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

Figure 7. Loss of hcf-1 Enhances the Enrichment of DAF-16 on the Promoters of Its Target Genes.

Figure 7

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.