phosphatase eyes absent

Developmental Biology 310 (2007) 416 – 429 www.elsevier.com/developmentalbiology

Genomes & Developmental Control

Identification of transcriptional targets of the dual-function transcription factor/phosphatase eyes absent Jennifer Jemc a , Ilaria Rebay b,⁎ a b

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA Ben May Department for Cancer Research, University of Chicago, Chicago, IL 60637, USA Received for publication 28 June 2007; revised 16 July 2007; accepted 21 July 2007 Available online 27 July 2007

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. © 2007 Elsevier Inc. All rights reserved. Keywords: Eye development; Microarray; Drosophila; Cell cycle

Introduction Regulation of gene expression is a primary means by which signaling networks control cell fate specification. Studies of the compound eye of Drosophila melanogaster have provided numerous insights into how multiple signaling pathways are integrated at the level of transcription to control proliferation and cellular differentiation during development. The Drosophila eye is composed of approximately 800 units, called ommatidia, which each contain 8 photoreceptor neurons and 12 accessory cells. The adult eye develops from a structure called the eye imaginal disc, which consists of cells set aside in the embryo that subsequently proliferate and differentiate during larval and pupal development (Wolff, 1993). In the third instar larval eye imaginal disc, a wave of differentiation, termed the morphogenetic furrow (MF), initiates at the posterior of the disc and moves anteriorly across the field, marking the transition ⁎ Corresponding author. Fax: +1 773 702 4476. E-mail address: [email protected] (I. Rebay). 0012-1606/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2007.07.024

from an asynchronously proliferating population of cells to G1arrested cells (Wolff, 1993). After specification of the initial five photoreceptors just posterior to the MF, the remaining undifferentiated cells undergo a final mitotic division, called the second mitotic wave, and subsequently differentiate to give rise to the additional photoreceptors and accessory cells (reviewed by Wolff, 1993). Vital to the process of eye development is a network of transcription factors, known as the retinal determination gene network (RDGN), which are required for early eye specification. The genes comprising the RDGN include eyeless (ey), twin of eyeless (toy), sine oculis (so), eyes absent (eya), optix and dachshund (dac). Loss of function of these genes results in the complete absence of eye tissue, while overexpression in non-retinal tissues often induces ectopic eye formation (Bonini et al., 1993, 1997; Chen et al., 1997; Cheyette et al., 1994; Czerny et al., 1999; Halder et al., 1995; Mardon et al., 1994; Pignoni et al., 1997; Quiring et al., 1994; Shen and Mardon, 1997; Weasner et al., 2006). Within the RDGN, genetic epistasis and expression analyses have positioned EY and TOY upstream

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of EYA, SO and DAC (Czerny et al., 1999; Halder et al., 1998; Niimi et al., 1999; Ostrin et al., 2006; Shen and Mardon, 1997; Zimmerman et al., 2000). Supporting the genetic data, EY has been shown to directly regulate transcription of eya and so, with regulation of the latter involving cooperation with TOY (Niimi et al., 1999; Ostrin et al., 2006; Punzo et al., 2002). A number of positive feedback loops, some of which operate at the level of direct transcriptional control, further reinforce expression of network components to drive eye development (Bonini et al., 1997; Bui et al., 2000b; Chen et al., 1997; Pauli et al., 2005; Pignoni et al., 1997; Shen and Mardon, 1997). While the regulatory relationships within the RDGN have been worked out, much less is known about how RDGN members modulate patterns of gene expression to yield specific developmental outcomes. As a downstream component of the RDGN, EYA provides a logical place to begin examining how transcriptional output from the RDGN leads to retinal specification. EYA family proteins are conserved from worms to humans and are defined by a conserved C-terminal domain, termed the EYA domain (ED). The ED is required for interaction with SO and DAC, and also contains a phosphatase catalytic motif (Chen et al., 1997; Li et al., 2003; Pignoni et al., 1997; Rayapureddi et al., 2003; Tootle et al., 2003; Zimmerman et al., 1997). EYA mediates transactivation function through its more divergent N-terminal half, which contains a second moderately conserved domain, the EYA domain 2 (ED2), embedded in a proline–serine–threonine (P/S/T)-rich stretch of amino acids (Zimmerman et al., 1997). The P/S/T-rich region is required for transactivation, while the role of the ED2 domain remains unclear (Silver et al., 2003; Xu et al., 1997). Given that EYA does not have DNA binding activity, it must bind a cofactor to be recruited to target DNA. While EYA binds both SO and DAC through the ED (Chen et al., 1997; Pignoni et al., 1997), only SO has been demonstrated to recruit EYA to target DNA (Ohto et al., 1999; Silver et al., 2003). Previous studies of EYA–SO transcriptional targets have focused on identifying SO binding sites in target genes and showing the ability of EYA to coregulate expression. These studies have lead to the identification of five EYA–SO targets in Drosophila: lozenge (lz), sine oculis (so), eyeless (ey), hedgehog (hh) and atonal (ato) (Pauli et al., 2005; Yan et al., 2003; Zhang et al., 2006), all of which are required for proper eye development. As mentioned above, EYA not only functions as a transcription factor, but also as a phosphatase. This unique juxtaposition of functions is intriguing and begs the question of whether phosphatase activity is required for transcriptional regulation of EYA–SO target genes or whether the two functions are independent. Experiments using transcriptional reporter assays in transfected cultured cells have yielded conflicting answers to this question (Li et al., 2003; Tootle et al., 2003), emphasizing the necessity of additional analysis. Although the relationship between these two EYA functions remains to be determined, it should be emphasized that both are required for eye development (Rayapureddi et al., 2003; Silver et al., 2003; Tootle et al., 2003). In order to begin exploring the relationship between EYA phosphatase and transcription functions, we needed first to

