Identification of novel direct targets of Drosophila Sine oculis and Eyes absent by integration of genome-wide data sets.

Drosophila eye development is a complex process that involves many transcription factors (TFs) and interactions with their cofactors and targets. The TF Sine oculis (So) and its cofactor Eyes absent (Eya) are highly conserved and are both necessary and sufficient for eye development. Despite their many important roles during development, the direct targets of So are still largely unknown. Therefore the So-dependent regulatory network governing eye determination and differentiation is poorly understood. In this study, we intersected gene expression profiles of so or eya mutant eye tissue prepared from three different developmental stages and identified 1731 differentially expressed genes across the Drosophila genome. A combination of co-expression analyses and motif discovery identified a set of twelve putative direct So targets, including three known and nine novel targets. We also used our previous So ChIP-seq data to assess motif predictions for So and identified a canonical So binding motif. Finally, we performed in vivo enhancer reporter assays to test predicted enhancers from six candidate target genes and find that at least one enhancer from each gene is expressed in the developing eye disc and that their expression patterns overlap with that of So. We furthermore confirmed that the expression level of predicted direct So targets, for which antibodies are available, are reduced in so or eya post-mitotic knockout eye discs. In summary, we expand the set of putative So targets and show for the first time that the combined use of expression profiling of so with its cofactor eya is an effective method to identify novel So targets. Moreover, since So is highly conserved throughout the metazoa, our results provide the basis for future functional studies in a wide variety of organisms.