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. 2010 Aug 10:6:399.
doi: 10.1038/msb.2010.54.

DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO

Eugene Schuster  1 , Joshua J McElwee, Jennifer M A Tullet, Ryan Doonan, Filip Matthijssens, John S Reece-Hoyes, Ian A Hope, Jacques R Vanfleteren, Janet M Thornton, David Gems
Affiliations

DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO

Eugene Schuster et al. Mol Syst Biol. .

Abstract

Insulin/IGF-1 signaling controls metabolism, stress resistance and aging in Caenorhabditis elegans by regulating the activity of the DAF-16/FoxO transcription factor (TF). However, the function of DAF-16 and the topology of the transcriptional network that it crowns remain unclear. Using chromatin profiling by DNA adenine methyltransferase identification (DamID), we identified 907 genes that are bound by DAF-16. These were enriched for genes showing DAF-16-dependent upregulation in long-lived daf-2 insulin/IGF-1 receptor mutants (P=1.4e(-11)). Cross-referencing DAF-16 targets with these upregulated genes (daf-2 versus daf-16; daf-2) identified 65 genes that were DAF-16 regulatory targets. These 65 were enriched for signaling genes, including known determinants of longevity, but not for genes specifying somatic maintenance functions (e.g. detoxification, repair). This suggests that DAF-16 acts within a relatively small transcriptional subnetwork activating (but not suppressing) other regulators of stress resistance and aging, rather than directly regulating terminal effectors of longevity. For most genes bound by DAF-16::DAM, transcriptional regulation by DAF-16 was not detected, perhaps reflecting transcriptionally non-functional TF 'parking sites'. This study demonstrates the efficacy of DamID for chromatin profiling in C. elegans.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
The DAM identification (DamID) procedure in C. elegans. (A) How DamID works. A fusion protein consisting of DNA adenine methyltransferase (DAM) and the protein of interest methylates GATC sites near binding sites. Genomic DNA is digested with DpnI, which cuts only methylated GATC sites. Adaptors are added, and DNA is digested with DpnII (which cuts at unmethylated GATC sites) to assure selective amplification of methylated DNA. A parallel DAM-only experiment is also performed to control for non-specific methylation. Samples are then labeled and hybridized to arrays. (B) Schematic of plasmid constructs used for preparation of transgenic strains. (C, D) Transgene expression. Nuclear localization of GFP was detected in UL1782 animals (expressing DAM∷DAF-16∷GFP) (marked with arrows in C) in body wall muscle and anterior bulb of pharynx (circled) following heat shock. UL1787 animals (expressing DAM∷GFP) (D) do not show nuclear localization. (E) DAF-16∷DAM methylation profile for ist-1, one of several evolutionarily conserved FoxO targets identified. (F) Average distribution of methylation (DAF-16∷DAM versus DAM) from peak center for 1135 peaks identified.
Figure 2
Figure 2
Features of DAF-16∷DAM methylation. (A) Percent of probes that can be mapped to intergenic, exonic and intronic regions that show DAF-16 binding (i.e. log2 ratio DAF-16∷DAM versus DAM methylation >1.5). Probes were also mapped to regions that were 0–2 kb upstream (5′) or 0–2 kb downstream (3′) of a gene. (B) Percent of genes with DAF-16 binding. Genes were identified by ChIP (Oh et al, 2006), or by microarray analysis as upregulated or downregulated in a daf-2 mutant relative to a daf-16; daf-2 mutant (McElwee et al, 2007) or a daf-16 mutant relative to wild type (Budovskaya et al, 2008). Note: daf-16 down refers to genes that are putative regulatory targets of DAF-16 (i.e. genes that are downregulated when daf-16 is not expressed). (C) Direct targets of DAF-16 are upregulated. Density of log2 ratio for mRNA (daf-2 versus daf-16; daf-2) for genes significantly upregulated or downregulated in reference data set (IIS reg. genes) (McElwee et al, 2007), DAF-16 targets identified by DamID, and the overlap between the two gene lists. (D) Mean log2 ratio (DAF-16∷DAM versus DAM methylation) for the above groups of genes at positions relative to the translational start site.
Figure 3
Figure 3
Cross-referencing of chromatin and transcriptome profiling. (A) Venn diagram showing relationship between genes identified by microarray analysis and DamID analysis as DAF-16 regulated. This figure also includes an interpretation of the relationship between DAF-16 and the genes within Sets A, B and C. This hypothesis will require further validation. (B) Overrepresentation of expression clusters (Cluster), protein domains and GO biological processes among genes in Sets A, B, and C. (C) Selected regulatory motifs that are overrepresented (P-value<0.0001) in Sets A, B, and C. (D) Fold enrichment of matches to motifs for genes in Sets A, B, and C.
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