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improve our understanding of EYA's role as a transcription factor by expanding the list of EYA transcriptional targets. Toward this goal, we used a microarray-based approach to identify genes whose expression was altered upon overexpression of a wild type eya (eyaWT) transgene. In parallel, we examined the ability of eya phosphatase mutant (eyaMUT) transgenes to regulate these potential target genes. From our microarray analysis, we identified 577 genes that underwent at least a twofold change in expression upon overexpression of wild type or phosphatase mutant eya. Comparison of the expression profiles for tissue overexpressing wild type or mutant eya reveals that the majority of genes have similar expression changes across all samples. Further comparison of the ability of EYAWT and EYAMUT to upregulate putative targets by quantitative PCR analysis confirms that reduced phosphatase activity may decrease EYA's transactivation potential in some contexts, but does not eliminate its transcriptional ability. Focusing on those genes that were upregulated in all of our experimental samples, we asked which might be directly regulated by EYA and SO by first identifying potential SO binding sites in silico and then experimentally confirming their functionality using chromatin immunoprecipitation (ChIP). Among the five validated targets that resulted from this analysis, we have focused on string (stg), a Cdc25a phosphatase that regulates the G2 to M phase transition of the cell cycle by dephosphorylating Cdk1. Our results suggest that EYA and SO might influence cell proliferation by transcriptionally regulating stg. Materials and methods Western blots Adult flies of the genotypes (1) w1118, (2) w1118; hspNeyaWT, (3) w1118; hspNeyaD493N, and (4) w1118; hspNeyaE728Q were heat-shocked at 37 °C for 1 h, followed by a 3-h incubation at 25 °C. Fly heads were prepared by collecting adult males and females into microcentrifuge tubes, freezing in liquid nitrogen, vortexing, and repeating three times. Fly heads were collected into a new tube and 50 μl of 2× reducing sample buffer (100 mM Tris–Cl (pH 6.8), 4% SDS, 20% glycerol, 200 mM DTT) was added. Tissue was homogenized, boiled and run on an 8% SDS–PAGE gel. Protein levels were detected with gpαEYA (1:10,000) and mαtubulin E7 (1:5000; Developmental Studies Hybridoma Bank) using the Li-COR imaging system and quantitated using ImageJ software (Abramoff et al., 2004).

Microarray sample preparation and hybridization Flies of the genotypes described above were used for microarray analysis. Adult males and females were collected into 15-ml falcon tubes, frozen in liquid nitrogen, and vortexed, repeating three times. Fly parts were poured onto an RNaseZap (Ambion)-treated glass plate and 50 heads were collected into an Eppendorf tube using a paintbrush. After homogenization in 50 μl of Trizol reagent (Invitrogen), an additional 200 μl of Trizol was added. Four samples were pooled for a total of 200 heads and RNA was isolated in Trizol according to the manufacturer's instructions and purified using the RNeasy Minikit (Qiagen). Total RNA concentration and 260/280 ratio was determined using a NanoDrop ND-1000 UV–Vis Spectrophotometer (NanoDrop Technologies). cDNA synthesis was carried out according to the Expression Analysis Technical Manual (Affymetrix, standard sample) using 2 μg of total RNA for each sample. cRNA reactions were carried out using one cycle cDNA synthesis according to the GeneChip Expression Analysis Technical Manual (Affymetrix). Ten micrograms of labeled cRNA were fragmented and hybridized to

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Affymetrix Drosophila Genome Array version 2.0 following the manufacturer's instructions.

Microarray data analysis and binding site identification Raw data were exported to Excel from the Affymetrix Microarray Suite, normalized and expression ratios and t-test values calculated. For genes whose expression was considered to be absent, the normalized filtered value was initially set to 1, and changes in expression were confirmed by repeated analysis in which the normalized filtered value was set to 50. Only the 577 genes which showed a twofold change in expression levels (based on a log2 value of 1) and had a t-test value of b0.05 in at least one of the experimental samples were used for further analysis. For binding site analysis, the publicly available program Genome Enhancer (http://www.genomeenhancer.org/fly; Markstein et al., 2002) was used. Each chromosome arm was examined individually to allow for identification of the maximal number of clusters. Binding sites for SO targets were identified using the following conditions: four YGATAY (Y = T/C) sites within a 50-bp window; a single copy of the longer, more degenerate sequence described by Pauli et al. (2005), GTAANYNGANAYS (Y = T/C, S = C/G, N = any nucleotide) in the forward and reverse direction; one to four copies of the short sequence in conjunction with binding sites for the transcription factors ETS (GGA(T/A)), TCF/LEF ((T/A)(T/A)CAAAG), STAT (TTCCCGGAA), MAD (GCCGNCG), Su(H) ((T/C)GTG(A/G)GAA) and GLI (TGGG(T/A)GGTC). See Supplementary materials and methods for further details regarding search parameters. Conservation of binding sites was assessed using the UCSC Genome Browser (http://www.genome.ucsc.edu/) and examining sequence alignments across twelve Drosophila species.

Quantitative real-time RT–PCR Adult flies of the genotypes w1118, w1118; hspNUAS eyaWT and w1118; hspNUAS eyaE728Q were heat-shocked, 25 heads collected, and RNA isolated in Trizol and resuspended in 16 μl nuclease-free H2O as described for microarray sample preparation. RNA was DNase (Invitrogen)-treated for 1 h in a total volume of 20 μl at 37 °C, followed by addition of 1 μl of 25 mM EDTA and inactivation at 70 °C for 10 min. cDNA was generated from 1 μg of DNasetreated total RNA using the Reverse Transcription System (Promega) according to the manufacturer's instructions. Quantitative PCR was carried out in triplicate on the Stratagene Mx4000 machine using 1.0 μl of the undiluted RT reaction, 0.5 μl each of 100 ng/μl sense and antisense primer, and 12.5 μl of Quantitect SYBR Green (Qiagen) in a total volume of 25 μl. For analysis in eye–antennal imaginal discs, 40 pairs of eye–antennal discs from third instar wandering larvae of the genotypes GMR-Gal4 and GMR-Gal4 N UAS–eya, which had been shifted to 29 °C, 2 days after egg laying, were dissected in Schneider's S2 cell medium and processed as described above. Quantitative real-time PCR was carried out as described above using 0.5 μl cDNA on the ABI Prism 7700. Relative quantification of real-time PCR product was performed using the comparative CT method (as described in the ABI Prism 7700 Sequence Detection System User Bulletin no. 2) and normalized by subtracting the CT value of the control gene ribosomal protein 17 (RpS17). Primers for this analysis are in Supplementary Table S1.

