extended by set-18 RNAi (B), but not by knockdown of set-10, set-32, or F54F7.7 (C). control, empty vector. The representative examples of three independent experiments are shown in (B) and (C). See also Figure S1. All data on the three repeats and statistical analysis are in Table S1.
(D) The kinetics of set-18 mRNA level in N2 throughout young (day 0 and day 3) and old age (day 7 and day 11). Set-18 mRNA levels of N2 worms in day 3, day 7, and day 11 were measured and normalized by that level in day 0. Error bars represent the SEM. act-1 was used as an internal control. ∗p < 0.05.
(E and F) Muscle-specific expression of SET-18::GFP was gradually increased from day 3 to day 11. The whole-body photos of worms expressing SET-18::GFP driven by set-18 promoter were taken at 100 × magnification under confocal microscope with the identical exposure settings. Their heads, bodies, and tails were detected at 400× magnification, respectively. According to previous publication (An et al., 2005; Tullet et al., 2008), a triple-band emission filter set was used in conjunction with a narrow-band excitation filter (484/14 nm) to discriminate intestinal autofluorescence (yellow/orange) from SET-18::GFP epifluorescence (green). The representative images of SET-18::GFP expression are shown in (E). Arrows indicated SET-18::GFP distribution in muscle. The intensities of SET-18::GFP fluorescence expressed in pharyngeal muscle and body wall muscle (close to vulva) were quantitatively analyzed by Image-Pro Plus and shown in (F). Error bars represent the SEM. n, the number of worms for quantitative analysis. ∗∗p < 0.01; ∗∗∗p <
(G) Distribution of SET-18::GFP overlaid well with muscle marker MYO-3::mCherry. Plasmids Pset-18::GFP and muscle reporter marker Pmyo-3::mCherry were co-injected into N2. The fluorescence of GFP and mCherry were detected and merged by confocal microscope.
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4. Figure 2
SET-18 Shortens Lifespan and Decreases Oxidative Stress Resistance Depending on daf-16 Activity and IIS Pathway
(A) Set-18(gk334) mutants lived longer than N2. (A)–(O) show the representative examples of three independent experiments. All data on the three repeats and statistical analysis were supplemented in Table S2 (lifespan assays) and Table S3 (oxidative stress resistance assays).
(B and C) Loss of set-18 (B) and set-18 RNAi (C) increased the resistance against oxidative stress. t-butyl hydrogen peroxide (tBHP) treatment was utilized as oxidative stress according to previous publication (Li et al., 2017). control, empty vector.
(D–G) Recovery of SET-18::GFP expression driven by set-18 or myo-3 muscle-specific promoters attenuated increases in lifespan and oxidative stress defense of set-18(gk334) mutants. 1# and 2# were two independent extrachromosomal arrays created by co-injecting plasmids Pset-18::SET-18::GFP::3′UTRset-18 or Pmyo-3::SET-18::GFP::3′UTRset-18 with rol-6 marker (pRF4) into set-18(gk334) worms. (D and E) The lifespan assays. (F and G) The oxidative stress resistance assays.
(H and I) Loss of set-18 did not change the lifespan of daf-2(e1368) (I) and daf-2(e1370) (H) mutants with reduced IIS signal.
(J and K) Loss of set-18 did not alter the resistance of daf-2(e1368) (K) and daf-2(e1370) (J) mutants against oxidative stress.
(L and M) Loss of set-18 prolonged the lifespan of glp-1(e2141) (L) mutants lacking of germlines and eat-2(ad1116) (M) mutants with caloric restriction. glp-1(e2141) mutants were shifted to 25°C from L1 stage and re-shifted to 20°C from young adult for lifespan assays.
(N and O) set-18 RNAi did not change the lifespan and oxidative stress resistance of daf-16(mu86). (N) The lifespan assays. (O) The oxidative stress resistance assays. control, empty vector. See also Figure S2.
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5. Figure 3 SET-18 Is a Histone H3K36 Dimethyltransferase in C. elegans
(A) Histone H3 was methylated by GST-SET-18 directly. In vitro methylation assays were performed by incubating GST-SET-18 or GST with histone H3 in the presence of 3H-SAM. GST was used as the negative control. The 3H-labeled methylation of histone H3 was detected by autoradiography (upper). Coomassie brilliant blue (CBB) staining (lower) was used as a loading control.
(B and C) Loss of set-18 significantly downregulated global H3K36me2 levels. The global H3K36me2/3, H3K4me2/3, H3K27me2/3, and H3K9me2/3 levels in the whole-worm extracts of N2 and set-18(gk334) young adults were measured by western blots (B), followed by quantitative densitometry analysis based on at least three independent replicates (C). Histone H3 was used as the internal reference. Error bars represented the SEM. ns, no significant; ∗p < 0.05; ∗∗p < See also Figure S4A.
