Figure S8 Co-expression of org-1 with Slou and Lbl. Expression of org-1 RNA in stage 11 (A) and stage 13 (B) embryos detected by in situ hybridization. Co-expression of org-1 RNA (purple) with Lbl protein (brown) in the Lbl-expressing SBM FC (stage 11; C) and myotube (stage 13; D). Co-expression of org-1 RNA (purple) with Slou protein (brown) in the LO1 and VT1 FCs (stage 11; E) and myotubes (stage 13; F). CVM: circular visceral muscle which expresses org-1 but neither lbl nor slou. (TIF) Figure S9 Motif distribution in FC enhancers. (A) The heatmap illustrates the occurrence of 11 motifs that have been shown to be relevant for FC regulation in the 18 sequences that have been positively assayed for FC enhancer activity. Columns and rows are clustered using Ward’s method and binary distances. (B) Maximum fraction of the 18 assayed FC enhancer sequences sharing N motifs that have been shown to be relevant for FC regulation, for N in {1, 2, …, 11}. (TIF) Table S1 Expression and genomic coordinates of D. melanogaster and orthologous enhancer regions used for training, and the list of FC genes considered in this study. (XLSX) Table S2 Genomic coordinates and ontology of REDfly D. melanogaster mesodermal enhancers. The majority of these enhancers have multiple activities. (XLSX) References 1. Carroll SB, Grenier JK, Weatherbee SD (2005) From DNA to Diversity. Molecular Genetics and the Evolution of Animal Design. MaldenMassachu- Massachusetts: Blackwell Publishing. 2. Davidson E (2006) The Regulatory Genome: Gene Regulatory Networks In Development And Evolution: Academic Press. 304 p. 3. Berman BP, Nibu Y, Pfeiffer BD, Tomancak P, Celniker SE, et al. (2002) Exploiting transcription factor binding site clustering to identify cis-regulatory modules involved in pattern formation in the Drosophila genome. Proc Natl Acad Sci U S A 99: 757–762. 4. Markstein M, Markstein P, Markstein V, Levine M (2002) Genome-wide analysis of clustered Dorsal binding sites identifies putative target genes in the Drosophila embryo. Proc Natl Acad Sci U S A 99: 763–768. 5. Philippakis AA, Busser BW, Gisselbrecht SS, He FS, Estrada B, et al. (2006) Expression-guided in silico evaluation of candidate cis regulatory codes for Drosophila muscle founder cells. PLoS Comput Biol 2: e53. doi:10.1371/ journal.pcbi.0020053. 6. Mahaffey JW (2005) Assisting Hox proteins in controlling body form: are there new lessons from flies (and mammals)? Curr Opin Genet Dev 15: 422–429. 7. Narlikar L, Sakabe NJ, Blanski AA, Arimura FE, Westlund JM, et al. (2010) Genome-wide discovery of human heart enhancers. Genome Res 20: 381–392. 8. MacArthur S, Li XY, Li J, Brown JB, Chu HC, et al. (2009) Developmental roles of 21 Drosophila transcription factors are determined by quantitative differences in binding to an overlapping set of thousands of genomic regions. Genome Biol 10: R80. 9. Biggin MD, McGinnis W (1997) Regulation of segmentation and segmental identity by Drosophila homeoproteins: the role of DNA binding in functional activity and specificity. Development 124: 4425–4433. 10. Ivan A, Halfon MS, Sinha S (2008) Computational discovery of cis-regulatory modules in Drosophila without prior knowledge of motifs. Genome Biol 9: R22. 11. Leung G, Eisen MB (2009) Identifying cis-regulatory sequences by word profile similarity. PLoS ONE 4: e6901. doi:10.1371/journal.pone.0006901. 12. Nazina AG, Papatsenko DA (2003) Statistical extraction of Drosophila cis- regulatory modules using exhaustive assessment of local word frequency. BMC Bioinformatics 4: 65. 13. Kantorovitz MR, Kazemian M, Kinston S, Miranda-Saavedra D, Zhu Q, et al. (2009) Motif-blind, genome-wide discovery of cis-regulatory modules in Drosophila and mouse. Dev Cell 17: 568–579. 14. Abnizova I, te Boekhorst R, Walter K, Gilks WR (2005) Some statistical properties of regulatory DNA sequences, and their use in predicting regulatory regions in the Drosophila genome: the fluffy-tail test. BMC Bioinformatics 6: 109. 