Chromatin immunoprecipitation Eye–antennal discs were dissected from third instar larvae in Drosophila S2 media. One hundred pairs of eye–antennal discs were fixed for 15 min at room temperature in 1 ml of S2 media with 40.5 μl of 37% formaldehyde. Glycine was added to 125 mM and incubated for 5 minutes on ice. Discs were washed 3 times in 1× PBS and resuspended in ChIP lysis buffer (50 mM K–HEPES pH 7.8, 140 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.1% Nadeoxycholate) with protease inhibitors. Samples were kept on ice and pooled for a total of 200 pairs of discs for each condition. Discs were homogenized with a pestle in an Eppendorf tube 3 times and syringe passed 10 times with a 25-gauge needle and 10 times with a 27-gauge needle, followed by incubation in lysis buffer for 20 min at 4 °C with rocking. Discs were sonicated on ice using a Branson digital sonifier for 15 rounds with two minutes rest in between at the

following settings: 15% amplitude, 15 s (.9 s ON/.1–.2 s OFF). Following sonication, samples were spun at 13.2 × 103 for 10 min at 4 °C. The supernatant from 400 total disc pairs was pooled in a new tube, and 10 μl was removed for the input and frozen. The remainder of the supernatant was divided into equal volumes for the control and the experimental sample (∼600 μl each). For the control sample, pre-immune serum was added at a concentration of 1:500. For the experimental sample, gpαSO antibody was added at a concentration of 1:500 (Mutsuddi et al., 2005). Samples were incubated overnight at 4 °C. On the second day, Gammabind G-Protein-Coupled Sepharose beads (Amersham Biosciences) beads were washed 2 times in 1× PBS and 20 μl of a 1:1 slurry of the beads and lysis buffer were added to each sample and incubated for 3 h. Samples were spun down for 2 min at 2 × 103 and the supernatant removed. Beads were washed 3 × 5 min with ChIP lysis buffer, 1 × 5 min with high-salt ChIP lysis buffer (same as above with 500 mM NaCl), 1 × 5 min in TE. Beads were resuspended in 150 μl TE/SDS (10 mM Tris pH 8, 1 mm EDTA, 1% SDS) and 140 μl TE/SDS was added to the input sample. Samples were incubated at 65 °C for 10 min, with vortexing every couple of minutes. Beads were pelleted at 14 × 103 rpm for 1 min and the supernatant was removed to a fresh tube. Tubes were sealed with parafilm and incubated at 65 °C overnight to reverse crosslinks. Qiagen PCR purification kit was used to extract DNA. Sample was eluted in 30 μl elution buffer and PCR analysis was performed with appropriate primers and 1–3 μl of eluted DNA. (See Supplementary Table S2 for a list of ChIP primers.) Chromatin enrichment in the IP sample over the mock-treated sample was determined using ImageJ (Abramoff et al., 2004).

Gel shift assays For labeling of probes, 25 pmol of annealed oligos (see Supplementary Table S3 for oligo sequences) were labeled with T4 polynucleotide kinase (New England BioLabs) and 75 μCi γ-32P-dATP in a total volume of 25 μl for 30 min at 37 °C. Kinase was inactivated by ∼ 20 min of incubation at 65 °C. Recombinant GST full-length SO was purified from bacteria as previously described (Sullivan and Ashburner, 2000). Binding reactions were carried out in 15 μl binding buffer (10 mM HEPES pH 7.9, 35 mM KCl, 10 mM NaCl, 4 mM MgCl2, 20% glycerol, 1 mM DTT, 1 mM EDTA, 0.1% BSA), 1 μg poly dI–dC, 1 μl labeled probe, 2 μl protein and H2O in a total volume of 20 μl. Samples were incubated for 20 min and analyzed on 6% non-denaturing polyacrylamide gels followed by autoradioagraphy. For competition experiments, samples were pre-incubated with 100× excess of unlabeled mutant or wild type probe for 10 min at room temperature before adding the labeled probe.

In situ hybridization stg fragments were generated by PCR amplification from genomic DNA using the following oligos: STG-Sense 5′-GATAATTTAGGTGACACTATAGAAGAG ATGACGGAGAGCAACACCAACAGC-3′ and STG-Antisense 5′GAATTAATACGACTCACTATAGGGAGAGAAGCGGGACATTTTCGGTC-3′. The PCR product was gel-purified using the QIAquick Gel Extraction kit (Qiagen) according to manufacturer's instructions. 1 μg of DNA was used in a total volume of 15 μl and was heated to 95 °C and cooled on ice. 2 μl of Dig DNA labeling mix (Roche), 2 μl hexanucleotide mix (Roche) and 1 μl of Klenow (New England BioLabs) were added and the labeling reaction was incubated overnight at room temperature. The labeled probe was precipitated and resuspended in 50 μl nuclease-free H2O. Eye–antennal or wing imaginal discs were dissected from third instar larvae in Schneider's S2 cell media. Discs were fixed in 4% paraformaldehyde for 20 min on ice, followed by incubation in 4% paraformaldehyde with 0.6% Triton X-100 for 15 min at room temperature. Discs were washed 3 × 5 min in 1× PBS with 0.3% Triton and 1 × 5 min in PBT (PBS + 0.1% Tween-20), post-fixed in 4% paraformaldehyde for 20 min at room temperature, washed 3 × 5 min in PBT, 1 × 5 min PBT/hybridization buffer (50% formamide, 5× SSC, 100 μg/ml salmon sperm DNA, 100 μg/ml tRNA, 50 μg/ml heparin, 0.1% Tween-20) and 1 × 5 min in hybridization buffer. Discs were prehybridized in hybridization buffer for at least 1 h at 48 °C. Probe was heated to 95 °C for 3 min, cooled and added at a concentration of 1:10 for overnight hybridization at 48 °C. Discs were washed in a PBT/hybridization buffer series, followed by 5 × 20 min washes in PBT and incubation with pre-absorbed alkaline phosphatase-conjugated anti-dioxygenin antibody (1:2000) (Roche)

J. Jemc, I. Rebay / Developmental Biology 310 (2007) 416–429 overnight at 4 °C. Development was carried out in AP buffer (100 mM Tris pH 9.5, 100 mM NaCl, 50 mM MgCl2, 0.1% Tween-20, 1 mM levamisol) with 3.375 μl NBT/3.5 μl BCIP (Roche).

