Identification of muscle-specific regulatory modules in Caenorhabditis elegans

Abstract

Transcriptional regulation is the major regulatory mechanism that controls the spatial and temporal expression of genes during development. This is carried out by transcription factors (TFs), which recognize and bind to their cognate binding sites. Recent studies suggest a modular organization of TF-binding sites, in which clusters of transcription-factor binding sites cooperate in the regulation of downstream gene expression. In this study, we report our computational identification and experimental verification of muscle-specific cis-regulatory modules in Caenorhabditis elegans. We first identified a set of motifs that are correlated with muscle-specific gene expression. We then predicted muscle-specific regulatory modules based on clusters of those motifs with characteristics similar to a collection of well-studied modules in other species. The method correctly identifies 88% of the experimentally characterized modules with a positive predictive value of at least 65%. The prediction accuracy of muscle-specific expression on an independent test set is highly significant (P<0.0001). We performed in vivo experimental tests of 12 predicted modules, and 10 of those drive muscle-specific gene expression. These results suggest that our method is highly accurate in identifying functional sequences important for muscle-specific gene expression and is a valuable tool for guiding experimental designs.

Figure 1.

Two examples of comparison between predicted modules and experimentally defined modules. The lines below the scale represent the experimentally defined modules with start and end positions labeled. The black filled box represents the DNA sequence of corresponding gene. The end at the right side is −1 position of the gene. The small filled boxes below represent predicted modules with start and end positions labeled below. Black filled triangles indicate translational start sites. (A) For C09D1.1a, −1 to −588 bp upstream of ATG is an experimentally defined module. Our method predicted three modules and two are located within the first 588 bp (−66 to −209, −235 to −280). (B) T18D3.4 has two experimentally defined modules, −17 to −239 and −370 to −764. We predicted three modules, and all three are located within experimentally defined regions.

Figure 2.

ROC curves of muscle gene prediction. Genomic genes are ranked by their muscle-specificity score. We plotted the ROC curves of the set of well-characterized muscle-specific genes that were not used for motif identification (44 test set). The diagonal line represents the result of random guessing. The Y-axis is the fraction of true positives exceeding the cutoff for every cutoff value. The X-axis is the fraction of true negatives that exceed the same cutoff.

Figure 3.

GFP expression pattern driven by DNA sequences encompassing the predicted modules. (A) C01B7.1::gfp expression in the pharyngeal muscle. (B) C10G11.7::gfp expression in neurons. F27D4.2::gfp expression in pharyngeal muscle (C) and body wall muscles (D, arrow). F45D3.2::gfp expression in the body wall muscle (E, arrows) and neurons (F). T28B8.1::gfp expression in the pharyngeal muscle (G), vulva muscle (H, arrow), and neurons (I). W06H8.6::gfp expression in the body wall muscle (J), vulva muscle (K), pharyngeal muscle (L), and H cells (data not shown). (M) A merge of DIC image and fluorescent image taken for the same worm showing W06H8.6::Δpes10::gfp expression in the pharyngeal muscle cells. (N) K10G6.3::gfp expression in the pharyngeal muscle (arrow) and neurons.