muscle identity TFs and downstream target genes [18,19]. Thus, 30 distinct cell states exist, each governed by a different but partially overlapping set of regulatory TFs. In contrast to the difficulties involved in dissecting regulatory codes at single cell resolution, shared features that direct activity to the general level of tissues and organs have been more readily identified using a machine learning approach, as was found here for enhancers having mesodermal, although not necessarily FC, activity. This likely reflects the dominant role that some TFs play in the regulatory network specifying the identities of numerous tissues [86–91]. Fifth, since there appears to be a regulatory signature for enhancers [16,92], it is likely that these aspects of enhancer structure will be more significantly over-represented than those features that specify individual FC activity patterns. Sixth, the use of phylogenetic profiling might have expanded the biological function of the training dataset by introducing additional enhancer functions acquired by the orthologs of the original D. melanogaster sequences during their evolution. While we have been able to show that the phylogenetic profiling approach improves the accuracy of the classifier, one drawback of its use might be that the final classifier recognizes a broader biological domain than the function of the original training set of sequences derived from the reference species. Finally, classifier predictions may represent cis-regulatory elements other than enhancers, for example, silencers and insulators [2], which would not be detected by our transgenic reporter assays. In summary, a number of confounding factors influenced our ability to identify an enhancer signature that is specific for individual muscle FCs. However, despite these challenges, our successful identification of novel TF binding motifs responsible for the cell type-specific activity of FC enhancers encourages us that this is a tractable problem that can be solved by an iterative approach to the computational analysis of this and other complex developmental systems. Thus, future studies must focus on obtaining a larger training set of sequences in which enhancers are categorized based on their activities at single cell resolution, combined with the appropriate weighting of newly validated motifs that contribute to the expression pattern of interest. In this manner, each experimental round would improve the accuracy of the classifier. Sequence Motifs Associated with FC Enhancers Are Functional and Can Be Used to Identify Novel Trans- Acting Factors The motifs ranked by our classifier as having the highest discriminatory power are part of a large regulatory network that is known to be critical for mesoderm specification and myogenesis. These motifs include binding sites for JAK/STAT [57], Ets [93], bHLH [94,95], Wingless/Tcf [49,96,97], Mef2 [66], homeodo- main ([19,20] 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) and forkhead (X. Zhu, S. M. Ahmad, A. Aboukhalil, B. W. Busser, Y. Kim, T. R. Tansey, A. Haimovich, N. Jeffries, M. L. Bulyk, and A. M. Michelson, unpublished data) proteins. Furthermore, we previously suggested that Ets is part of a transcriptional code regulating the C1 subset of FC genes [5], which we validated here using site-directed mutational analysis of the Ndg enhancer, a previously character- ized regulatory element associated with a C1 FC gene. To extend the components of the myogenic regulatory network beyond these known TFs and motifs, we examined the function of the classifier-defined sequence motifs recognized by POU homeodomain and Myb proteins, transcription factors having no previously known role in Drosophila myogenesis. Mutagenesis of Machine Learning Transcriptional Regulators POUHD motifs attenuated the activity of the Ndg enhancer in many mesodermal cells. However, a zygotic loss-of-function mutation in acj6, the only POUHD that we found to be expressed in the mesoderm, had no effect on Ndg gene expression (data not shown). Given the strong maternal contribution to this gene [98], we used RNAi to knock down both maternal and zygotic acj6 transcripts, but this manipulation had no effect on Ndg-GFP reporter activity (data not shown). These findings leave unresolved the identity of the TF that binds to the motif in question. The future characterization of this TF, including exploring the possibility that it is not a POUHD protein, will require searching functional motifs against larger TF databases [99] or with STAMP [60], combined with analysis of the embryonic expression and function of any new candidates that emerge. Inactivating mutations of the Myb binding sites in the Ndg enhancer led to extensive de-repression of the reporter in other mesodermal cells. Myb is a ubiquitously-expressed DNA binding protein which plays a critical role in controlling regulatory decisions during proliferation and differentiation of progenitor cells [100]. Identifying a putative role for Myb in myogenesis documents the power of this approach, since functional studies tend to focus on genes with restricted expression patterns. However, a definitive assessment requires examining the effect of loss-of-function mutations in Myb. In any event, as myogenesis in Drosophila occurs through a series of asymmetric and symmetric cell divisions [101], a role for Myb in regulating FC gene expression is entirely consistent with a transcriptional regulator acting at the interface between replication and transcription [102,103]. Alternatively, Myb may cooperate with other TFs to activate cell or tissue-specific gene expression [104]. Interestingly, T-box motifs scored well in the classification, yet no role for T-box TFs has previously been described in Drosophila somatic muscle development, despite widespread functions of this TF class in mesoderm specification and myogenesis in vertebrates [105,106], as well as cardiogenesis in Drosophila and vertebrates [62,63,107]. Here we show using both cis and trans tests of TF function, along with gene co-expression, that Org-1 is a muscle identity TF. In particular, the cis effects of Org-1 were documented in the FC enhancers associated with two known muscle identity TFs, Slou and Lbl, and org-1 expression localizes to the SBM and VT1, muscles in which the lb genes and slou, respectively, are the only previously described determinants of muscle identity [54,108]. Slou function is critical for the proper development of muscles LO1 and VT1 and is further required to repress the lb genes in these cells, suggesting a co-regulatory relationship between slou and lb [54]. It is likely that org-1 acts upstream of slou and lb in this regulatory hierarchy since org-1 expression precedes slou and lb, and the ectopic expression of org-1 causes increased expression of slou and lb (Figure 7 and data not shown). In addition, the essential role of org-1 in this regulatory network is revealed by the effects of org-1 overexpression and RNAi knockdown on development of lb- and slou-expressing muscles. Interestingly, the mouse orthologs of org-1 and lb genes, Tbx1 and Lbx1, respectively, have been suggested to regulate myogenic differentiation in the limb [109–111]. Given the high degree of sequence similarity, and the close correspondence of expression patterns and functions in Drosophila and mouse, the collaborative roles of these two TFs in myogenesis appear to have been conserved through evolution. Motif Combinatorics in FC Enhancers Computational prediction of regulatory elements requires a thorough understanding of the TFs and motifs that orchestrate gene co-expression patterns. In prior studies, we established that 5- PLoS Genetics | www.plosgenetics.org 15 March 2012 | Volume 8 | Issue 3 | e1002531