Immunostaining Wing imaginal discs from wandering third instar larvae were dissected in Drosophila S2 media, fixed for 10 min in 4% paraformaldehyde with 0.1% Triton X-100, washed 3× in PT (1× PBS, 0.1% Triton), and blocked for 30 min in PNT (1× PBS, 0.1% Triton, 1% normal goat serum). Samples were incubated with mouse anti-dac (1:10; Developmental Studies Hybridoma Bank) in PNT at 4 °C overnight. Samples were washed in PT, incubated with goat anti-mouse Cy3 (1:2000; Jackson ImmunoResearch, West Grove, PA) for 2 h at room temperature, washed in PT and mounted.

Genetics Stocks used for microarray analysis have been previously described: HSP70Gal4 (Bloomington), UAS-eyaWT, UAS-eyaD493N and UAS-eyaE728Q (Tootle et al., 2003). GMR-Gal4 (S. L. Zipursky) was used to drive eya expression in the eye disc for qRT-PCR analysis. For genetic interactions and in vivo expression analysis, the following stocks were used in addition to those above: DPP-Gal4 (40C6) (Staehling-Hampton et al., 1994), UAS-eyaWT/Cyo actin-GFP; stg02135/ TM3Ser actin-GFP, UAS-ey, eya2/Cyo actin-GFP; DPP-Gal4/TM6B, eya2/ Cyo actin-GFP; UAS-ey/TM6B, UAS-eyaWT, UAS-soWT/Cyo actin-GFP, and UAS-eyaE728Q, UAS-soWT/Cyo Dfd-YFP.

Results Understanding the role of EYA activity in Drosophila eye development and the extent to which EYA's phosphatase activity influences transcriptional output requires knowledge of its downstream transcriptional targets. At the time this project was initiated, only a single EYA target gene, lozenge (lz), had been identified; therefore, we used a microarray-based approach to isolate additional genes with altered expression levels upon eya overexpression. In parallel, we profiled tissue in which an eya transgene mutant for phosphatase activity was overexpressed, reasoning that if phosphatase activity is required for transcriptional activity, then the patterns of altered gene expression would be quite different. Despite the inherent limitations of an overexpression-based analysis, we selected this approach first because the eya mutant alleles predicted to have reduced phosphatase activity behave as embryonic lethal nulls and therefore are not suitable for array analysis in the eye (Bui et al., 2000a; Mutsuddi et al., 2005), and second because the requirement for EYA phosphatase activity for normal eye development precludes us from setting up a genetic rescue context in which eya MUT transgenes are expressed in a background lacking endogenous eya expression (Rayapureddi et al., 2003; Tootle et al., 2003). Specifically, we compared the expression profile of wild type control tissue to that of tissue overexpressing an eyaWT transgene or one of two eya phosphatase mutant transgenes, eyaD493N and eyaE728Q, collectively referred to as eyaMUT (Li et al., 2003; Rayapureddi et al., 2003; Tootle et al., 2003), under the control of the HSP70-Gal4 driver in the adult head. Adult heads were selected both for the ease in isolating significant quantities of RNA, and because so, which encodes the DNA binding moiety of the EYA–SO transcription factor, is endogenously expressed

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(data not shown). Eya transgenes with similar expression levels were chosen based on quantification of protein levels on Western blots (Fig. 1). Tissue was harvested 3 h post-induction in order to maximize chances that resulting changes in gene expression would reflect direct transcriptional control, rather than downstream secondary effects. RNAwas extracted from adult heads in duplicate for each genotype, processed and hybridized to Drosophila Affymetrix chips (Materials and methods). Linear regression analysis demonstrated that the replicates of each genotype were highly similar, with control, eyaWT and eyaE728Q samples having R2 values N 0.99 and eyaD493N samples an R2 value of 0.96. Comparison of the eyaWT, eyaD493N and eyaE728Q expression profiles to the control sample resulted in a list of 577 genes that demonstrated a twofold or greater change in expression with a t-test value of b 0.05 in at least one of the experimental conditions. In order to determine if reducing phosphatase activity altered transactivation, we compared the expression level changes of the 577 genes in the eyaWT and eyaE728Q samples. For this comparative analysis, we relaxed the p value to 0.10 and the fold change to ∼1.87 in order to survey a slightly larger data set. Using these parameters, out of the 577 genes with altered expression in at least one of our array conditions, 452 genes had statistically significant changes in gene expression in both the eyaWT and eyaE728Q. Of these genes, 80% were regulated similarly between the eyaWT and eyaE728Q samples, suggesting that reducing EYA phosphatase activity does not globally alter transcriptional output. These genes were also similarly regulated in eyaD493N samples, but due to the lower correlation coefficient, often had higher p values. Of the remaining 89 genes that were not similarly regulated among all three array conditions, in only 9 cases were the expression changes more similar between the two mutant samples than between either mutant to wild type. Because this latter pattern would be the expected signature of a gene whose transcriptional regulation depended on EYA phosphatase activity, our microarray-based survey suggests that loss of phosphatase activity does not broadly alter EYA's transcriptional output. In silico identification of SO binding sites in putative EYA target genes Given that EYA functions as a transcriptional activator (Ohto et al., 1999; Silver et al., 2003), we focused our analysis of potential targets on the 226 genes out of the initial set of 577

Fig. 1. Eya transgenes are expressed at equivalent levels. Western blot of head lysates from flies overexpressing eyaWT, eyaD493N, and eyaE728Q.

