way and 3-way ‘‘AND’’ combinations of 3 signal-activated (Tcf, Mad and Pnt) plus 2 tissue-restricted (Twi and Tin) TFs constitute distinct regulatory models for different FC enhancers [5,28,29]. The present study significantly extend these prior combinatorial codes for FC gene regulation by identifying four additional classes of TFBSs that are critical for accurate FC enhancer activity, namely POUHD, Myb, Fkh and T-box motifs. Moreover, these findings provided us with an opportunity to examine the complete spectrum of regulatory motif usage across a collection of regulatory elements that are active in different muscle FCs, which led to the identification of 18 unique combinations of 11 TFBSs for the entire set of 18 known FC enhancers. Thus, unlike other cases that have been studied, a single enhancer archetype does not appear to exist for this subpopulation of myoblasts [23–25,50]. This finding likely reflects the fact that although these elements all display FC activity, with some overlap at the level of individual cells, no two FC gene expression patterns directed by this enhancer set are identical. The marked heterogeneity of FC enhancer architecture uncovered here reflects not only distinct combinations of various TF classes (including signal-activated, ubiquitous and both tissue- and cell type-specific TFs), but also diversity at other biological levels, including the unique identities of the thirty muscle FCs and their differentiated derivatives in each abdominal hemisegment, and the different gene expression patterns exhibited by those particular cells. Thus, TFBS combinatorics provide a plausible molecular explanation for the functional complexity of enhancers having related but non-identical activites at the resolution of individual cells in the context of the developing embryo. Conclusions We have investigated the transcriptional regulatory network specifying individual muscle FCs using an integrated genomics approach that includes identification of orthologous enhancers, de novo motif discovery, classification of enhancer sequence features, empirical testing of candidate enhancers, and cis-trans tests of target gene regulation. We also have established that a small set of training sequences can be expanded with orthologous sequences [76]. Moreover, motifs learned by the classifier were empirically found to be critical for the appropriate spatio-temporal activities of FC enhancers, and suggested new candidate TFs in the myogenic regulatory network. Using this approach, we identified one such candidate TF, Org-1, as a novel muscle identity TF, and further found that no two enhancers with related activities contain the same combination of TFBSs. The tools and strategy used here can be readily applied to other cell types to identify the motifs and trans-acting factors regulating a set of co-expressed genes. Finally, we anticipate that an iterative application of this approach, which could include training on datasets of different epigenetic marks associated with active enhancers [18,80,112,113] or previous ChIP studies of known mesodermally-relevant TFs [114], will lead to further refinements in the determination of cell type-specific transcriptional codes. Materials and Methods Fly Stocks Drosophila stocks containing the following transgenes and mutant alleles were used: UAS-org-1 (gift of G. Pflugfelder, Univ. Wurzburg, Germany), attP40 and nos-phiC31intNLS [115] (gift of N. Perrimon, Harvard University, USA), lbl-lacZ and Ndg-lacZ [5], ap-lacZ (gift of J. Botas, Baylor College, USA) [35], acj66 (gift of J. Carlson, Yale University, USA) [116], and twi-Gal4 [49]. Machine Learning Transcriptional Regulators Analysis of Transgenic Reporter Constructs and Embryo Staining Enhancer regions were either synthesized in vitro (Integrated DNA Technologies, Coralville, IA, USA) or PCR-amplified and sequence-verified and then subcloned into the reporter vector pWattB-GFP (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) or pWattB-nlacZ. The pWattB-nlacZ vector was constructed by cloning the EcoRI-SpeI fragment from a version of pH-pelican [117] in which nuclear lacZ replaced cytoplasmic lacZ into the EcoRI-SpeI sites in the pWattB-GFP vector. All constructs were targeted to attP40 [47] with phiC31-mediated integration [46], and homozygous viable insertion lines were obtained. Whole-embryo immunohistochem- istry, in situ hybridization and fluorescent in situ hybridization with tyramide signal amplification (Invitrogen, Carlsbad, CA, USA) followed standard protocols [28]. Embryo collections for twi-Gal4 UAS-org-1 were incubated at 25uC. For fluorescent staining, the following antibodies were used: mouse anti-Ladybird early (Lbe) (1:2500, gift of K. Jagla; Lbe and Lbl are co-expressed in the same mesodermal cells), rabbit anti-Slou (1:200, gift of M. Baylies), chicken anti-GFP (1:2000, Abcam, Cambridge, MA), mouse anti- bgal (1:500, Promega, Madison, WI), rabbit anti-Kirre (1:200, gift of K. Fischbach), rabbit anti-MHC (1:500, gift of D. Kiehart), and guinea pig anti-Eve (1:200, gift of D. Kosman). RNA Interference Assay Embryo RNAi was performed as previously described [22]. Using SnapDragon (http://www.flyrnai.org/cgi-bin/RNAi_find_ primers.pl), two independent gene segments for synthesis of org-1 double-stranded RNA (dsRNA) were selected with lengths of 570 and 473 bp and with less than 20 and 22 bp of identity to any other predicted gene, respectively. These segments of the org-1 gene were PCR-amplified from primary embryonic cDNA using the primers, CGTCCAAAAAGTTCAAGGGA and GCTCGTTCTCATC- CAAGGAG (570 bp) and GCTCCAACAGAGCCAGAATC and CCGAACCGTAAAAACTTGGA (473 bp), and transcribed in vitro using the MEGAscript RNAi kit (Ambion, USA). lbl-GFP, slou-GFP or MHC-tauGFP embryos were dechorionated and injected with negative control (lacZ) or org-1 dsRNA at the syncytial blastoderm stage and allowed to develop to stage 14 or 16 before examination by fluorescence microscopy for assessment of reporter GFP expression. A similar protocol was used to assess the function of acj6 in Ndg-GFP embryos. FC Enhancer and Control Sequences The sixteen sequences in the training set of Drosophila melanogaster FC enhancers range in length from 311 to 2068 bp (average length 1232 bp), in GC-content from 39% to 49% (average GC-content 43%), and in repeat-content from 0% to 7% (average 1.5%). The twenty-four orthologs have similar characteristics, with an average length of 1311 bp, GC-content of 43% and repeat content of 5%. The control set comprised 1000 randomly selected D. melanogaster noncoding genomic sequences with length, GC- and repeat- content matching those of the enhancer set. Identification of TFBSs Putative TFBSs were identified by searching the sequences with MAST [118] for motifs in TRANSFAC Release 2009.2 [119], in addition to binding sequences for Tin, Twi and Pnt from the literature [5]. MAST was run independently on each individual sequence with default setup and parameters. In particular, for the final analysis of the TFBS composition of FC enhancers, we PLoS Genetics | www.plosgenetics.org 16 March 2012 | Volume 8 | Issue 3 | e1002531