Machine Learning Transcriptional Regulators Figure 2. The enhancer classifier performs with high specificity and sensitivity. (A) Over-representation of TFBSs in the training set including only D. melanogaster enhancers and in the set extended using phylogenetic profiling, as compared with background sequence. P-values were adjusted for multiple testing using the method of Benjamini and Hochberg (BH) [120]. (B) Average ROC curve for the 10-fold cross-validation. Our method achieves an area under the ROC curve of 0.89 (shaded in gray). FPR: false-positive rate; TPR: true-positive rate. (C) Distribution of FC enhancer scores for the genome-wide scan. Scores assigned by the classifier for each evaluated sequence are shown in red. We used a FPR of 5% to define a cut-off for putative enhancers (dotted blue line; see Materials and Methods for details). (D) Fold-enrichment in 180 validated FC genes in the neighborhood of putative FC enhancers, as determined for different FPRs. Intergenic putative FC enhancers were associated with the closest gene, whereas intronic sequences were associated with their host gene. P-values were computed using the binomial test. doi:10.1371/journal.pgen.1002531.g002 for an increase in the size of the training set [43–45], and support the use of phylogenetic profiling for expanding the training set. To verify that the orthologous sequences function as FC enhancers, we randomly chose 5 examples to test for transcrip- tional activity in D. melanogaster embryos using transgenic reporter assays. Each enhancer construct was introduced into the same attP site in the D. melanogaster genome using a custom vector containing a green fluorescent protein (GFP) reporter and an attB site allowing phiC31 integrase-mediated integration ([46,47] and B. W. Busser, L. Shokri, S. A. Jaeger, S. S. Gisselbrecht, A. Singhania, M. F. Berger, B. Zhou, M. L. Bulyk and A. M. Michelson, unpublished data). All of the tested sequences drove similar, although not always identical, expression patterns as their orthologous D. melanogaster enhancers (Figure 3). PLoS Genetics | www.plosgenetics.org 5 March 2012 | Volume 8 | Issue 3 | e1002531