(D and E) Overexpression of SET-18::GFP rescued global H3K36me2 of set-18(gk334) to the levels of N2. 1# and 2# were two independent extrachromosomal arrays created by injecting plasmids Pset-18::SET-18::GFP::3′UTRset-18 with rol-6 marker (pRF4) into set-18(gk334). Global H3K36me2 levels were measured by western blots (D), followed by quantitative densitometry analysis based on at least three independent replicates (E). Histone H3 was used as the internal reference. Error bars represented the SEM. ns, no significant; ∗∗p < 0.01.
(F) GST-SET-18 dimethylated K36 on histone H3 in vitro in a dose-dependent manner. GST-tagged SET-18 proteins were expressed in bacteria and purified. Different amounts of GST-SET-18 (0, 3.5, 7, and 14 μg) were incubated with identical amount of histone H3 in the presence of SAM, followed by western blot experiments. Coomassie brilliant blue (CBB) staining was used as a loading control. The relative H3K36me2 level quantified by densitometry analysis was shown in Figure S4D. Histone H3 was used as the internal reference.
(G) The H3K36 dimethyltransferase activity of GST-SET-18 was decreased by Y245F and D247A single-site mutations on its conserved enzymatic domain. Wild-type (WT) and mutated (Y245F and D247A) GST-tagged SET-18 were expressed in bacteria, purified, and then incubated with histone H3 and SAM in the reaction system, respectively. The mutation sites are shown in Figure S3. Coomassie brilliant blue (CBB) staining was used as a loading control. The relative H3K36me2 level quantified by densitometry analysis was shown in Figure S4E. Histone H3 was used as the internal reference.
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6. Figure 4 Expression of daf-16 Is Repressed by SET-18
(A) The mRNA levels of upstream genes in IIS pathway were not changed by loss of set-18. The mRNA levels of daf-2, age-1, akt-1, and akt-2 in N2 and set-18(gk334) were measured by RT-qPCR. act-1 was used as an internal control. Error bars represented the SEM. ns, no significant.
(B) Loss of set-18 upregulated the mRNA levels of daf-16 and DAF-16 target genes. RT-qPCR experiments were conducted as in (A). Error bars represented the SEM. ns, no significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p <
(C) Rescue of SET-18::GFP expression attenuated the increases in mRNA levels of daf-16 and DAF-16 target genes of set-18(gk334) mutants. Two independent extrachromosomal arrays (1# and 2#) were created by injecting plasmids Pset-18::SET-18::GFP::3′UTR set-18 with rol-6 marker (pRF4) into set-18(gk334). RT-qPCR experiments were conducted as in (A). Error bars represented the SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p <
(D) Loss of set-18 increased DAF-16::GFP expression in the intestines and muscles. The DAF-16::GFP expression in N2 and set-18(gk334) at young adults was monitored by confocal microscope with the identical camera gain and exposure settings. Arrows indicated the location of DAF-16::GFP expression in muscles (M) and intestines (I). See also Figure S5.
(E) Nuclear translocalization of DAF-16::GFP was slightly increased in set-18(gk334) mutants. The representative images of distinct DAF-16::GFP cellular localization patterns named nuclear (Nuc), intermediate (Inter), and cytoplasmic (Cyt) are shown in upper. The percentages of distinct patterns were analyzed according to previous publication (Lin et al., 2001; Padmanabhan et al., 2009). n, the number of worms for quantitative analysis. ∗p < 0.05.
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7. Figure 5
SET-18 Represses the Activity of daf-16a Promoter by H3K36me2 Modification
(A) The extended lifespan of set-18(gk334) was diminished by daf-16a RNAi, but not daf-16d/f RNAi. According to previous publication (Kwon et al., 2010), daf-16a and daf-16d/f isoform-specific RNAi were designed for treating N2 and set-18(gk334) mutant. Shown here is the representative example of three independent experiments. All data on the three repeats and statistical analysis are in Table S4. See also Figure S6 and Table S5.
(B) Rescue of SET-18::GFP expression attenuated the increase in daf-16a mRNA level of set-18(gk334)mutants. Two independent extrachromosomal arrays (1# and 2#) were created by injecting plasmids Pset-18::SET-18::GFP::3′UTR set-18 with rol-6 marker (pRF4) into set-18(gk334). By utilizing isoform-specific primers, the mRNA levels of daf-16a and daf-16d/f were measured by RT-qPCR. act-1 was used as an internal control. Error bars represented the SEM. ns, no significant; ∗∗p < 0.01.
