To quantitatively model the developmental functions of cis-regulatory modules (CRMs), Eileen Furlong and colleagues used synthetic engineering to systematically interrogate the individual and combinatorial contributions of transcription factor binding site motifs to enhancer activity in Drosophila melanogaster development (PLoS Genet. 10, e1004060, 2014). The authors engineered 63 synthetic cis-regulatory elements from 10 binding site motifs recognized by transcription factors of the Wingless and Dpp pathways and stably integrated them into the Drosophila genome. Each synthetic CRM was composed of six motifs and neutral spacer sequences, and enhancer potential was assessed by reporter expression during embryonic development. They determined that, for some motifs, homotypic clusters were able to drive specific spatiotemporal expression patterns that recapitulate in vivo expression of the corresponding transcription factor. To interrogate the importance of motif organization, the authors generated heterotypic CRMs composed of motifs for transcription factors known to interact and found that altering the spacing and organization of some motifs can affect enhancer activity in a tissue-specific manner. The authors quantified variability in CRM-driven expression and used fractional site occupancy modeling to determine that transcription factor cooperativity has an important role in CRM activity in some tissues.