Genome-wide identification of direct targets of the Drosophila retinal determination protein Eyeless

Abstract

The discovery of direct downstream targets of transcription factors (TFs) is necessary for understanding the genetic mechanisms underlying complex, highly regulated processes such as development. In this report, we have used a combinatorial strategy to conduct a genome-wide search for novel direct targets of Eyeless (Ey), a key transcription factor controlling early eye development in Drosophila. To overcome the lack of high-quality consensus binding site sequences, phylogenetic shadowing of known Ey binding sites in sine oculis (so) was used to construct a position weight matrix (PWM) of the Ey protein. This PWM was then used for in silico prediction of potential binding sites in the Drosophila melanogaster genome. To reduce the false positive rate, conservation of these potential binding sites was assessed by comparing the genomic sequences from seven Drosophila species. In parallel, microarray analysis of wild-type versus ectopic ey-expressing tissue, followed by microarray-based epistasis experiments in an atonal (ato) mutant background, identified 188 genes induced by ey. Intersection of in silico predicted conserved Ey binding sites with the candidate gene list produced through expression profiling yields a list of 20 putative ey-induced, eye-enriched, ato-independent, direct targets of Ey. The accuracy of this list of genes was confirmed using both in vitro and in vivo methods. Initial analysis reveals three genes, eyes absent, shifted, and Optix, as novel direct targets of Ey. These results suggest that the integrated strategy of computational biology, genomics, and genetics is a powerful approach to identify direct downstream targets for any transcription factor genome-wide.[Supplemental material is available online at www.genome.org. The sequence data from this study have been submitted to GEO under accession no. GSE4008.]Transcriptional regulation plays a key role in complex biological processes such as development. This occurs when combinations of nuclear transcription factors (TFs) acting with the nuclear effectors of signal transduction pathways bind DNA to activate and repress target genes. The structure of most gene regulatory networks is highly complex and often involves cooperative interactions and feedback regulation. While genetic dissection of such networks has been very productive, the discovery of direct targets of TFs remains largely underexplored, due mostly to technical limitations. Techniques to discover direct targets of TFs on a genomic scale include gene expression profiling using microarrays (Tavazoie et al. 1999), in silico prediction of binding sites (Halfon et al. 2002), and direct chromatin profiling (Ren et al. 2000). However, microarray analysis is limited by an inability to distinguish direct targets of TFs from indirect targets; in silico prediction of binding sites is limited by poor characterization of most TF binding sequences and by lack of experimental confirmation of most TF binding events in vivo; and direct chromatin profiling using techniques such as chromatin immu...

Direct control of neurogenesis by selector factors in the fly eye:regulation ofatonalby Ey and So

“…It is possible that the mechanisms of transcriptional regulation uncovered between Pax and Six genes (Niimi et al, 1999;Pauli et al, 2005;Ostrin et al, 2006) and between Pax/Six and ato (this work) may have arisen early during evolution. Such regulatory interactions may have favored the continued association of Pax/Six/Ath as various modifications of their genetic cascades led to the development of more complex and diverse sensory organs.…”

“…Over the last few years, Ey and So have been shown to play a crucial role in the deployment and maintenance of the RD network by directly regulating the transcription of several eye-specification genes [ey, so, eya, dachshund (dac) and optix] (Niimi et al, 1999;Punzo et al, 2002;Ostrin et al, 2006;Pappu et al, 2005;Pauli et al, 2005). However, little is known about downstream targets of the RD cascade.…”

“…4A and see Fig. S2 in the supplementary material) (Niimi et al, 1999;Ostrin et al, 2006;Pauli et al, 2005). The presence of potential binding sites raised the possibility that Ey and/or So directly regulate ato expression.…”

“…The base pair changes introduced had been reported to reduce binding affinity to consensus sites (Ostrin et al, 2006;Pauli et al, 2005;Punzo et al, 2002;Niimi et al, 1999). In all cases, reporter gene expression was severely affected, from strongly reduced to completely absent (Fig.…”

Generating patterned arrays of photoreceptors

“…Since the vertebrate and insect eyes are likely to be evolved independently [21••], the common use of Pax6 likely reflects a common ancestral function in regulating PR fate in both lineages [9,22]. ey and toy sit at the top of the transcriptional hierarchy of eye development, while the remaining retinal determination genes so, eya, and dac comprise the downstream regulators that are all required for eye development [1,13,23]. Interestingly, extensive crosstalk occurs between these factors, with multiple feedback loops, thus allowing downstream members of the pathway to retain their ability to induce ectopic eyes [24] (Figure 1).…”