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. 2008 Sep 30;6(9):e233.
doi: 10.1371/journal.pbio.0060233.

Caenorhabditis elegans HCF-1 functions in longevity maintenance as a DAF-16 regulator

Ji Li  1 , Atsushi Ebata, Yuqing Dong, Gizem Rizki, Terri Iwata, Siu Sylvia Lee
Affiliations

Caenorhabditis elegans HCF-1 functions in longevity maintenance as a DAF-16 regulator

Ji Li et al. PLoS Biol. .

Abstract

The transcription factor DAF-16/forkhead box O (FOXO) is a critical longevity determinant in diverse organisms, however the molecular basis of how its transcriptional activity is regulated remains largely unknown. We report that the Caenorhabditis elegans homolog of host cell factor 1 (HCF-1) represents a new longevity modulator and functions as a negative regulator of DAF-16. In C. elegans, hcf-1 inactivation caused a daf-16-dependent lifespan extension of up to 40% and heightened resistance to specific stress stimuli. HCF-1 showed ubiquitous nuclear localization and physically associated with DAF-16. Furthermore, loss of hcf-1 resulted in elevated DAF-16 recruitment to the promoters of its target genes and altered expression of a subset of DAF-16-regulated genes. We propose that HCF-1 modulates C. elegans longevity and stress response by forming a complex with DAF-16 and limiting a fraction of DAF-16 from accessing its target gene promoters, and thereby regulates DAF-16-mediated transcription of selective target genes. As HCF-1 is highly conserved, our findings have important implications for aging and FOXO regulation in mammals.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. hcf-1 Modulates Lifespan by Acting Upstream of daf-16, but in Parallel to the IIS and Germline Signaling Pathways
Lifespan of (A) wild-type worms treated with hcf-1 RNAi, (B) the hcf-1(ok559) and hcf-1(pk924) deletion mutants, (C) the daf-16(mgDf47);hcf-1(pk924) double mutant, (D) the daf-2(e1370);hcf-1(pk924) double mutant, (E) the age-1(mg44);hcf-1(ok559) double mutant, (F) the glp-1(e2141);hcf-1(pk924) double mutant worms. Each of the lifespan experiments was repeated at least two independent times with similar results. Data from representative experiments are shown. Quantitative data and statistical analyses for the experiments shown here are included in Table 1.
Figure 2
Figure 2. HCF-1 Is a Ubiquitously Expressed Nuclear Protein
(A) Gravid adults of wild-type and hcf-1(pk924);sur-5::gfp mutant worms were immunostained using an affinity-purified HCF-1 antibody. To ensure identical staining conditions, worms from both strains were processed on the same slide. The hcf-1(pk924) mutants were marked by SUR-5::GFP to distinguish them from wild-type worms. Endogenous HCF-1 was found to localize in the nucleus of most, if not all, somatic and germline cells in wild-type worms. The hcf-1(pk924) mutant worms marked by SUR-5::GFP showed only background signal. DAPI staining was used to indicate the nucleus. Photos were taken at 200× magnification. (B) A magnified image of the head region of the wild-type worm shown in (A).
Figure 3
Figure 3. Loss of hcf-1 Results in Heightened Resistance to Specific Environmental Stresses
(A) The hcf-1(pk924) mutant worms exhibited increased survival in 200-mM paraquat compared to wild-type worms. (B) The enhanced paraquat resistance of hcf-1(pk924) was dependent on daf-16. (C) The hcf-1(pk924) mutant worms showed increased survival in CdCl2 (18mM) that was daf-16 dependent. (D) The hcf-1(pk924) and hcf-1(ok559) mutants and wild-type worms showed similar survival kinetics when cultured at 35 °C. The age-1(hx546) and daf-16(mgDf47) worms were included as controls as they have been previously reported to be either resistant or sensitive to paraquat, heavy metal, or heat shock, respectively [–43]. For the stress assays, duplicate to quadruplicate samples were examined for each strain. Mean fraction alive indicates the average survival among the multiplicates and error bars represent the standard deviation of the multiplicates. p-Value was calculated using Student's t-test. *, p < 0.05 when compared to wild-type (wt). **, p < 0.05 when compared to hcf-1(pk924). Each of the stress assays was repeated at least two independent times with similar results, and the data of representative experiments are shown.
