Machine Learning Transcriptional Regulators Figure 3. Orthologous sequences are functional enhancers. GFP (green) expression in transgenic stage 11 D. melanogaster embryos containing the indicated GFP reporter constructs driven by the D. ananassae lbl (A), D. mojavensis ap (B), D. persimilis Ndg (C), D. ananassae Ndg (D), and D. persimilis eve (E) enhancers. Co-expression of GFP driven by the D. ananassae lbl enhancer with endogenous Lbl protein (magenta, A9) and D. persimilis eve enhancer with endogenous Eve protein (magenta, E9). b-Gal driven by the D. melanogaster versions of the Ndg (C9, D9) and ap (B9) enhancer co-expresses in some but not all mesodermal cells with GFP driven by the orthologous sequences. doi:10.1371/journal.pgen.1002531.g003 Of note, in several cases, we observed differences in the organization of TFBSs within D. melanogaster FC enhancers and their orthologs, which did not appear to affect their transcriptional activities. For example, the D. melanogaster lbl FC enhancer contains multiple binding sites for each of Pnt, Twi and Tin [5], whereas there are several Pnt, only one Twi and no Tin binding sites in the candidate D. ananassae lbl enhancer (Figure S3A). Nevertheless, both the D. melanogaster and D. ananassae enhancers direct reporter expression in the same two adult muscle precursors and single embryonic muscle FC in which endogenous lbl is expressed (Figure 3A). In this case, the cellular specificity achieved by the orthologous enhancer might be accounted for by the perfect conservation of a single binding site that is preferred by the Slouch (Slou) homeodomain TF, which we have recently shown to be critical in repressing activity of the D. melanogaster lbl enhancer in two Slou-expressing FCs (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L. Shokri, S. A. Jaeger, S. S. Gisselbrecht, A. Singhania, M. F. Berger, B. Zhou, M. L. Bulyk and A. M. Michelson, unpublished data). In other examples, the expression patterns driven by ortholo- gous enhancers were similar but did not precisely replicate those of their D. melanogaster counterparts. For example, the ap muscle enhancer is active in a subset of endogenous ap-expressing muscles and was previously shown to depend on the input of Hox TFs [35]. Interestingly, only 3 out of the 5 known functional Hox binding sites are conserved between the D. melanogaster and the D. mojavensis orthologous sequences (Figure S3B). To compare activities of the orthologous enhancers, we generated a D. melanogaster transgenic line containing a D. mojavensis ap-GFP reporter construct and crossed it to a D. melanogaster ap-lacZ reporter strain. This experiment revealed that the candidate D. PLoS Genetics | www.plosgenetics.org 6 March 2012 | Volume 8 | Issue 3 | e1002531