H3K9me1/2 methylation limits the lifespan of daf-2 mutants in C. elegans

  1. Meng Huang
  2. Minjie Hong
  3. Xinhao Hou
  4. Chengming Zhu
  5. Di Chen
  6. Xiangyang Chen
  7. Shouhong Guang
  8. Xuezhu Feng

  • Curated by eLife

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    Evaluation Summary:

    This manuscript examines how putative C. elegans H3K9me methyltransferases affect aging by investigating their effects on long-lived daf-2 mutants. They surprisingly find that modifiers of H3K9me1/2, but not H3K9me3, can synergistically extend the lifespan of daf-2 (in some cases, to three times as long as wild-type). They demonstrate that this synergistic effect on lifespan requires the DAF-16 transcription factor and some of its downstream regulatory targets, and compellingly, they show that the effects on lifespan are phenocopied by a small molecular inhibitor known to target a conserved H3K9me1/2 HMT.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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Abstract

Histone methylation plays crucial roles in the development, gene regulation, and maintenance of stem cell pluripotency in mammals. Recent work shows that histone methylation is associated with aging, yet the underlying mechanism remains unclear. In this work, we identified a class of putative histone 3 lysine 9 mono/dimethyltransferase genes ( met-2, set-6, set-19, set-20, set-21, set-32, and set-33 ), mutations in which induce synergistic lifespan extension in the long-lived DAF-2 (insulin growth factor 1 [IGF-1] receptor) mutant in Caenorhabditis elegans . These putative histone methyltransferase plus daf-2 double mutants not only exhibited an average lifespan nearly three times that of wild-type animals and a maximal lifespan of approximately 100 days, but also significantly increased resistance to oxidative and heat stress. Synergistic lifespan extension depends on the transcription factor DAF-16 (FOXO). mRNA-seq experiments revealed that the mRNA levels of DAF-16 Class I genes, which are activated by DAF-16, were further elevated in the daf-2;set double mutants. Among these genes, tts-1 , F35E8.7 , ins-35 , nhr-62 , sod-3 , asm-2, and Y39G8B.7 are required for the lifespan extension of the daf-2;set-21 double mutant. In addition, treating daf-2 animals with the H3K9me1/2 methyltransferase G9a inhibitor also extends lifespan and increases stress resistance. Therefore, investigation of DAF-2 and H3K9me1/2 deficiency-mediated synergistic longevity will contribute to a better understanding of the molecular mechanisms of aging and therapeutic applications.

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  1. Version published to 10.7554/elife.74812 on eLife
  2. eLife

    Author Response

    Reviewer #2 (Public Review):

    In this paper, Huang and colleagues investigate whether putative C. elegans H3K9me methyltransferases are involved in aging by investigating their effects on long-lived daf-2 mutants. They find that modifiers of H3K9me1/2, but not H3K9me3, can synergistically extend the lifespan of daf-2 (in some cases, to three times as long as wild-type). They demonstrate that this synergistic effect on lifespan requires the DAF-16 transcription factor and some of its downstream regulatory targets. Like other mutations that extend lifespan, mutations in these HMTs also protect against heat and oxidative stress. Compellingly, they show that the effects on lifespan are phenocopied by a small molecular inhibitor known to target a conserved H3K9me1/2 HMT - this experiment strengthens their claim that the effects on lifespan are due to changes in H3K9me1/2 specifically, and are unlikely to be caused by non-enzymatic effects of mutating the SET-domain proteins.

    This work contributes a new regulatory layer to the well-studied DAF-2/DAF-16 pathway for stress resistance and aging - it implicates a functional role for H3K9me1/2 at several DAF-16 target genes, and identifies possible HMTs. The conclusions of this paper are generally supported by the data presented. However, I have concerns regarding technical aspects of the experiments & analysis, and find some interpretations to be overstated.

    1. The effects on lifespan reported in this manuscript are highly dependent on experimental technique. However, data are presented in this manuscript in a way that makes it difficult to evaluate the reproducibility of their results, which is important for effects on lifespan that may be statistically significant, but small. The following changes will improve the rigor of their findings. First, each lifespan assay should be replicated at least twice, if not three times, and results reported in the summary data table suggested below. Second, major results, like those of the daf-2; set-21 double mutants or the G9a inhibitor, should be performed blinded to further validate their findings. Finally, summary data for each experiment should be included in supplementary table(s), with conditions examined per assay, N, animals censored, median lifespan (along with average lifespan), and comparison used for determination of significance, which is most commonly calculated using a log rank test (which captures distinctions in survival for the entirety of the survival assay).