Figure 4
Figure 4. Loss of hcf-1 Does Not Result in Altered DAF-16 Subcellular Localization or a Change in DAF-16 Expression Level
(A) Transgenic worms over-expressing DAF-16::GFP (daf-16(mgDf47);xrIs87) were treated with empty vector L4440 control RNAi, hcf-1 RNAi, or daf-2 RNAi at 16 °C for 5 d. DAF-16::GFP exhibited diffuse expression pattern in both the control RNAi and the hcf-1 RNAi knock down worms. hcf-1 RNAi was able to substantially reduce HCF-1 levels (bottom right panel). daf-2 RNAi was included as a positive control as it is known to stimulate robust nuclear localization of DAF-16::GFP. Photos showed the DAF-16::GFP expression pattern and DIC images of live day 2 gravid adults. Nuclear localization was verified using DIC. A total of ∼60–70 worms were scored, and the percentage of worms showing DAF-16::GFP nuclear localization was shown in the photo. Worm extracts made from the DAF-16::GFP worms treated with control or hcf-1 RNAi were immunoblotted using anti-HCF-1 antibody ([A], bottom right panels). (B) The RNA levels of daf-16, daf-2, age-1, and akt-1 in wild-type, hcf-1(ok559), and hcf-1(pk924) worms were quantified using qRT-PCR. The data for three independent experiments were pooled, and the mean normalized RNA level and standard error of the mean (SEM) for each gene in the hcf-1 mutant and wild-type worms are shown. act-1 was used as an internal control, and the RNA level of each gene was normalized to the act-1 level. The mean normalized RNA level for each gene in wild-type (wt) worms was set as 1. None of the genes tested showed any significant expression change in the hcf-1 mutants compared to wild-type worms.
Figure 5
Figure 5. Loss of hcf-1 Promotes the DAF-16 Transcriptional Regulation of Several Target Genes
(A) The expression of sod-3, mtl-1, and F21F3.3 was elevated and that of C32H11.4 was repressed in the hcf-1 mutants. The elevated expression of sod-3 and mtl-1 in the hcf-1 mutants was completely dependent on daf-16; that of F21F3.3 and C32H11.4 was partially dependent on daf-16. *, p < 0.05 when compared to wild-type (wt). **, p < 0.05 when compared to hcf-1(ok559). (B) The expression of sod-3 and mtl-1 in the daf-2(e1370);hcf-1(pk924) double mutant showed synergistic up-regulation when compared to the expression in either hcf-1(pk924) or daf-2(e1370) single mutant. *, p < 0.05 when compared to wild-type (wt). **, p < 0.05 when compared to daf-2(e1370). The quantitative data are summarized in Tables 3–5. The RNA levels of sod-3, mtl-1, F21F3.3, and C32H11.4 were quantified using qRT-PCR and normalized to the internal control act-1. The data for at least three independent experiments were pooled, and the mean normalized RNA level and SEM for each gene in the indicated strains are shown. The mean normalized RNA level for each gene in wt worms was set as 1. p-Value was calculated using Student's t-test. For sod-3 or mtl-1 expression, we analyzed for a synergistic effect in daf-2(e1370);hcf-1(pk924) compared to daf-2(e1370) or hcf-1(pk924) using two-way ANOVA analysis.
Figure 6
Figure 6. HCF-1 Forms a Protein Complex with DAF-16 in C. elegans
