Whole-genome analysis of temporal gene expression during foregut development

Abstract

We have investigated the cis-regulatory network that mediates temporal gene expression during organogenesis. Previous studies demonstrated that the organ selector gene pha-4/FoxA is critical to establish the onset of transcription of Caenorhabditis elegans foregut (pharynx) genes. Here, we discover additional cis-regulatory elements that function in combination with PHA-4. We use a computational approach to identify candidate cis-regulatory sites for genes activated either early or late during pharyngeal development. Analysis of natural or synthetic promoters reveals that six of these sites function in vivo. The newly discovered temporal elements, together with predicted PHA-4 sites, account for the onset of expression of roughly half of the pharyngeal genes examined. Moreover, combinations of temporal elements and PHA-4 sites can be used in genome-wide searches to predict pharyngeal genes, with more than 85% accuracy for their onset of expression. These findings suggest a regulatory code for temporal gene expression during foregut development and provide a means to predict gene expression patterns based solely on genomic sequence.

Figure 1. Strategy to Identify Temporal Regulatory Elements

(A) Flowchart of the strategy used. (B) Northern blot of pha-4. pha-4 transcripts were approximately 25- to 100-fold enriched in par-1 compared to skn-1 embryos, but only approximately 5- to 10-fold enriched in wild-type compared to skn-1 embryos. Arrowheads indicate the three different pha-4 isoforms. (C) The same blot was probed with a fragment of the act-1 gene to demonstrate equal loading of RNA between lanes.

Figure 2. Different Predicted Products of the Temporal Groups

The Ph-E group is enriched for predicted transcription factors, while the Ph-L group is enriched for predicted structural or muscle proteins (see Table 1). Muscle proteins are proteins known or predicted to be involved in muscle function, including myosins, tropomyosins, and troponins. Table S2 provides a complete listing of the catergorization of the Ph-E and Ph-L genes.

Figure 3. Candidate Pharyngeal Motifs Identified by Improbizer

Improbizer represents motifs as PWMs; the PWMs for the motifs are shown in Dataset S1. We converted these matrices to the “sequence logos” shown here (Schneider and Stephens 1990; Crooks et al. 2004). The threshold scores used by Cluster-Buster (Frith et al. 2003) for each motif are shown below the sequence logos. “% Ph-E Genes” and “% Ph-L Genes” are the percentage of Ph-E and Ph-L genes that contain occurrences of a given motif within 500 bp upstream of the predicted ATG start codon, above the threshold shown. “% Neg Genes” is the percentage of DNA synthesis genes that contain occurrences of a given motif (above the threshold score) within 500 bp upstream of the predicted ATG start codon. Two other negative groups (carbohydrate synthesis genes [Kim et al. 2001] and a set of 194 randomly selected genes) yielded similar results (data not shown). “Enhancer Activity” is the relative strength of expression generated by a motif present in three copies upstream of the Δpes-10::GFP::HIS2B reporter. ND, not determined.

Figure 4. Five Newly Identified Motifs Function as Pharyngeal Enhancers

(A–C) Nomarski differential contrast interference images of embryos representing three different stages of embryonic development: (A) “early” development, when the pharynx primordium is formed, (B) “mid” development, when the pharynx has completed cell division and attached to the presumptive buccal cavity, and (C) “late” development, when pharynx development is almost complete and the embryo is about to hatch. Images on the left are of “early” embryos, images in the middle are of “mid” embryos, and images on the right are of “late” embryos. (D–U) Representative transgenic embryos showing expression from reporter constructs containing the Δpes-10 promoter alone (D–F) or with insertion of three copies of Early-1 (G–I), Early-2 (J–L), Late-2 (M–O), P-1 (P–R), or P-2 (S–U). Dashed lines indicate the outline of the developing pharynx.