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whose expression was upregulated in both eyaWT and eyaMUT samples. To identify potential direct targets of the EYA–SO complex, two consensus sequences were used to detect the presence of potential SO binding sites in genomic regions surrounding candidate targets: a short sequence, (T/C)GATA(T/C), identified through in vitro binding studies (Hazbun et al., 1997); and a longer consensus sequence, GTAAN(T/C)NGANA(T/C) (C/G) (N = any nucleotide), generated by comparison of SO binding sites in the Drosophila transcriptional targets lz (Yan et al., 2003) and so with sequences from targets of the mammalian Six family genes (Pauli et al., 2005). Three independent computational strategies, all using the Genome Enhancer tool (Markstein et al., 2002), were implemented. We first searched for the presence of the short consensus site (Hazbun et al., 1997) (exact search parameters described in Materials and methods). Due to the short and somewhat palindromic nature of this sequence, we searched for the presence of four binding sites within a 50-bp window, but found only two candidate targets with predicted binding sites nearby. In a separate analysis, we searched for one copy of the larger consensus sequence in regions proximal to genes upregulated in our microarray analysis, yielding an additional nine targets. Finally, considering the limitations of searching for such a short binding site and the fact that the longer consensus sequence was based only on a handful of Drosophila SO targets and is likely to evolve further, we developed a third approach based on the knowledge that binding sites for different transcription factors often cluster in cis-regulatory elements (Stanojevic et al., 1991). Specifically, we searched for the short SO binding site (Hazbun et al., 1997) in combination with identified consensus binding sites for transcription factors downstream of canonical signaling pathways. Our search included the binding sites for the Wingless pathway effector TCF/LEF/PAN (van de Wetering et al., 1997), the Hedgehog effector GLI (Kinzler and Vogelstein, 1990), the Epidermal Growth Factor Receptor Pathway ETS family effectors (Sharrocks et al., 1997), the JAK/STAT pathway effector STAT (Yan et al., 1996), the Notch pathway effector Suppressor of Hairless (Su (H)) (Rebeiz et al., 2002), and the Decapentapalegic (DPP) pathway effector MAD (Kim et al., 1997). Searching for these binding sites in combination with the SO binding site allowed us to search for fewer copies of the SO binding site and to cover a larger genomic window, thereby isolating an additional 32 potential candidate target genes. The combined results for all three searches produced a list of 43 potential direct EYA–SO targets that showed similar upregulation in the eyaWT and eyaMUT array samples (Supplementary Table S4). For many of our potential targets, more than one SO binding region was predicted proximal to the gene of interest, suggesting the possibility of complex or tissue-specific regulation. qRT-PCR validation of EYA targets confirms impaired phosphatase activity has minimal impact on transcriptional output We next implemented a number of secondary tests to distinguish relevant candidates from the inevitable background.

First, in order to validate our microarray results independently, we performed quantitative real-time PCR (qRT-PCR) on cDNA derived from adult head tissue, prepared in the same manner as that used for the microarray analysis. Genes were chosen for analysis if they had an ∼threefold change in expression and/or a potentially relevant molecular function. Using qRT-PCR, we observed the upregulation of fourteen of seventeen tested target genes upon overexpression of eyaWT (Fig. 2A and data not shown). Although the fold changes in expression were not identical between the microarray and qRT-PCR data, the trends in upregulation were consistent, confirming our microarray results. As discussed above, our microarray data suggest that eyaMUT transgenes retain transcriptional activity at levels similar to eyaWT. To further validate this result, we compared the ability of an eyaWT and an eyaMUT transgene to upregulate expression of the genes confirmed above by quantitative real-time PCR (Fig. 2A). All ten targets tested were significantly upregulated in response to eyaMUT, consistent with the microarray data, and six showed statistically comparable fold activation upon expression of either eyaWT or eyaMUT transgenes. Two, mal and CG6560, showed a modest, less than twofold reduction in induction by eyaMUT versus eya WT transgenes, while stg and CG8211 exhibited a more striking ∼ threefold reduction in induction by eyaMUT relative to eyaWT transgenes. However, even in the latter two cases, upregulation of target gene expression by eyaMUT was significantly (three- to fourfold) greater than the control (Fig. 2A). These results suggest that the ability of an EYA phosphatase mutant to activate expression of target genes is overall quite similar to that of wild type EYA, but that there may be some context dependency, consistent with transcription assay data from cultured cells in which EYAMUT activated some reporters as robustly as EYAWT, but others to a lesser degree (Li et al., 2003; Tootle et al., 2003). In addition, we compared the ability of eyaWT and eyaMUT transgenes co-overexpressed with so to induce ectopic expression of dac, a gene predicted to be a direct transcriptional target of EYA–SO, based on previous studies (Anderson et al., 2006; Chen et al., 1997; Kenyon et al., 2005; Pignoni et al., 1997). Both wild type EYA and the phosphatase mutant induced strong ectopic expression of dac when overexpressed with so (Figs. 2B–D), demonstrating that EYA phosphatase activity is not required for the expression of this downstream EYA–SO target. Thus, loss of EYA phosphatase activity does not appear to globally or drastically alter direct downstream transcriptional output. As one of the goals of this study was to identify transcriptional targets of EYA relevant to eye development, we asked if these targets could be similarly upregulated in response to overexpressing eya in the third instar eye–antennal imaginal disc. Using GMR-Gal4 to drive eya expression in all cells in and behind the morphogenetic furrow, we observed upregulation of twelve out of fourteen target genes by qRT-PCR (Fig. 2E, Table 1 and data not shown). These data suggest that our adult head microarray analysis was successful in identifying target genes that could also be regulated by EYA in the developing eye.

