expected by chance (P-value = 5.8610210; Figure 2D). The latter result suggests that the sequence features learned by the classifier have specificity for FC enhancer function. Functional Assessment of Enhancers Predicted by the Classifier To test the functions of the classifier-predicted enhancers, we first asked if the presence of a high-scoring putative FC enhancer could be used to predict expression in FCs [52]. To more readily associate an enhancer with its putative target gene, we examined the expression patterns of genes with a high-scoring intronic enhancer, which was assumed to control the gene in which it is located. In total, 5 genes out of 20 tested (25%) were actually expressed in FCs (Table S3). This is 8-times higher than would be predicted by chance (P,0.002), given that only 3% of Drosophila genes have been estimated to be expressed in FCs [22]. As an example, defective proventriculus (dve) was identified and validated as a novel FC gene using this approach (Figure 4A). In summary, since the presence of putative FC enhancers is strongly associated with FC gene expression, it is likely that a large fraction of FC enhancer Machine Learning Transcriptional Regulators predictions represent authentic FC regulatory sequences, including FC-specific enhancers and possibly silencers and insulators [2]. To directly assess the in vivo functions of these candidate enhancers, we used site-specific transgenic reporter assays to test 12 enhancer predictions associated with known FC genes. We assayed the activities of genomic regions with varying scores in the classifier ranking (Table S3). Whereas 9 out of the 12 candidates were found to have enhancer activity, 4 of these were functional in the mesoderm, with 2 directing reporter expression in muscle FCs (Figure 4). Forty-four percent (4/9) of enhancers driving expression in mesoderm represents a validation rate comparable with p300 based ChIP-Seq discovery of tissue-specific enhancers [53], while 2/9 FC enhancers in the set was below expected. These findings presumably reflect the limitations of the training set which, as previously noted, contain only 2 enhancers with specificity restricted only to FCs. Other factors contributing to this outcome are considered in the Discussion. One informative example of a newly identified FC enhancer is that associated with slou (Figure 4B). This enhancer is found upstream of the gene in a region previously shown to recapitulate the complete FC expression of slou [54], but it is active in only a Figure 4. Candidate enhancers predicted by the classifier are active in FCs. In situ hybridization of dve in wild-type (WT) embryos and embryos over-expressing Ras (Twi.Ras) in the mesoderm (A). Note the increased activity of dve in Twi.Ras embryos, indicative of a FC gene [22]. GFP driven by the classifier-predicted enhancers associated with the upstream sequences of slou (arrows in B) and slp1 (arrows in C). Slou protein (magenta) co-expresses with GFP (green) in slou-GFP embryos (B). Duf (magenta), which marks all FCs, co-expresses with slp1-GFP (green) (C). GFP (D) driven by the classifier-predicted intronic sequence associated with the dve gene co-expresses with Mef2 (D9) in myotubes at stage 15 in dve-GFP embryos. doi:10.1371/journal.pgen.1002531.g004 PLoS Genetics | www.plosgenetics.org 8 March 2012 | Volume 8 | Issue 3 | e1002531