The Caenorhabditis elegans Myc-Mondo/Mad complexes integrate diverse longevity signals

Abstract

The Myc family of transcription factors regulates a variety of biological processes, including the cell cycle, growth, proliferation, metabolism, and apoptosis. In Caenorhabditis elegans, the "Myc interaction network" consists of two opposing heterodimeric complexes with antagonistic functions in transcriptional control: the Myc-Mondo:Mlx transcriptional activation complex and the Mad:Max transcriptional repression complex. In C. elegans, Mondo, Mlx, Mad, and Max are encoded by mml-1, mxl-2, mdl-1, and mxl-1, respectively. Here we show a similar antagonistic role for the C. elegans Myc-Mondo and Mad complexes in longevity control. Loss of mml-1 or mxl-2 shortens C. elegans lifespan. In contrast, loss of mdl-1 or mxl-1 increases longevity, dependent upon MML-1:MXL-2. The MML-1:MXL-2 and MDL-1:MXL-1 complexes function in both the insulin signaling and dietary restriction pathways. Furthermore, decreased insulin-like/IGF-1 signaling (ILS) or conditions of dietary restriction increase the accumulation of MML-1, consistent with the notion that the Myc family members function as sensors of metabolic status. Additionally, we find that Myc family members are regulated by distinct mechanisms, which would allow for integrated control of gene expression from diverse signals of metabolic status. We compared putative target genes based on ChIP-sequencing data in the modENCODE project and found significant overlap in genomic DNA binding between the major effectors of ILS (DAF-16/FoxO), DR (PHA-4/FoxA), and Myc family (MDL-1/Mad/Mxd) at common target genes, which suggests that diverse signals of metabolic status converge on overlapping transcriptional programs that influence aging. Consistent with this, there is over-enrichment at these common targets for genes that function in lifespan, stress response, and carbohydrate metabolism. Additionally, we find that Myc family members are also involved in stress response and the maintenance of protein homeostasis. Collectively, these findings indicate that Myc family members integrate diverse signals of metabolic status, to coordinate overlapping metabolic and cytoprotective transcriptional programs that determine the progression of aging.

Figure 1. The C. elegans Myc-Mondo/Mad complexes have opposing functions in longevity.

(A) The C. elegans Myc-Mondo/Mad interaction network consists of four proteins that form two heterodimeric complexes, with opposing effects on transcription and longevity. The Myc-Mondo transcriptional activation complex consists of MML-1 and MXL-2, and the Mad transcriptional repression complex consists of MDL-1 and MXL-1 . The mammalian homologues are listed beside each protein. (B) The mdl-1(tm311) and mxl-1(tm1530) null mutations significantly increase lifespan (compare black (wild-type) to purple and blue, respectively). (C) The mml-1(ok849) and mxl-2(tm1516) null mutations significantly shorten lifespan (compare to wild-type (black)). (D) RNAi inactivation of mxl-2 abolishes the lifespan extension conferred by mdl-1(tm311) and mxl-1(tm1530) null mutations (dark purple and dark blue, versus purple and blue traces respectively). Similar results were obtained using mml-1 RNAi (Dataset S1). Graphs illustrate the combined data from multiple trials. Complete information regarding the number of trials, total number of animals examined, and statistical significance for each experiment can be found in Dataset S1.

Figure 2. The Myc-Mondo/Mad complexes intersect with ILS in longevity.

