Identification of Transcriptional Targets Dual Functions Transcription Factor/Phosphatase Eyes Absent

Drosophila eye specification an development relies on a collection of transcription factors termed the retinal determination gene network (RDGN). Two members of this network, Eyes absent (EYA) and Sine oculis (SO), form a transcriptional complex in which EYA provides the transactivation function while SO provides the DNA binding activity. EYA also function as a protein tyrosine phosphatase, raising the question of whether transcriptional output is dependent or independent of phosphatase activity. To explore this, we used microarrays together with binding site analysis, quantitative real-time PCR, chromatin immunoprecipitation, genetics, and in vivo expression analysis to identify new EYA-SO targets. In parallel, we examined the expression profiles of tissue expressing phosphatase mutant eya and found that reducing phosphatase activity did not globally impair transcriptional output. Among the targets identified by our analysis was the cell cycle regulatory gene, string (stg), suggesting that EYA and SO may influence cell proliferation through transcriptional regulation of stg. Future investigation into the regulation of stg and other EYA-SO targets identified in this study will help elucidate the transcriptional circuitries whereby output from the RDGN integrates with other signaling inputs to coordinate retinal development. Keywords: gain of function, expression comparison We compared gene expression across duplicate samples for controls in which eya was not overexpressed to duplicate samples in which a wildtye overexpressed eya transgene, an eya mutant transgene with the D493N mutation, which reduces phosphatase activity, and a second eya mutant transgene with the E728Q mutation, which has little is any posphatase activity.