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Fig. 2. Overexpression of eya induces expression of putative target genes. (A) Quantitative RT–PCR analysis of target expression in adult heads overexpressing eyaWT and eyaMUT under control of the HSP-Gal4 driver compared to adult heads of control flies. (B–D) Dac antibody staining in wing imaginal discs. (B) Wild type. (C) DPP-Gal4 driving coexpression of eyaWT and so transgenes. (D) DPP-Gal4 driving coexpression of the phosphatase dead eyaEQ and so transgenes. (C, D) Dac expression is induced strongly by eyaWT and eyaMUT when overexpressed with so. (E) Quantitative RT–PCR of target expression in third instar larval eye–antennal imaginal discs overexpressing eyaWT under the control of the GMR-Gal4 driver compared to discs carrying the driver alone.

In vivo and in vitro confirmation of predicted SO binding sites in EYA target genes To distinguish direct EYA transcriptional targets from genes upregulated several steps downstream, we looked for the ability of SO, which contributes DNA binding activity to the EYA–SO transcription factor, to bind predicted target sequences both in vivo and in vitro. First, we investigated whether endogenous SO protein in the eye imaginal disc associated with chromatin regions encompassing predicted binding sites using chromatin immunoprecipitation (ChIP). Out of the ten candidate target genes examined, five, plus the positive control, lz, showed

enrichment of SO at one or more computationally predicted binding sites (Fig. 3). CG8449 and CG15879 each had enrichment at a single predicted binding region, stg showed enrichment at each of its two predicted binding regions, while maroonlike (mal) and CG12030 showed significant occupancy at each of three predicted regulatory elements (Fig. 3). We observed SO enrichment at binding sites predicted by all three computational strategies described above, demonstrating the efficacy of this approach in identifying potential targets. The association of endogenous SO with these genomic regions during the course of normal eye development suggests that we have identified five new direct targets of EYA–SO transcriptional regulation, while

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Table 1 Predicted direct targets of EYA and SO Gene

Full name and predicted function

stg mal CG12030 CG15879 CG8449

String; phosphatase Maroon-like; molybdopterin sulfurase Galactose metabolism Hydrolase fold Related to human RabGAP-related protein Novel GTPase activity Novel Oxysterol binding protein; cholesterol biosynthesis Novel Ecdysdone-inducible protein

CG8211 CG6560 CG15203 osbp CG17002 Eip63E a

sequences contribute to regulatory specificity in vivo remains an open question. Fold change a WT/MUT 2.72/2.24 2.83/2.63 5.45/6.88 6.11/3.12 3.85/3.77 4.56/4.09 2.95/2.73 2.70/2.89 2.94/3.01 2.54/2.62 14.83/15.19

Fold change is indicated for eyaWT and eyaE728Q samples.

the remaining five negatives could still be relevant direct targets in other developmental contexts. Because our computational methods for identifying SO binding sites involved searching for clustered sequence motifs, most of the ChIP confirmed regions contained multiple potential SO binding sequences. In order to confirm SO binding to the predicted elements within these genomic regions and to determine how these sequences compare with the previously generated SO consensus binding motif, we performed electrophoretic mobility shift assays (EMSAs) using the two stg genomic regions bound by SO as a test case. In these experiments, purified recombinant full-length GST–SO fusion protein was tested for its relative ability to bind radiolabeled wild type oligonucleotide probes covering the predicted binding sites versus mutant probes that no longer matched the consensus sequence. Two predicted SO binding sites within the stg-A region and three probes covering five potential SO binding sites within the stg-B region were tested. While none of the mutant probes formed a complex with SO, all wild type probes demonstrated association with SO as evidenced by a strong mobility shift that was competed away by excess unlabeled wild type competitor DNA, but not by excess mutant competitor (Fig. 4). However, SO did not bind to all probes with equal strength. Site stg-A2 showed strongest association with SO within the stg-A region, while stg-B3 was the most effective in complexing with SO in the stg-B region (Fig. 4A). Assuming comparable affinities in vivo, these data predict that stg-A2 and stg-B3 should be the primary sites of SO recruitment to the stg genomic locus during eye development. To determine if the strength of SO binding could be correlated with a specific sequence, we aligned the probe sequences and looked for conserved motifs. From this analysis, only the short SO binding site (T/C)GATA(T/C) (Hazbun et al., 1997) emerged (Fig. 4B). Direct comparison of this sequence and other binding sites identified in this study to the longer sequence GTAAN(T/C)NGANA(T/C)(C/G) (N = any nucleotide) (Pauli et al., 2005) revealed no obvious conservation of flanking sequences, suggesting that the core sequence requirement for SO binding is (T/C/G)GA(A/T/G)A(T/C). Thus, how flanking

Stg is an in vivo target of EYA–SO In order to determine if EYA and SO regulation of stg expression is required during development, we first tested the ability of eya and stg to interact genetically. Specifically, we took advantage of the fact that misexpression of EYA can induce ectopic eye formation (Bonini et al., 1997; Pignoni et al., 1997). In this assay, modulating the expression level of a gene predicted to cooperate with eya in eye specification can be tested for its ability to dominantly modify the frequency of ectopic eye induction (Hsiao et al., 2001). Therefore, we examined the frequency of EYA ectopic eye induction when one copy of stg was removed. Heterozygosity for stg resulted in a 40% decrease in the frequency of ectopic eye induction by EYA (Fig. 5), suggesting that eya and stg function cooperatively during the specification of eye tissue. We next examined the ability of EYA and SO to regulate stg expression in vivo. First, using a series of stg-lacZ enhancer trap lines, two of which, pstgβ-E4.9 and pstgβ-E6.7, contained our identified SO binding sites (Lehman et al., 1999), we tested the ability of DPP-Gal4 driven EYA and SO to induce lacZ

Fig. 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.