Figure 5. Late-1 Represses Early PHA-4-Dependent Expression

Percent values indicate the percentage of transgenic animals exhibiting pharyngeal GFP expression. A reporter construct with three copies of a high-affinity PHA-4 site (TGTTTGC) upstream of the Δpes-10 promoter (A) expresses GFP in pharyngeal cells of most transgenic embryos (B) and roughly one-third of transgenic larvae (C). The addition of three copies of the Late-1 element from R07B1.9 (CCTTGGCGGCGC) to this transgene (D) drastically reduces expression in transgenic embryos (E) but has no observable effect on transgenic larvae (F). Dashed lines indicate the outline of the pharynx.

Figure 6. Early-1 and Early-2 Elements Are Required for K07C11.4 Expression

(A) A portion of the promoter sequence of K07C11.4 from C. elegans (bottom) aligned with its ortholog from C. briggsae (top). Boxed regions show conserved predicted PHA-4 binding sites and Early-1 and Early-2 elements. Site-directed mutations that disrupt Early-1 and Early-2 (“E2 + E1 Mut”) are shown below their respective wild-type (“E2 + E1 WT”) sequence from K07C11.4. (B–E) Confocal images of mid-stage embryos expressing GFP under the control of the wild-type K07C11.4 promoter (B) or promoters with a mutation in Early-1 (C), Early-2 (D), or both Early-1 and Early-2 (E). Percentages are the fraction of transgenic embryos expressing GFP; the remainder of embryos do not express GFP. (F) Expression of the wild-type K07C11.4 reporter in a subset of somatic gonad cells in an L4 animal (arrowheads). (G) Mutation of the Early-1 element eliminates gonadal expression but does not strongly affect expression in other tissues, such as intestinal cells (arrows). Dashed lines indicate the outline of the developing pharynx.

Figure 7. The Late-1 Element Negatively Regulates R07B1.9

(A) A portion of the promoter sequence of R07B1.9 from C. elegans (bottom) aligned with its ortholog from C. briggsae (top). Boxed regions show conserved predicted PHA-4 binding sites and a Late-1 element. The site-directed mutation that disrupts Late-1 (“Mut”) is shown below the respective wild-type sequence from R07B1.9. (B and C) Confocal images of representative early and late embryos expressing GFP under the control of the wild-type R07B1.9 promoter. (D and E) Confocal images of representative early and late embryos expressing GFP under the control of the R07B1.9 promoter with a mutation in the Late-1 element. Note the early activation of R07B1.9 when Late-1 is inactivated. Dashed lines indicate the outline of the developing pharynx.

Figure 8. High-Affinity PHA-4 Sites Activate Pharyngeal Expression Earlier Than Low-Affinity Sites

Percent values indicate the percentage of transgenics exhibiting pharyngeal GFP expression. Dashed lines indicate the outline of the developing pharynx. (A–C) A reporter construct with three copies of a high-affinity PHA-4 site (TGTTTGC) upstream of the Δpes-10 promoter reproducibly activates pharyngeal expression from the time of pharynx primordium formation (“early”) through embryogenesis. (D–F) A reporter construct with three copies of a low-affinity PHA-4 site (TATTTGT) upstream of the Δpes-10 promoter activates pharyngeal expression from the time of attachment of the pharynx to the mouth (“mid”) through embryogenesis.

Figure 9. Temporal Elements Combined with PHA-4 Sites Regulate the Onset of Pharyngeal Expression

“Early,” “mid,” and “late” are as defined in Figure 3. E1, Early-1; L2, Late-2; High, high-affinity PHA-4 site (TGTTTGC); Low, low-affinity PHA-4 site (TATTTGT). Percent values indicate the percentage of transgenics exhibiting pharyngeal GFP expression; the remainder of embryos do not express GFP. A reporter construct with one copy of the Early-1 element and one copy of a high-affinity PHA-4 site is expressed in “early” to “late” embryos (A–C). In contrast, a reporter with one copy of the Early-1 element and one copy of a low-affinity PHA-4 site is not consistently expressed until the “mid” to “late” stages (D–F). Reporters with one copy of Late-2 and one copy of either a high-affinity (G–I) or low-affinity (J–L) PHA-4 site are expressed in “mid” and “late” stage embryos. Dashed lines indicate the outline of the developing pharynx.