(C) Overexpression of SET-18 inhibited the activity of daf-16a promoter, but not the daf-16d/f promoter. The luciferase reporter constructs containing promoter of daf-16a or daf-16d/f were co-transfected with the empty vector (control) or Flag-tagged SET-18 overexpression plasmid in human 293T cells, followed by luciferase assay. The Flag-SET-18 expression level was confirmed by western blot. β-actin was used as an internal control (shown in upper). The Firefly luciferase activity was normalized to groups of control. Renilla was used as the internal reference. Error bars represented the SEM. ∗∗p < 0.01; ns, no significant.
(D) The primers used for ChIP-qPCRs on daf-16a and daf-16d/f genes. The distribution of H3K36me2 and H3K36me3 marks on daf-16a and d/f gene analyzed from public H3K36me2 (GEO: GSE49721) and H3K36me3 (GEO: GSE28776) ChIP-seq data in modENCODE are shown in upper. The gene structures of isoform daf-16a and daf-16 df are shown in lower. Three pairs of primers on daf-16a promoter (primers a1, a2, and a3), a pair of primers on daf-16d/f promoter (primers d/f), and a pair of primers on their shared intron (primers Intron) were designed for ChIP-qPCRs. The primers of 3′ UTR (primers 3′UTR) was utilized as a negative control. See also Table S6.
(E) Loss of set-18 significantly decreased H3K36me2 on the daf-16a promoter. H3K36me2 and H3K36me3 ChIP-qPCR experiments were performed by using N2 and set-18(gk334) mutants. The levels of histone H3, H3K36me2 and H3K36me3 on daf-16a and daf-16d/f genes were presented as percentage of the qPCR signal in total input DNA. IgG was used as the negative control. Histone H3 level was used as the internal reference. Error bars represent the SEM. ns, no significant; ∗∗p < 0.01.
(F) Loss of set-18 increased panacetylation of H3 (H3Ac) on daf-16a promoter and decreased the recruitment of a HDAC1 homolog on this region. ChIP-qPCR experiments were conducted by using primers a2. The anti-human HDAC3 antibody was used, as its immunogen sequence is conserved to C. elegans HDAC1 homolog HDA-3 that is most similar to HDAC1 and affects radiation sensitivity (Boulton et al., 2002). The levels of histone H3 and H4 were utilized as the internal references for H3Ac and H4Ac ChIP-qPCRs. Error bars represent the SEM. ns, no significant; ∗p < 0.05; ∗∗∗p <
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8. Figure 6
Activation of SET-18 Expression in Old-Aged Worms (day 7 and day 11) Results in Elevation of Global H3K36me2 Levels and Inhibition of daf-16a Expression
(A and B) Loss of set-18 attenuated the elevation of global H3K36me2 levels in old age (day 7 and day 11). The global H3K36me2 levels of N2 and set-18(gk334) in day 3, day 7, and day 11 were detected by western blot (A) and quantified by densitometry analysis (B). Histone H3 was used as an internal reference. Error bars represented the SEM. ns, no significant; ∗p < 0.05; ∗∗p < 0.01.
(C and D) Knockdown of mes-4 decreased global H3K36me2 levels of N2 in day 3, but not day 7 and day 11. N2 worms in day 3, day 7, and day 11 were treated with mes-4 RNAi. control, empty vector. Global H3K36me2 levels were detected by western blot (C) and quantified by densitometry analysis (D). Histone H3 was used as an internal reference. Error bars represented the SEM. ns, no significant; ∗p < 0.05.
(E) Loss of set-18 increased daf-16a mRNA level in old-aged worms (day 7 and day 11). The daf-16a mRNA level of N2 and set-18(gk334) worms in day 3, day 7, and day 11 was detected by RT-qPCR and normalized to that level of N2 in day 0. act-1 was used as an internal reference. Error bars represented the SEM. ns, no significant; ∗p < 0.05; ∗∗p < 0.01.
(F) daf-16a mRNA level was not altered by mes-4 RNAi throughout young (day 3) to old age (days 7 and 11). N2 worms in day 0, day 3, day 7, and day 11 were treated with mes-4 RNAi. control, empty vector. RT-qPCRs were conducted as in (E). Error bars represented the SEM. ns, no significant.
(G) The schematic model illustrates that the activation of muscle-specific SET-18 expression in old-aged worms (day 7 and day 11) shortens longevity by increasing H3K36me2 levels and repressing daf-16a transcription.
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