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Fig. 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.

expression in the wing imaginal disc. No induction in lacZ expression in the region where EYA and SO are overexpressed was observed (data not shown). Examination of eight enhancer trap lines spanning the 5′UTR of stg (Lehman et al., 1999) also failed to reveal an expression pattern consistent with that of stg in the eye imaginal disc (Alphey et al., 1992) (data not shown). Together these results suggest that stg expression requires input from multiple cis-regulatory regions and multiple transcription factors. This is consistent with other studies which have shown that stg expression is directly regulated by the epidermal growth factor transcriptional regulators, Pointed and Tramtrack69 (Baonza et al., 2002), and directly or indirectly by Notch and Wingless signaling (Deng et al., 2001; Johnston and Edgar, 1998). Therefore, as an alternative to in vivo reporter analysis, we overexpressed eya and so singly or in combination using a DPPGal4 driver in third instar wing and antennal imaginal discs, both tissues that normally lack expression of these genes, and followed stg expression by in situ hybridization. While overexpression of either eya or so alone did not induce stg (data not shown), co-overexpression of both genes resulted in increased stg along the dorsal/ventral margin of the wing and in the ventral part of the antennal disc (Figs. 6A–D).

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Using the same assay, we next examined the ability of the eyaMUT to activate stg expression when co-expressed with so. Similar to eyaWT, eyaMUT also induced ectopic stg expression in both the wing and antennal discs (Figs. 6E, F). The results support our earlier microarray and qPCR data, suggesting that reducing EYA phosphatase activity does not severely compromise its transcriptional ability. However, the overproliferation and resulting tissue distortion observed upon coexpression of so and eyaMUT (Figs. 6C, D) was much less pronounced than that observed with so and eyaWT (Figs. 6E, F), suggesting that although the immediate transcriptional output might not be significantly compromised, reduced phosphatase activity can alter the downstream developmental program. Although the above results are consistent with EYA and SO directly regulating stg expression, overexpression of ey, itself a transcriptional target of EYA–SO (Pauli et al., 2005; Pignoni et al., 1997), has also been shown to result in upregulation of stg (Michaut et al., 2003; Ostrin et al., 2006). Thus it was formally possible that EYA–SO-mediated regulation of stg might occur indirectly through EY. To rule out this possibility, we compared the ability of EY to induce stg expression in wild type wing and antennal imaginal discs versus discs homozygous for the eyespecific allele eya2. This allele deletes 0.3 kb of an eye enhancer element through which EY regulates eya expression (Zimmerman et al., 2000). As expected based on the results of Michaut et al. (2003), overexpression of ey using a DPP-Gal4 driver resulted in increased stg expression in wing and antennal discs (Figs. 6G, H). Antibody staining revealed induction of eya and so expression in these tissues (data not shown), suggesting that stg upregulation could be directed by EYA and SO. In contrast, in discs overexpressing ey, but lacking eya, we no longer observed stg induction in wing or antennal discs, indicating that EYA and SO function downstream of EY to directly regulate stg transcription (Figs. 6G–J). The ability of EYA and SO to induce ectopic stg expression demonstrates that EYA and SO are sufficient for stg expression, and the fact that EY cannot induce stg in the absence of eya suggests that eya is also necessary. Further supporting the

Fig. 5. Heterozygosity for stg reduces the frequency of ectopic eye induction upon eya misexpression under the DPP-Gal4 driver.

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Fig. 6. EYA and SO regulation of stg expression. (A–J) In situ hybridization for stg. Wild type eye–antennal (A) or wing imaginal disc (B) show strong stg expression. Eye–antennal (C) or wing (D) imaginal discs overexpressing eyaWT 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 eyaMUT and so are overexpressed under the control of the DPP-Gal4 driver demonstrate ectopic stg expression. Arrows in panels 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.

argument that eya is necessary for stg expression, loss of eya also compromises the normal pattern of stg expression at the morphogenetic furrow (Figs. 6A, I), demonstrating that eya is required for stg expression during eye development. However, we still observe tissue overgrowth in wing discs overexpressing ey and mutant for eya (Figs. 6I, J), indicating that EY may act through additional parallel pathways to influence cell proliferation and organ size. This may also be the case for EYA and SO,

as heterozygosity for stg was unable to rescue tissue overgrowth in wing discs overexpressing eya and so (data not shown). These observations are consistent with previous data demonstrating that overexpression of stg alone does not increase cell proliferation (Neufeld et al., 1998). Thus, the ability of EYA and SO to induce stg outside of its normal expression region, coupled with our demonstration that eya is necessary for stg expression in the eye imaginal disc, suggest that a direct

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mechanism through which EYA and SO might regulate cell cycle progression during eye development. Discussion We have used microarray analysis combined with computational and experimental validation to identify potential EYA– SO transcriptional targets. Two general conclusions have resulted from this work: first, the similarity in expression profiles between tissue overexpressing wild type and phosphatase-dead eya transgenes and analysis of gene expression by quantitative PCR suggest that EYA's phosphatase activity is not generally required for EYA transcriptional activity, although it may be required for maximal transactivation of some target genes; and second, the short sequence (T/C/G)GA(A/T/G)A(T/C) appears to be the only recognizable motif shared among all SO binding sites. As exemplified by our in vivo validation of EYA–SOmediated regulation of the cell cycle regulator stg, further analysis of the target genes identified in this study will likely shed new light into the mechanisms underlying EYA–SO function during development. Identification of new EYA–SO targets The main goal of this study was to identify new targets of EYA transcriptional activity. Although we used adult head tissue for our overexpression experiments, our 86% success rate in confirming changes in expression of our potential targets in developing Drosophila eye–antennal imaginal discs overexpressing eya supports the ability of this system to identify similar data sets in different developmental stages. Out of the ten genes upregulated by eya overexpression in both adult head tissue and eye–antennal imaginal discs, five demonstrated enrichment of endogenous SO at one or more predicted binding sites. These predicted binding regions were conserved across a minimum of two and up to nine Drosophila species, emphasizing their likely biological relevance (data not shown; see methods for details). Two binding sites that did not demonstrate SO enrichment were not conserved across other Drosophila species, while binding sites in the remaining three genes were conserved across multiple species and could be EYA–SO targets in other tissues. The core sequence shared by all of these targets is (T/C/G)GA (A/T/G)A(T/C), a pared down version of the previously proposed GTAAN(T/C)NGANA(T/C)(C/G) SO binding sequence (Pauli et al., 2005). In Drosophila EYA–SO targets, the sequence flanking the core (T/C/G)GA(A/T/G)A(T/C) has only been shown to be important in the case of the target so (Pauli et al., 2005), and is absent in one of the two SO binding sites identified in the lz locus (Yan et al., 2003) and in the binding sites in stg we confirmed by gel shifts. While specific flanking sequences may further stabilize SO–DNA interactions, characterization of such a flanking sequence consensus awaits further analysis. Confirmation of additional targets predicted by our microarray and binding site analysis should provide for further characterization of the SO binding sequence. Out of the remaining 31 potential targets, all except one have binding sites