Worm extracts were made from mixed stage worms and subjected to immunoprecipitation. Extracts from wild-type (wt), Psod-3::gfp, daf-16::gfp (daf-16(mu86);muIs71), and daf-16::gfp;hcf-1(-) (daf-16(mu86);hcf-1(ok559);muIs71) worms were either immunoprecipitated using anti-GFP antibody (A) or anti-HCF-1 antibody (B). The immunoprecipitated protein complexes were subsequently immunoblotted using anti-HCF-1, anti-DAF-16, or anti-GFP antibodies. Psod-3::gfp worms were used as a negative control to indicate that there was no interaction between the GFP tag and HCF-1. For input, 50 μg of total protein was loaded per lane. For immunoprecipitation, 2 mg of total protein was used.
Figure 7
Figure 7. Loss of hcf-1 Enhances the Enrichment of DAF-16 on the Promoters of Its Target Genes
ChIP was performed using daf-2(e1370);daf-16(mgDf47);daf-16::gfp worms treated with control RNAi (L4440) or hcf-1 RNAi. Synchronized adult worms of the different strains were incubated at 25 °C for ∼6 h to inactivate daf-2 and to induce robust DAF-16::GFP nuclear localization. Worm extracts were subjected to immunoprecipitation using anti-GFP, anti-HCF-1, or anti-Rabbit IgG. The recovered DNA was quantitated using qPCR. Regions around the DAF-16 binding element (DBE) at the sod-3 or mtl-1 promoters, as well as a putative noncoding region of Chromosome IV not containing any DBE were monitored. The figure shows one representative experiment. Error bars represent the SEM of the duplicated reactions in qPCR. Similar results were obtained for three independent experiments. (A) DAF-16 enrichment at the promoters of sod-3 or mtl-1 was enhanced upon hcf-1 RNAi. DAF-16 was robustly enriched at the sod-3 or mtl-1 promoters after anti-GFP ChIP compared to that of anti-Rb (anti-GFP/anti-Rabbit at sod-3 promoter: ∼9-fold; anti-GFP/anti-Rabbit at mtl-1 promoter: ∼11-fold). The fold enrichment of DAF-16 at sod-3 or mtl-1 was consistently greater than that at the nonspecific Chromosome IV region: sod-3/chr IV: ∼2.9-fold; mtl-1/chr IV: ∼2.4-fold. As a control, anti-GFP ChIP in wild-type (wt) worms (not expressing daf-16::gfp) showed background signal that was very similar to that of anti-Rabbit. Upon hcf-1 RNAi knockdown, DAF-16 enrichment at the sod-3 or mtl-1 promoters was greatly increased. Anti-GFP/anti-Rabbit at sod-3 promoter: ∼33-fold (versus ∼9-fold for L4440 RNAi); anti-GFP/anti-Rabbit at mtl-1 promoter: ∼33-fold (versus ∼11-fold for L4440 RNAi). In contrast, for the nonspecific Chromosome IV region, anti-GFP/anti-Rabbit: ∼4-fold (versus ∼3-fold for L4440 RNAi). These data indicated that in the absence of HCF-1, a greater amount of DAF-16 becomes recruited to the sod-3 or mtl-1 promoters, but the nonspecific binding of DAF-16 to the Chromosome IV region is not substantially changed. (B) HCF-1 was greatly enriched at the efl-1 promoter, but not at sod-3 or mtl-1 promoters. efl-1 is the C. elegans homolog of E2f1, which has been shown to be a direct target of HCF-1 in mammalian cells [53]. The region of the efl-1 promoter containing a conserved E2F1 binding element was included as a positive control for anti-HCF-1 ChIP. Whereas HCF-1 was found to be greatly enriched at the efl-1 promoter, it was not substantially enriched at the promoters of sod-3 or mtl-1, or the Chromosome IV noncoding region. Fold change of anti-HCF-1/anti-Rabbit at efl-1, sod-3, mtl-1, Chromosome IV noncoding region: ∼30.8, ∼2.1, ∼3.6, ∼2.3, respectively. As a control, when hcf-1 was knocked down by RNAi, the enrichment of HCF-1 on the promoter of efl-1 was greatly reduced.
Figure 8
Figure 8. The Model
We propose that in wild-type C. elegans, HCF-1 associates with a fraction of DAF-16 in the nucleus and limits the recruitment of some DAF-16 to its target gene promoters. Inactivation of hcf-1 allows more DAF-16 to access its target gene promoters and enforces DAF-16-mediated regulation of selective target genes, which likely contributes to the prolonged lifespan and enhanced stress resistance phenotypes of the hcf-1 mutants.
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