    Thanks very much for the comments. We have revised the materials and methods section (lines 549-559) and figure legends to include the information of each lifespan assay. We also included a new Table S1 to summarize all lifespan experiments.

    1. The transcriptomic analysis is important to link the synergistic extension of lifespan to the known DAF-16 pathway. However, the analysis was superficial -the authors used the mRNA-seq data to primarily validate their hypothesis that DAF-16 targets are most affected in HMT; daf-2 double mutants. Transcriptomic data are never used in an unbiased manner to identify other potential pathways, or even to demonstrate that DAF-16 Class I/II genes are the most affected in these genetic backgrounds. For example, it is important to show that there is more misregulation observed among Class I and Class II genes when compared to all transcriptomic changes caused by the mutations. The cursory approach to genomic analysis is also seen by how methods are explain, making it difficult to tell what comparisons are being drawn to identify misregulation. More analysis is required before the authors can fully support their claim that the effects of removing an HMT in a daf-2 background occur primarily through DAF-16 Class I gene regulation.

    Thanks very much for the suggestions. We used two approaches to analyze the mRNA-seq data.

    First, the depletion of DAF-2 reduces the insulin signaling pathway, promotes DAF-16 nuclear translocation, and leads to both upregulation and downregulation of large sets of genes, referred to as Class I and II genes, respectively. Class I genes are induced in daf-2 mutants but are repressed in daf-2;daf-16 double mutants. Class II genes are not induced in daf-2 mutants but are induced in daf-2; daf-16 double mutants. 1663 genes are classified as positive (class I) DAF-16 targets and 1733 genes are classified as negative (class II) DAF-16 targets of DAF-16. Class I genes are enriched for the Gene Ontology categories including oxidation, reduction, and energy metabolism, whereas class II genes are enriched for genes involved in biosynthesis, growth, reproduction, and development. We performed mRNA-seq and analyzed Class I and II DAF-16 genes to identify the mis-regulated genes in the daf-2;set mutants. Interestingly, the mRNA levels of DAF-16 Class I, but not Class II, genes are consistently activated in long-lived daf-2;set-19, daf-2;set-21 and daf-2;set-32 worms, than in the control daf-2 and daf-2;set-25 animals (Figure 5E-F and new Figure 5-figure supplement 2).

    Second, to identify the target genes in the group of lifespan extension daf-2;set mutants, we re-analyzed the mRNA expression profile in the double mutants via an unbiased method (new Figure 6, Figure 6-figure supplement 1). We have identified 49 co-upregulated genes and 11 co-downregulated genes that are specifically enriched in the long-lived double mutants daf-2;set-19, daf-2,set-21, daf-2;set-32, but not in daf-2 and daf-2;daf-25 animals (Figure 6A-B and Figure 6-figure supplement 1A). Interestingly, among the 49 co-upregulated genes, 27 of them are also DAF-16 Class I genes (new Figure 6-figure supplement 1B). 22 co-upregulated genes are not DAF-16 Class I genes, suggesting the existence of additional regulations.

    We then analyzed a number of known transcription factors for their binding to the co-regulated targeted genes (new Figure 6C-D, Figure 6-figure supplement 1C-E). Among them, we found that DAF-16 and NHR-80 were specifically enriched at the transcription start sites (TSS) of the 49 co-upregulated genes. NHR-80 is a homolog of mammalian hepatocyte nuclear factor 4 and is an important nuclear hormone receptor involved in the control of fat consumption and fatty acid composition in C. elegans. Among the genes targeted by DAF-16 and NHR-80, twelve of them are co-regulated by both factors, which is consistent with previous report that daf-16 and nhr-80 function in parallel pathway for lipid metabolism.

    We revised the text to include this information.

    1. The findings presented here are interesting and uncover a new avenue of research for understanding longevity and stress resistance. However, for the most part, the effects on lifespan and stress resistance are seen in a daf-2 mutant background. This genetic background already experiences a significant lifespan increase, and therefore has many molecular & physiological differences from wild-type animals (which are well-characterized). Therefore, many of the broad statements in the abstract and discussion overstate the generality of their findings. This work clearly demonstrates that HMTs act to limit the lifespan of daf-2 mutants. Little effect, if any, was seen in HMT mutants in an otherwise wild-type background. In fact, some HMT mutants, like met-2, have a decreased lifespan, indicating that H3K9me1/2 may be protective for lifespan in some circumstances. Furthermore, the authors claim that these HMTs regulate Class I DAF-16 target genes, but no effort was made to demonstrate that this class of genes was more affected than any other class. Care should be taken to ensure that the claims made are fully supported by the data presented here.

