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, 6 (2), e1000848

Genome-wide Identification of Binding Sites Defines Distinct Functions for Caenorhabditis Elegans PHA-4/FOXA in Development and Environmental Response

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Genome-wide Identification of Binding Sites Defines Distinct Functions for Caenorhabditis Elegans PHA-4/FOXA in Development and Environmental Response

Mei Zhong et al. PLoS Genet.

Abstract

Transcription factors are key components of regulatory networks that control development, as well as the response to environmental stimuli. We have established an experimental pipeline in Caenorhabditis elegans that permits global identification of the binding sites for transcription factors using chromatin immunoprecipitation and deep sequencing. We describe and validate this strategy, and apply it to the transcription factor PHA-4, which plays critical roles in organ development and other cellular processes. We identified thousands of binding sites for PHA-4 during formation of the embryonic pharynx, and also found a role for this factor during the starvation response. Many binding sites were found to shift dramatically between embryos and starved larvae, from developmentally regulated genes to genes involved in metabolism. These results indicate distinct roles for this regulator in two different biological processes and demonstrate the versatility of transcription factors in mediating diverse biological roles.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Experimental pipeline for identification of transcription factor binding sites in C. elegans.
Individual transcription factors encoded within fosmids are tagged with a dual green fluorescent protein (GFP) and 3xFLAG tag at its C-terminus. A construct is then bombarded into worms to generate a series of integrated transgenic lines expressing the tagged factor. The expression of each transcription factor is confirmed through both fluorescence imaging and immunoblot analysis. The binding sites of each transcription factor are then identified using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq).
Figure 2
Figure 2. Binding patterns of GFP-tagged AMA-1 are highly similar to that of native AMA-1.
(A) Signal tracks of AMA-1 binding profiles for a representative stretch of chromosome I. The top track represents binding of AMA-1:GFP as detected by anti-GFP. The middle track represents binding of AMA-1 and AMA-1:GFP as detected by anti-RNA Pol II (8WG16). The bottom track represents input chromatin. (B) Signal values (relative abundance of sequencing tags in ChIP DNA versus input) for each binding site (p<0.001) in the anti-GFP and anti-8WG16 IPs were subjected to Pearson correlation coefficient analysis across 600 bp windows. The linear correlation coefficient between the two samples is 0.934.
Figure 3
Figure 3. PHA-4 is required for starvation survival.
(A) Loss of pha-4 leads to reduced starvation survival of first stage (L1) larvae. Wild-type (WT) worms were soaked in no RNAi, Cherry double-stranded RNA (dsRNA), or pha-4 dsRNA without food for the indicated times. To determine viability, triplicate samples were transferred to plates with food. Numbers of worms surviving past L1 were counted after 2 days. Results are an average of three independent experiments, n = 300–500 worms counted for each strain per experiment, error bars represent standard error. * = p<0.05. (B) Overexpression of pha-4 increases L1 starvation survival. PHA-4:GFP and outcrossed WT worms were subjected to starvation in liquid. Survival was determined as in (A). Results are an average of two independent experiments, n = 500–1900 worms counted for each strain per experiment, error bars represent standard error, p<0.0001 log rank (Mantel-Cox) test.
Figure 4
Figure 4. Identification of PHA-4 binding sites in embryos and starved L1 larvae.
(A) PHA-4:GFP is expressed primarily in the pharynx and gut in embryos and L1s. (B) PHA-4:GFP is enriched upon immunoprecipitation by anti-GFP relative to input and is not immunoprecipitated by a control IgG antibody. (C) Signal tracks demonstrating specific PHA-4:GFP binding sites on chromosome V. Green track – PHA-4:GFP (GFP antibody); maroon track – RNA Pol II (8WG16 antibody); blue track – input control; purple track – mapped reads from RNA sequencing data. Embryonic data set shown on top, L1 larval dataset shown below. (D) Close-up of smk-1 locus showing that PHA-4 binding changes between stages, although the gene appears to be expressed at both stages. Other examples of PHA-4 binding are shown in Figure S5.
Figure 5
Figure 5. Characterization of PHA-4 binding patterns and gene targets.
(A) The distribution of the distance between PHA-4 binding sites and candidate gene targets (Figure S3 for algorithm for assigning gene targets). (B) Scatter plot comparing similarity and uniqueness of PHA-4 binding profile in embryos and L1 larvae. Signal strength is sequenced reads normalized against input in the peak region (p<10−5). (C,D) Gene Ontology (GO) categories showing the highest level of enrichment for the candidate target genes of PHA-4 specific to embryos (C) and L1 larvae (D). Fold enrichment is defined as the increase in abundance in the immunoprecipitated sample relative to total input chromatin.
Figure 6
Figure 6. PHA-4 binding correlates with gene expression levels.
The expression levels of PHA-4 targets show that binding correlates with increased gene expression. Genes bound by PHA-4 specifically in embryos tend to have higher expression (indicated by increasing red intensity) in embryos than in L1s, whereas genes bound specifically in L1s have higher expression in larvae than in embryos. Genes bound at both stages show a mix of expression levels.
Figure 7
Figure 7. Genes displaying RNA Pol II stalling are preferentially bound by PHA-4.
Pie charts showing the fraction of genes with an RNA Pol II stalling index >4 bound by PHA-4 for genes with either stage-specific PHA-4 binding or shared PHA-4 binding.

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