Identification of transcriptional targets of the dual-function transcription factor/phosphatase eyes absent

Abstract

Drosophila eye specification and 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 functions 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.

Figure 1

Eya transgenes are expressed at equivalent levels. Western blot of head lysates from flies overexpressing eya^WT, eya^D493N, and eya^E728Q.

Figure 2

Overexpression of eya induces expression of putative target genes. (A) Quantitative RT-PCR analysis of target expression in adult heads overexpressing eya^WT and eya^MUT under control of the HSP-Gal4 driver compared to adult heads of flies carrying the driver alone. (B-D) Dac antibody staining in wing imaginal discs. (B) Wild type. (C) DPP-Gal4 driving coexpression of eya^WT and so transgenes. (D) Wing imaginal disc overexpressing eya^MUT and so under the control of the DPP-Gal4 driving coexpression of the phosphatase dead eya^EQ and so transgenes. (C-D) Dac expression is induced strongly by eya^WT and eya^MUT when overexpressed with so. (E) Quantitative RT-PCR of target expression in third instar larval eye-antennal imaginal discs overexpressing eya^WT under the control of the GMR-Gal4 driver compared to discs only carrying the driver alone.

Figure 3

Chromatin immunoprecipitation analysis of SO association with genomic regions containing predicted SO binding sites. PCR amplified product obtained from input chromatin is in the first lane, from mock treated sample incubated with pre-immune serum is in the second lane, and from sample immunoprecipitated using the SO antibody is in the third lane. Chromatin enrichment in the IP sample over the mock treated sample is given below each panel.

Figure 4

SO binding analysis. (A) Electrophoretic mobility shift assays with recombinant GST-full-length SO on double-stranded oligonucleotides containing predicted wild type or mutant SO binding sites. (B) Alignment of predicted SO binding sites highlights the core consensus sequence.

Figure 5

Heterozyosity for stg reduces the frequency of ectopic eye induction upon eya misexpression under the DPP-Gal4 driver.

Figure 6

EYA and SO regulation of stg expression. (A-H) In situ hybridization for stg. Wild type eyeantennal (A) or wing imaginal disc (B) show strong stg. Eye-antennal (C) or wing (D) imaginal discs overexpressing eya^WT and so under the control of the DPP-Gal4 driver. In both tissues stg expression is induced in the region where eya and so are being overexpressed. Eye-antennal (E) and wing (F) imaginal discs in which eya^MUT and so are overexpressed under the control of the DPP-Gal4 driver demonstrate ectopic stg expression. Arrows in C-F indicate regions of increased stg expression. Overexpression of ey using a DPP-Gal4 driver induces stg expression in eye-antennal (G) and wing imaginal discs (H). Discs overexpressing ey, but homozygous for the eya² allele no longer exhibit induction of stg expression (I, J). In eya²/eya²; DPP-Gal4/ UAS-ey, wing discs significant folding is observed, which results in the darker stg staining in the region of ey overexpression but does not reflect an overall increase in stg expression.