    Thanks very much for the comments.

    eat-2 mutant is a genetic model in dietary restriction (DR) research in C. elegans. The mutation of eat-2 renders a non-efficient pharynx in grinding bacteria and results in DR on regular media plates. eat-2 animals exhibit phenotypes similar to those observed in other species subjected to DR, including a ~36% longer lifespan (new Figure 1C). Strikingly, knocking out set-21 further extended the lifespan of eat-2(ad465) mutant worms (new Figure 1C). The average lifespan of eat-2(ad465);set-21(ust68) were 16% longer than that of eat-2(ad465) animals, suggesting a broader effect of these lifespan limiting SET proteins (lines 123-130).

    To identify the target genes in the group of lifespan extension daf-2;set mutants, we re-analyzed the mRNA expression profile in the double mutants via an unbiased method (new Figure 6, Figure 6-figure supplement 1). We have identified 49 coupregulated genes and 11 co-downregulated genes that are specifically enriched in the long-lived double mutants daf-2;set-19, daf-2,set-21, daf-2;set-32, but not in daf-2 and daf-2;daf-25 animals (Figure 6A-B and Figure 6-figure supplement 1A). Interestingly, among the 49 co-upregulated genes, 27 of them are also DAF-16 Class I genes (new Figure 6-figure supplement 1B). 22 co-upregulated genes are not DAF-16 Class I genes, suggesting the existence of additional regulations.

  3. eLife

    Evaluation Summary:

    This manuscript examines how putative C. elegans H3K9me methyltransferases affect aging by investigating their effects on long-lived daf-2 mutants. They surprisingly find that modifiers of H3K9me1/2, but not H3K9me3, can synergistically extend the lifespan of daf-2 (in some cases, to three times as long as wild-type). They demonstrate that this synergistic effect on lifespan requires the DAF-16 transcription factor and some of its downstream regulatory targets, and compellingly, they show that the effects on lifespan are phenocopied by a small molecular inhibitor known to target a conserved H3K9me1/2 HMT.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    In the manuscript "H3K9me1/2 methylation limits the lifespan of C. elegans" Huang M, Hong M et al performed a genetic analysis of putative H3K9 methyltransferases and their involvement in the lifespan regulation of daf-2 mutant worms. They build on an initial finding that set-21 mutation leads to a further extension of daf-2 lifespan and stress resistance and expand this to analyze all other putative H3K9 methyltransferases in the context of daf-2. They identify several putative methyltransferases which all extend daf-2 lifespan and stress resistance and show that a H3K9me inhibitor also has the same effect. They also perform some transcriptional analyses to examine how gene expression changes in these double mutant strains and take some daf-16 target genes and examine their effects on lifespan in the daf-2;set-21 double mutant. They show that deletion of some of those daf-16 target genes decreases the lifespan of daf-2;set-21 double mutants.

    All in all I thought this was an interesting paper and the data appears to be strong but there are several critical missing pieces that I think need to be added to bolster the manuscript and shore up the findings. Most importantly I think that the authors need to examine how daf-2 mutation affects H3K9me1/2? Why are these putative methyltransferases actually important here? Also since the paper is so focused on H3K9me1/2 it seems important to show that the enzymes that are being studied do actually directly regulate H3K9 methylation. Finally some experiments should attempt to address why there are 6 enzymes which the authors believe are modifying the same mark all have the exact same effect on lifespan in daf-2 mutant worms. Usually it is believed that these chromatin modifying enzymes have some specificity (either by being expressed in different tissues or modifying different chromatin regions) but here they all have the exact same consequence! That is quite surprising and an attempt should be made to explain this finding (and best if backed up by some experiments!).

  5. eLife

    Reviewer #2 (Public Review):

    In this paper, Huang and colleagues investigate whether putative C. elegans H3K9me methyltransferases are involved in aging by investigating their effects on long-lived daf-2 mutants. They find that modifiers of H3K9me1/2, but not H3K9me3, can synergistically extend the lifespan of daf-2 (in some cases, to three times as long as wild-type). They demonstrate that this synergistic effect on lifespan requires the DAF-16 transcription factor and some of its downstream regulatory targets. Like other mutations that extend lifespan, mutations in these HMTs also protect against heat and oxidative stress. Compellingly, they show that the effects on lifespan are phenocopied by a small molecular inhibitor known to target a conserved H3K9me1/2 HMT - this experiment strengthens their claim that the effects on lifespan are due to changes in H3K9me1/2 specifically, and are unlikely to be caused by non-enzymatic effects of mutating the SET-domain proteins.

