EMBO Conference: From Functional Genomics to Systems Biology location:Heildelberg date:17-20 November 2012
Methods for the prediction of cis-regulatory elements can take advantage of comparative genomics to increase signal-to-noise levels. However, gene expression data are usually derived from only one species. Here we investigate tissue-specific cross-species gene expression profiling by high-throughput sequencing, combined with cross-species motif discovery. We performed Tag-seq profiling across three Drosophila species, namely D.melanogaster, D.yakuba and D.virilis in two tissues, eye-antennal and wing imaginal discs. First, we optimized the pipeline to obtain gene expression values and eye/wing ratios for each species individually. Next, the eye/wing rankings were integrated by order statistics, defining a highly conserved gene set, which is strongly enriched in genes involved in compound eye photoreceptor development. On this conserved gene set we performed motif discovery analysis, using the tool i-cisTarget. Out of 3731 PWMs the Glass motif appeared as the highest overrepresented motif, yielding 96 predicted Glass targets including lz –its only previously know target. In addition, we identified other enriched motifs for eye-related TFs, such as SOXN, SCRT, EY and SU(H). To validate the Glass target predictions we performed differential expression analysis between wild-type and glass mutant eye-antennal discs using RNA-Seq. From 96 predicted Glass targets 62 were validated by RNA-Seq data, as they show significant down-regulation in the glass mutant. Finally, we performed in vivo reporter assays, both using cloned enhancers and using the D.melanogaster enhancer-GAL4 lines and the GAL4-UAS system in Drosophila. We achieved a success rate of 77% on Glass binding site predictions, with seven out of nine tested enhancers showing GFP expression in the eye disc and co-localizing with Glass expression.