Machine Learning Transcriptional Regulators Figure 5. DNA binding domains of the TFs most relevant to FC enhancer classification. Only DNA binding domains for the fifty most relevant TFs have been included. TFs were ranked according to the SVM weights of their respective motifs, which represent their discriminating power. Only the highest scoring motif for each TF was considered (median ranks computed across 10 random partitions of the training data varied between 12 and 117). De novo motifs were explicitly excluded from this analysis. TF domains and sequences have been clustered using average linkage and Euclidean distance. The dendogram on top of the heatmap represents the relationships among the sequences in the training data, built on the presence/absence of TFBSs recognized by a specific class of TF DNA binding domain. The dendogram on the left of the heatmap shows the relationships among the different TF DNA binding domains. doi:10.1371/journal.pgen.1002531.g005 expressed with Slou in the FCs corresponding to muscles LO1 and VT1, and with Lbl in the FC for the segment border muscle (SBM) (Figure S8). The previous co-expression studies raise the possibility that org-1 may directly regulate slou and lbl. To test this hypothesis, we identified potential T-box binding sites in the lbl and slou FC enhancers (Table S4). The previously described lbl muscle enhancer is active in the SBM and in two adult muscle precursor cells [5], while the slou FC enhancer identified in the present work is active in the two FCs which become muscles LO1 and VT1 (Figure 4B). Of note, the slou FC enhancer was predicted by the classifier due to the presence of a combination of motifs, including those that bind to T-box TFs. Targeted mutagenesis of the T-box sites in otherwise wild-type lbl (Figure 7D and 7E) and slou (Figure 7H and 7I) enhancers revealed that these sites are essential for full enhancer activity (compare to the wild-type versions in Figure 7B, 7C and 7F, 7G respectively). These results suggest that Org-1 is a direct activator of slou and lbl expression in these three FCs. If this is the case, then org-1 loss- and gain-of-function should lead to decreased and increased expression, respectively, of the putative target genes [65]. In agreement with this expectation, RNAi-mediated knockdown of org-1 causes loss of lbl-GFP (Figure 7K) and slou-GFP (Figure 7M) activity, whereas panme- sodermal overexpression of org-1 is associated with ectopic activation of both the endogenous lbl gene and the lbl enhancer- driven reporter (Figure 7O), as well as duplication of the SBM in late-stage embryos (Figure 7Q). These results suggest that Org-1 is a direct regulator of lbl and that it also contributes to the development of the lbl-expressing muscle. Consistent with the latter prediction, RNAi-mediated knockdown of org-1 in embryos expressing tau-GFP under control of a myosin heavy chain enhancer revealed a loss of both the SBM and muscle LO1 (Figure 7S). In summary, our computational enhancer classifica- tion not only led to the discovery of a T-box regulatory motif, but also facilitated the identification of org-1 as encoding a TF critical for FC enhancer activity and for determining muscle FC identity. TFBS Composition of FC Enhancers Having identified and experimentally validated the functions of 4 novel TFBSs that we found to be over-represented in FC enhancers—POUHD, Myb, Fkh and T-box—we were next PLoS Genetics | www.plosgenetics.org 10 March 2012 | Volume 8 | Issue 3 | e1002531