(A) RNAi inactivation of daf-16 ablates the lifespan extension conferred by mxl-1(tm1530) null mutation (dark blue versus blue traces), and does not further shorten the lifespan of mxl-2(tm1516) null mutants (pink versus red traces). Reciprocal experiments show that RNAi inactivation of mxl-1 or mdl-1 do not extend the lifespan of daf-16(mgDf47) null mutant animals, nor does RNAi inactivation of mxl-2 or mml-1 further shorten the lifespan of daf-16(mgDf47) null mutant animals (Dataset S1). (B) RNAi inactivation of mxl-2 partially suppresses the lifespan of daf-2(e1370) mutant animals (pink versus gray traces). Similar results were obtained with mml-1 RNAi (Figure S2A). (C) The mxl-2(tm1516) null mutation partially suppresses the extended lifespan observed in daf-2(e1370) mutant animals (pink versus gray traces). (D) RNAi inactivation of daf-2 does not further extend the lifespan of mxl-1(tm1530); daf-2(e1370) mutant animals (darker blue versus light blue traces). Traces represent the combined data from multiple separate trials. Complete information regarding the number of trials, total number of animals examined, and statistical significance for each experiment can be found in Dataset S1. Experiments shown within this figure were performed simultaneously with those shown in Figure 3 and were split into multiple figures for readability.

Figure 3. The Myc-Mondo/Mad complexes intersect with dietary restriction in longevity.

(A) RNAi inactivation of pha-4 ablates the lifespan extension conferred by the mxl-1(tm1530) null mutation (light blue versus blue traces), and does not further shorten the lifespan of mxl-2(tm1516) null mutant animals (pink versus red traces). (B) RNAi inactivation of mxl-2 partially suppresses the extended lifespan observed in eat-2(ad465) mutants (pink versus green traces). Similar results were obtained with mml-1 RNAi (Dataset S1). (C) The mxl-2(tm1516) null mutation partially suppresses the extended lifespan observed in eat-2(ad465) mutant animals (pink versus green traces). (D) The mxl-1(tm1530) null mutation does not further extend the lifespan of eat-2(ad465) mutant animals (dark blue versus green traces). Similar results were obtained with mxl-1 and mdl-1 RNAi (Dataset S1). Traces represent the combined data from multiple separate trials. Complete information regarding the number of trials, total number of animals examined, and statistical significance for each experiment can be found in Dataset S1. Experiments shown within this figure were performed simultaneously with those shown in Figure 2 and were split into multiple figures for readability.

Figure 4. ILS and DR pathways differentially regulate mml-1 gene expression and MML-1 localization.

(A) Nuclear accumulation of MML-1::GFP is increased in daf-2(e1370) and eat-2(ad465) mutant animals. Loss of pha-4, but not daf-16, blocks increases in MML-1::GFP nuclear accumulation in daf-2(e1370) and eat-2(ad465), but does not affect MML-1::GFP localization under basal conditions. Representative images of Types I-V are found below the graph. Additional high-resolution images of MML-1::GFP expressing animals can be found in File S1 (* denotes p < 0.05). (B) mml-1 mRNA levels are significantly increased in daf-2(e1370), but not in mdl-1(tm311) or eat-2(ad465) mutant animals, as compared to N2 (* denote p<0.05). (C) mxl-2, mxl-1, and mdl-1 mRNA levels are unaffected in daf-2(e1370) mutants. A daf-16 null mutation suppresses the increases in mml-1 mRNA observed in daf-2(e1370) mutants. (* denotes p<0.01) (D) mdl-1 mRNA levels are significantly increased in daf-2(e1370);daf-16(mgDf47) mutants (* denotes p<0.01).

Figure 5. ChIP-seq data shows significant overlap amongst MDL-1, DAF-16, and PHA-4 promoter binding.

