Machine Learning Transcriptional Regulators Figure 7. The T-box TF org-1 is a regulator of the lbl and slou FC enhancers. (A) TRANSFAC position weight matrix for Tbx5 (V$Tbx5_Q5), a vertebrate homolog of the Drosophila T-box TF org-1 and a top-scoring feature derived by the FC enhancer classifier. (B, C) In stage 11 embryos containing the lblWT-lacZ transgene, b-Gal (green) co-expresses with endogenous Lb (red) in three cells (arrow) but is absent from Slou-expressing FCs (blue, arrowhead). (D, E) Mutagenesis of T-box motifs in the lbl FC enhancer (lblTbox-lacZ) results in an attenuation of b-Gal (green) reporter activity in the three Lbl-expressing cells (red, arrow). Wild-type and mutant T-box binding sites in the lbl FC enhancer are described in Table S4. (F, G) GFP (green) co-expresses with endogenous Slou (red) in two cells (arrowhead) but not in the three Lbl- expressing cells (blue, arrow) in stage 11 embryos containig the slouWT-gfp transgene. (H, I) Mutagenesis of T-box motifs in the slou FC enhancer (slouTbox-lacZ) results in a marked attenuation of b-Gal (green, arrow) expression in two Slou-expressing cells (red, arrowhead). The asterisks denote de-repression of the lacZ reporter in cells of unknown identity. Wild-type and mutant T-box binding sites in the slou FC enhancer are described in Table S4. (J) GFP (green) fluorescence expression in living stage 14 lbl-GFP embryos is visible in the SBM (arrowhead), in two adult muscle precursors and in several cells of the central nervous system (asterisks) injected with control lacZ dsRNA. (K) Loss of GFP fluorescence from cells corresponding to the wild-type positions of the SBM and two adult muscle precursors but not in cells of the central nervous system (asterisks) in living stage 14 lbl-GFP embryos injected with org-1 dsRNA. (L) GFP (green) fluorescence expression in living stage 14 slou-GFP embryos is visible in muscles LO1 (arrow) and VT1 (arrowhead) injected with control lacZ dsRNA. (M) Loss of GFP fluorescence from cells corresponding to the wild-type positions of LO1 and VT1 in living stage 14 slou-GFP embryos injected with org-1 dsRNA. (N) Co-expression in the segment border muscle (SBM; arrowhead) of endogenous Lbl (red) and b-Gal in stage 14 lblWT-lacZ embryos containing the lblWT-lacZ transgene. (O) Panmesodermal expression of org-1 (Twi.org-1) in stage 14 lblWT-lacZ embryos induces ectopic activation of both endogenous Lb (red) and the b-Ggal reporter reporter (green). (P) Stage 16 wild-type (WT) embryo stained with antibodies directed against myosin heavy chain (MHC; green), Lb (red) and Slou (blue) showing expression of Lb in the single SBM (arrowhead) in each hemisegment. (Q) Panmesodermal expression of org-1 (Twi.org-1) induces duplication of the SBM in some but not all hemisegments (arrowheads). (R) GFP (green) fluorescence expression in living stage 16 MHC-tauGFP embryos is visible in the SBM (arrowhead) and muscle LO1 (arrow) injected with control lacZ dsRNA. (S) Loss of GFP fluorescence from cells corresponding to the wild-type positions of the SBM muscle LO1 in living stage 16 MHC-tauGFP embryos injected with org-1 dsRNA. doi:10.1371/journal.pgen.1002531.g007 except for the small number of cases where individual motifs have been functionally validated [28,29,35,67], each motif occurrence corresponds to a computational prediction without a verified assignable function. Second, the probability of finding a motif match is increased in longer genomic sequences, whereas the minimally active region has not been determined for most enhancers in this set. One notable exception is the enhancer directing FC expression of eve, where a minimally active regulatory element has been defined. In the case of eve, an approximately 300 bp sequence contains multiple instances of 6 different TFBSs (Figure 8 and Table S6), all 6 of which have been functionally validated as contributing to FC activity [28,29]. Third, PWMs, which are critically dependent on particular thresholds to limit false positives and negatives, were used to identify motifs within each enhancer. Fourth, the various PWMs have different relative information contents (Table S6), a parameter which affects the likelihood that a match will be found in any given sequence. Fifth, since many of these enhancers are active in cell types other than FCs, not all motifs that are present will necessarily contribute to FC activity. Notwithstand- ing these potential limitations, the present results suggest that the specificity of enhancer activities observed at the level of individual muscle FCs is reflected in the diversity of the TFBS compositions of these regulatory elements. PLoS Genetics | www.plosgenetics.org 12 March 2012 | Volume 8 | Issue 3 | e1002531