CisTarget X is a novel computational method that accurately predicts Atonal governed regulatory networks in the retina of the fruit fly.
A comprehensive systems-level understanding of developmental programs requires the mapping of the underlying gene regulatory networks. While significant progress has been made in mapping a few such networks, almost all gene regulatory networks underlying cell-fate specification remain unknown and their discovery is significantly hampered by the paucity of generalized, in vivo validated tools of target gene and functional enhancer discovery. We combined genetic transcriptome perturbations and comprehensive computational analyses to identify a large cohort of target genes of the proneural and tumor suppressor factor Atonal, which specifies the switch from undifferentiated pluripotent cells to R8 photoreceptor neurons during larval development. Extensive in vivo validations of the predicted targets for the proneural factor Atonal demonstrate a 50% success rate of bona fide targets. Furthermore we show that these enhancers are functionally conserved by cloning orthologous enhancers from Drosophila ananassae and D. virilis in D. melanogaster. Finally, to investigate cis-regulatory cross-talk between Ato and other retinal differentiation transcription factors (TFs), we performed motif analyses and independent target predictions for Eyeless, Senseless, Suppressor of Hairless, Rough, and Glass. Our analyses show that cisTargetX identifies the correct motif from a set of coexpressed genes and accurately predicts target genes of individual TFs. The validated set of novel Ato targets exhibit functional enrichment of signaling molecules and a subset is predicted to be coregulated by other TFs within the retinal gene regulatory network.
Tens of thousands of regulatory elements determine the spatiotemporal expression pattern of protein-coding genes in the metazoan genome. Each regulatory element, when bound by the appropriate transcription factors, can affect the temporal transcription of a nearby target gene in a particular cell type. Annotating the genome for regulatory elements, as well as determining the input transcription factors for each element, is a key challenge in genome biology. In this study, we introduce a computational method, cisTargetX, that predicts transcription factor binding motifs and their target genes through the integration of gene expression data and comparative genomics. We first validate this method in silico using public gene expression data and, then, apply cisTargetX to the developmental program governing photoreceptor neuron specification in the retina of Drosophila melanogaster. Particularly, we perturbed predicted key transcription factors during the initial steps of neurogenesis; measure gene expression by microarrays; identify motifs and predict target genes; validate the predictions in vivo using transgenic animals; and study several functional and evolutionary aspects of the validated regulatory elements for the proneural factor Atonal. Overall, we show that cisTargetX efficiently predicts genetic regulatory interactions and provides mechanistic insight into gene regulatory networks of postembryonic developmental systems.