15. Nazina AG, Papatsenko DA (2003) Statistical extraction of Drosophila cis- regulatory modules using exhaustive assessment of local word frequency. BMC Bioinformatics 4: 65. Machine Learning Transcriptional Regulators Table S6 (XLSX) Text S1 and TFBS Enhancers. (DOCX) Table S3 Classifier predictions and the activity and genomic coordinates of the tested enhancer predictions. (XLSX) Table S4 Motifs identified by the classifier and the mapping of TFBSs in the Ndg, lbl, slou and ap FC enhancers. (XLSX) Table S5 Summary of in situ hybridization analysis of T-box and POUHD family members. (XLSX) Mapping of TFBSs in all FC enhancers. Conservation Profile of Candidate FC Enhancers Distribution Among Orthologs of Candidate FC Acknowledgments We thank N. Perrimon, G. Pflugfelder, J. Botas, M. Baylies, K. Fischbach, and K. Jagla for providing fly strains and antibodies; S. Gisselbrecht for the pWattB-GFP vector; and C. Sonnenbrot for technical assistance. Author Contributions Conceived and designed the experiments: BWB LT YK TT IO AMM. Performed the experiments: BWB LT YK TT. Analyzed the data: BWB LT YK TT IO AMM. Contributed reagents/materials/analysis tools: MJB. Wrote the paper: BWB LT IO AMM. 16. Li L, Zhu Q, He X, Sinha S, Halfon MS (2007) Large-scale analysis of transcriptional cis-regulatory modules reveals both common features and distinct subclasses. Genome Biol 8: R101. 17. Busser BW, Bulyk ML, Michelson AM (2008) Toward a systems-level understanding of developmental regulatory networks. Curr Opin Genet Dev 18: 521–529. 18. Ruiz-Gomez M (1998) Muscle patterning and specification in Drosophila. Int J Dev Biol 42: 283–290. 19. Baylies MK, Bate M, Ruiz Gomez M (1998) Myogenesis: a view from Drosophila. Cell 93: 921–927. 20. Tixier V, Bataille L, Jagla K (2010) Diversification of muscle types: recent insights from Drosophila. Exp Cell Res 316: 3019–3027. 21. Berkes CA, Tapscott SJ (2005) MyoD and the transcriptional control of myogenesis. Semin Cell Dev Biol 16: 585–595. 22. Estrada B, Choe SE, Gisselbrecht SS, Michaud S, Raj L, et al. (2006) An Integrated Strategy for Analyzing the Unique Developmental Programs of Different Myoblast Subtypes. PLoS Genet 2: e16. doi:10.1371/journal. pgen.0020016. 23. Crocker J, Potter N, Erives A (2010) Dynamic evolution of precise regulatory encodings creates the clustered site signature of enhancers. Nature communi- cations 1: 99. 24. Crocker J, Tamori Y, Erives A (2008) Evolution acts on enhancer organization to fine-tune gradient threshold readouts. PLoS Biol 6: e263. doi:10.1371/ journal.pbio.0060263. 25. Erives A, Levine M (2004) Coordinate enhancers share common organizational features in the Drosophila genome. Proc Natl Acad Sci USA 101: 3851–3856. 26. Ludwig M, Bergman C, Patel N, Kreitman M (2000) Evidence for stabilizing selection in a eukaryotic enhancer element. Nature 403: 564–567. 27. Swanson CI, Evans NC, Barolo S (2010) Structural rules and complex regulatory circuitry constrain expression of a Notch- and EGFR-regulated eye enhancer. Dev Cell 18: 359–370. 28. Halfon MS, Carmena A, Gisselbrecht S, Sackerson CM, Jime´nez F, et al. (2000) Ras pathway specificity is determined by the integration of multiple signal-activated and tissue-restricted transcription factors. Cell 103: 63–74. 29. Halfon MS, Grad Y, Church GM, Michelson AM (2002) Computation-based discovery of related transcriptional regulatory modules and motifs using an experimentally validated combinatorial model. Genome Res 12: 1019–1028. 30. Han Z, Fujioka M, Su M, Liu M, Jaynes JB, et al. (2002) Transcriptional integration of competence modulated by mutual repression generates cell-type specificity within the cardiogenic mesoderm. Dev Biol 252: 225–240. 31. Knirr S, Frasch M (2001) Molecular integration of inductive and mesoderm- intrinsic inputs governs even-skipped enhancer activity in a subset of pericardial and dorsal muscle progenitors. Developmental Biology 238: 13–26. PLoS Genetics | www.plosgenetics.org 19 March 2012 | Volume 8 | Issue 3 | e1002531