Members of the H3K4 trimethylation complex regulate lifespan in a germline-dependent manner in C. elegans

The plasticity of ageing suggests that longevity may be controlled epigenetically by specific alterations in chromatin state. The link between chromatin and ageing has mostly focused on histone deacetylation by the Sir2 family, but less is known about the role of other histone modifications in longevity. Histone methylation has a crucial role in development and in maintaining stem cell pluripotency in mammals. Regulators of histone methylation have been associated with ageing in worms and flies, but characterization of their role and mechanism of action has been limited. Here we identify the ASH-2 trithorax complex, which trimethylates histone H3 at lysine 4 (H3K4), as a regulator of lifespan in Caenorhabditis elegans in a directed RNA interference (RNAi) screen in fertile worms. Deficiencies in members of the ASH-2 complex—ASH-2 itself, WDR-5 and the H3K4 methyltransferase SET-2—extend worm lifespan. Conversely, the H3K4 demethylase RBR-2 is required for normal lifespan, consistent with the idea that an excess of H3K4 trimethylation—a mark associated with active chromatin— is detrimental for longevity. Lifespan extension induced by ASH-2 complex deficiency requires the presence of an intact adult germline and the continuous production of mature eggs. ASH-2 and RBR-2 act in the germline, at least in part, to regulate lifespan and to control a set of genes involved in lifespan determination. These results indicate that the longevity of the soma is regulated by an H3K4 methyltransferase/demethylase complex acting in the C. elegans germline. Genome-wide RNAi screens for genes that regulate lifespan in C. elegans have been performed previously in worms in which progeny production was inhibited, which could mask the effect of some genes on lifespan. We performed a targeted RNAi screen in fertile worms by selecting genes that encode known worm methyltransferases, proteins containing the enzymatic domain of methyltransferases (SET domain), or orthologues of regulators of histone methylation. As reported previously, set-9 and set-15 knockdown extended worm lifespan (Fig. 1a). We also found that ash-2, set-2 and set-4 knockdown extended fertile worm lifespan, with ash-2 knockdown having the most significant effect (23.1–30.9%, P, 0.0001) (Fig. 1a, Supplementary Fig. 1a). ASH-2 is a member of an H3K4 trimethylation (H3K4me3) complex in yeast, flies and mammals and is important for the conversion of H3K4 dimethylation (H3K4me2) to H3K4me3 in mammals. ash-2 knockdown decreased global H3K4me3 levels at larval stage L3 (Fig. 1b), indicating that ASH-2 promotes histone H3 trimethylation at lysine 4 in C. elegans. WDR-5/TAG-125 is a WD40-repeat protein that interacts with ASH-2 in mammals, and is important for the mono-, diand trimethylation of H3K4 in mammals and worms. wdr-5/tag125 knockdown or wdr-5/tag-125 deletion also decreased H3K4me3 levels (Fig. 1b) and significantly extended C. elegans lifespan (,30%, P, 0.0001 and 16–28%, P, 0.0001, respectively) (Fig. 1c, Supplementary Fig. 1b). Thus, ASH-2 and WDR-5 promote H3K4 trimethylation and normally limit lifespan in C. elegans. In mammals, ASH-2 and WDR-5 form a complex with several H3K4me3 methyltransferases of the SET1/mixed lineage leukaemia (MLL) family. There are four SET1/MLL orthologues in C. elegans: SET-1, SET-2, SET-12 and SET-16 (Supplementary Fig. 1c). Of these, only SET-2, which is similar to mammalian SET1A/SET1B, regulated worm lifespan (set-2 knockdown: ,20%, P, 0.0001; set-2 deletion set-2(ok952): 17–26%, P, 0.0001) (Fig. 1a, d; Supplementary Fig. 1d, e). As reported previously, set-2(ok952) mutant worms or worms treated with set-2 RNAi had reduced H3K4me3 levels (Fig. 1e). SET-2 was relatively specific in regulating lifespan and H3K4me3 in worms, as neither SET-9 nor SET-15 affected global H3K4me3 levels (Supplementary Fig. 1f), even though they both regulate lifespan. The worm SET-2 methyltransferase domain expressed in bacteria directly methylated histone H3 at lysine 4 in vitro (Fig. 1f). SET2 generated H3K4me2 but not H3K4me3 in vitro (Supplementary Fig. 1g), consistent with the fact that ASH-2 is required for the conversion of H3K4me2 to H3K4me3 (ref. 18). To test if ASH-2, WDR-5 and SET-2 act together to regulate lifespan, we performed epistasis experiments. Lifespan extension by ash-2 or wdr-5/tag-125 knockdown was significantly less pronounced in set-2(ok952) mutants than in wildtype worms (Fig. 1g, combined two-way ANOVA P, 0.0001). Similarly, ash-2 knockdown did not extend further the lifespan of wdr-5/tag-125(ok1417) mutant worms (Supplementary Fig. 1h, P 5 0.1534). Thus, ASH-2, WDR-5 and SET-2 probably act in the same pathway, perhaps in a complex, to limit lifespan. As ash-2 and wdr-5/tag-125 knockdown slightly extended set-2(ok952) mutant worm lifespan (Fig. 1g, Supplementary Table 2), other methyltransferases may also complex with ASH-2 and WDR-5 to regulate lifespan. RBR-2 is an H3K4me3 demethylase involved in vulva formation in worms and is homologous to human RBP2 and PLU-1 (also known as KDM5A and KDM5B, respectively), two H3K4me3 demethylases of the JARID family. Consistent with a published report, rbr-2(tm1231) mutant worms showed increased H3K4me3 levels (Fig. 2a). Both rbr2(tm1231) mutation and rbr-2 knockdown decreased lifespan significantly (15–25%, P, 0.0001 and 10–24.2%, P, 0.0001, respectively) (Fig. 2b, Supplementary Fig. 2a), indicating that RBR-2 is necessary for normal longevity. The decrease of H3K4me3 induced by ash-2 knockdown was less pronounced in rbr-2(tm1231) mutant worms than in wild-type worms (Fig. 2c). In addition, ash-2 knockdown no longer extended rbr-2(tm1231) mutant worm lifespan (Fig. 2d, P 5 0.4673). Similarly, the lifespan of wdr-5/tag-125(ok1415); rbr-2(tm1231) and set-2(ok952); rbr-2(tm1231) double mutants was similar to that of