    This work contributes a new regulatory layer to the well-studied DAF-2/DAF-16 pathway for stress resistance and aging - it implicates a functional role for H3K9me1/2 at several DAF-16 target genes, and identifies possible HMTs. The conclusions of this paper are generally supported by the data presented. However, I have concerns regarding technical aspects of the experiments & analysis, and find some interpretations to be overstated.

    1. The effects on lifespan reported in this manuscript are highly dependent on experimental technique. However, data are presented in this manuscript in a way that makes it difficult to evaluate the reproducibility of their results, which is important for effects on lifespan that may be statistically significant, but small. The following changes will improve the rigor of their findings. First, each lifespan assay should be replicated at least twice, if not three times, and results reported in the summary data table suggested below. Second, major results, like those of the daf-2; set-21 double mutants or the G9a inhibitor, should be performed blinded to further validate their findings. Finally, summary data for each experiment should be included in supplementary table(s), with conditions examined per assay, N, animals censored, median lifespan (along with average lifespan), and comparison used for determination of significance, which is most commonly calculated using a log rank test (which captures distinctions in survival for the entirety of the survival assay).

    2. The transcriptomic analysis is important to link the synergistic extension of lifespan to the known DAF-16 pathway. However, the analysis was superficial -the authors used the mRNA-seq data to primarily validate their hypothesis that DAF-16 targets are most affected in HMT; daf-2 double mutants. Transcriptomic data are never used in an unbiased manner to identify other potential pathways, or even to demonstrate that DAF-16 Class I/II genes are the most affected in these genetic backgrounds. For example, it is important to show that there is more misregulation observed among Class I and Class II genes when compared to all transcriptomic changes caused by the mutations. The cursory approach to genomic analysis is also seen by how methods are explain, making it difficult to tell what comparisons are being drawn to identify misregulation. More analysis is required before the authors can fully support their claim that the effects of removing an HMT in a daf-2 background occur primarily through DAF-16 Class I gene regulation.

    3. The findings presented here are interesting and uncover a new avenue of research for understanding longevity and stress resistance. However, for the most part, the effects on lifespan and stress resistance are seen in a daf-2 mutant background. This genetic background already experiences a significant lifespan increase, and therefore has many molecular & physiological differences from wild-type animals (which are well-characterized). Therefore, many of the broad statements in the abstract and discussion overstate the generality of their findings. This work clearly demonstrates that HMTs act to limit the lifespan of daf-2 mutants. Little effect, if any, was seen in HMT mutants in an otherwise wild-type background. In fact, some HMT mutants, like met-2, have a decreased lifespan, indicating that H3K9me1/2 may be protective for lifespan in some circumstances. Furthermore, the authors claim that these HMTs regulate Class I DAF-16 target genes, but no effort was made to demonstrate that this class of genes was more affected than any other class. Care should be taken to ensure that the claims made are fully supported by the data presented here.

  6. eLife

    Reviewer #3 (Public Review):

    Previous work in C. elegans and other systems had suggested that H3K9 methylation is required for enhanced longevity. However, the authors discovered an interesting phenomenon in which decreased H3K9me2 methylation, but not H3K9me3 in a daf-2 mutant background is associated with increased longevity. This suggests that in the context of reduced insulin signaling, reduced H3K9me2 methylation has the opposite effect on longevity. By performing RNAseq, the authors show that daf-16 targets are increased in expression. This suggests that in a daf-2 mutant where daf-16 translocates into the nucleus, H3K9me2, but not H3K9me3 normally prevents daf-16 from fully activating it's transcriptional targets.

    Overall, this is a well written, comprehensive paper examining an interesting phenomenon, wherein the manipulation of H3K9 levels has a context dependent effect on longevity. The authors do a very nice job in convincingly showing that this effect is likely through H3K9me2 preventing the full activation of daf-16 targets. This work adds significantly to our understanding of the relationship between repressive chromatin and longevity.

  7. Version published to 10.1101/2021.10.27.466082v1 on bioRxiv