(A) Analysis of binding sites throughout the genome indicates that MDL-1 (blue) and PHA-4 (red) bind predominantly in a region located –700 to +100 bp (dashed lines) relative to the transcriptional start site (TSS). (B) Venn diagram based on ChIP-Seq data showing gene promoters bound by MDL-1, DAF-16, and PHA-4. Numbers indicate the total number of genes belonging to each group. Parenthesis denote the number of genes belonging to each group that have previously been annotated to be involved in aging (based on published studies, gene ontology and description). The inset box shows the total number of genes in the genome that are not bound by MDL-1, DAF-16, and PHA-4. (C) GO terms related to aging that are enriched in genes bound by MDL-1, DAF-16, and PHA-4 versus the entire genome (p <0.05). The total number of common target genes annotated with each GO term is shown to the right of each bar. Enrichment was determined as described in Materials and Methods. Details of this analysis can be found in Dataset S2 (D) Analysis of metabolic pathways contained within the GO term “carbohydrate metabolic process” (GO: 0005975). The total number of genes annotated within each pathway is shown. Some genes belong to more than one category, notably several genes function in glycolysis and gluconeogenesis. Details can be found in Dataset S2 (E) An analysis of stress response pathways contained with the GO term “response to stress” (GO: 0006950). The total number of genes annotated to be involved in each pathway is shown. Many genes are important for resistance to multiple stresses. Details can be found in Dataset S2. (F) qRT-PCR analysis of Class I genes known to be bound by MDL-1, DAF-16, and PHA-4 in daf-2(e1370), daf-2(e1370);daf-16(mgDf47), and daf-2(e1370);mxl-2(tm1516) animals. Class I genes were significantly upregulated (* denotes p<0.01) in daf-2(e1370) in a daf-16 dependent manner as previously described , . Additionally, icl-1, hsp-12.6, and stdh-1 failed to be induced in daf-2(e1370);mxl-2(tm1516) mutants as well.

Figure 6. Loss of the Myc-Mondo complex impairs resistance to oxidative and thermal stress.

(A) Loss of mxl-2 impairs resistance to oxidative stress imposed by the exposure to tert-butylhydroperoxide (tBOOH) in wild-type and daf-2(e1370) mutant animals. Animals were exposed to 7.7mM tBOOH for the denoted time and survival was assessed. Loss of mxl-2 in wild-type and daf-2(e1370) mutant animals impaired tBOOH survival to a similar extent. (B) Loss of mxl-2 significantly impairs intrinsic thermotolerance (ITT) in wild-type and daf-2(e1370) mutant animals. (C) Loss of mxl-2 does not further impair ITT in daf-2(e1370);daf-16(mgDf47) mutants. (D) mxl-2 is dispensable for acquired thermotolerance in daf-2(e1370) mutants. Graphs were generated from the combined data from multiple experiments performed as described in Materials and Methods. Statistical analyses and experimental details can be found in Dataset S3.

Figure 7. Myc-Mondo/Mad transcription factors influence proteostasis.

(A) RNAi inactivation of mxl-2 and mml-1 leads to early accumulation of Q35::YFP foci (red and orange versus black). RNAi inactivation of mxl-1, mdl-1, and daf-2 fail to delay accumulation of Q35::YFP foci (compare vector control RNAi treated animals (black) to blue, purple, and gray respectively. Traces are the combined data from three trials (*denotes p<0.01, standard deviations and statistical analysis can be found in Dataset S3). Images are from empty vector control and mxl-2 RNAi treated animals of Day 2 of adulthood. (B) RNAi inactivation of mxl-2 and mml-1 leads to premature paralysis (compare red and orange to vector RNAi control (black)). RNAi inactivation of mxl-1(blue), mdl-1(purple), and daf-2(gray) delays paralysis induced by Q35::YFP expression in the body wall muscles in comparison to animals treated with empty vector control RNAi (black). Traces are representative of data from two trials performed in duplicate. Complete statistical analysis for data represented in this figure can be found in Dataset S3.

Figure 8. Model for Myc-Mondo/Mad transcription factors in longevity control under basal conditions the Myc-Mondo activation complex (MML-1:MXL-2) is largely inactive, and transcription of genes encoding functions related to aging is limited by the Mad transcriptional repression complex (MDL-1:MXL-1).

Conditions of reduced ILS and DR promote Myc-Mondo complex activity, likely by regulating cellular localization and transcription of mml-1. Myc-Mondo/Mad transcription factors may cooperate with DAF-16 and PHA-4 to modulate the expression of key metabolic and cytoprotective genes to influence aging.