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conserved across multiple Drosophila species, suggesting that additional EYA–SO targets will be confirmed within this data set. While none of the previously identified EYA–SO targets were included in our final list, two targets, so and lz were upregulated upon eya overexpression, although less than our twofold cutoff. The expression of the previously identified targets hh, ato and ey was either absent or changes were not statistically significant. One explanation for this observation is that other signaling pathways required for the expression of these genes may not be activated, or, conversely, inhibitory signaling pathways could be activated in adult head tissue. In addition to examining expression levels of previously identified EYA–SO targets, we also compared our list of upregulated EYA–SO target candidates to genes that were upregulated by ey overexpression in microarray analyses (Halder et al., 1998; Niimi et al., 1999; Ostrin et al., 2006; Pauli et al., 2005; Punzo et al., 2002). Because ey both induces eya and so expression and is itself transcriptionally regulated by EYA–SO, one would expect to see a number of genes similarly regulated by overexpression of either ey or eya; however, as detailed below, pairwise comparisons between our data set to lists of candidate ey targets derived from two independent array studies (Michaut et al., 2003; Ostrin et al., 2006), reveals a surprisingly limited overlap. Michaut et al. identified 371 genes with at least 1.5-fold upregulation across two array experiments, only 55 of which were similarly upregulated in both arrays (Michaut et al., 2003). Comparison of this data set to a more recent report by Ostrin et al. of 300 candidate genes upregulated in response to ey overexpression revealed only 24 common targets. Comparison to our list of potential eya–so targets yielded 10 shared with the Michaut et al. data set and 3 common to the Ostrin et al. results. Encouragingly, despite this limited overlap, stg, a gene we have shown here to be transcriptionally regulated by eya and so, was one of the two targets consistently upregulated in all three studies (Michaut et al., 2003; Ostrin et al., 2006). As we continue to confirm additional targets, it is important to note that EYA may also associate with transcription cofactors other than SO to regulate gene expression. Although EYA can associate with DAC (Chen et al., 1997), and X-ray crystallographic analysis suggests that DAC can bind DNA (Kim et al., 2002), targets of an EYA–DAC complex or a consensus DAC binding site have not been identified. In addition, EYA also contains an engrailed homology 1 (eh1) domain (Goldstein et al., 2005), suggesting that it may be able to bind to the transcriptional repressor Groucho (GRO). However, as current in vivo data only supports a role for EYA as a transactivator complexed to SO, identification of additional EYA cofactors in vivo will be necessary to explore the potential of SOindependent EYA transcriptional functions further. Interplay between EYA transcription and phosphatase activities Previous studies using transcriptional reporter assays in transfected cultured cells have yielded conflicting results as to

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whether phosphatase activity is important for proper transcriptional output from EYA–SO (Li et al., 2003; Tootle et al., 2003). However, the emerging consensus from these experiments is that missense mutations in the ED that reduce phosphatase activity also compromise the ability of EYA to transactivate targets (Li et al., 2003; Mutsuddi et al., 2005). Importantly, these mutations do not affect SO binding (Mutsuddi et al., 2005; Tootle et al., 2003), suggesting that it is actually loss of EYA phosphatase activity that alters transcriptional output, presumably as a consequence of improper phosphoregulation of critical substrates, although altered protein–protein interactions cannot be ruled out. How faithfully these cultured cell-based reporter assays mirror in vivo regulation remains an open question. In order to examine further the relationship between EYA's dual functions as transcriptional coactivator and protein tyrosine phosphatase in vivo, we compared the expression profiles of tissue overexpressing wild type versus phosphatase mutant eya transgenes. We expected that if phosphatase activity is required for transactivation, then direct transcriptional targets showing altered expression in eyaWT samples would exhibit reduced changes in eyaMUT samples. Our microarray results demonstrated that ∼80% of a set of 452 candidate targets were similarly regulated among eyaWT and eyaMUT samples, supporting the alternate hypothesis that phosphatase activity is not broadly required for EYA's transcriptional activity. Only 2%, or 9 genes, exhibited a consistent change in expression in response to the two eyaMUT transgenes that differed from the response to eyaWT. Our follow-up analysis of a subset of these genes by quantitative PCR confirmed that an EYA phosphatase mutant has transcriptional activity quite similar to wild type EYA. In the cases in which phosphatase mutant EYA induced expression at a significantly lower level that wild type EYA, one hypothesis would be that those genes might represent indirect targets; arguing against this, stg, one of two genes whose pattern of induction fell into this category, was confirmed as a direct EYA–SO target. In addition, ectopic expression of dac, a likely direct downstream EYA–SO target, was induced by the eyaMUT as efficiently as eyaWT. In conclusion, while mutating EYA phosphatase activity has a minimal impact on the direct downstream transcriptional profile overall, at certain target genes it may reduce, but not eliminate transcriptional output. Given these results, future exploration of in vivo roles for EYA phosphatase activity independent of its transcriptional functions may be warranted. Disease and developmental implications of EYA–SO targets Many of the genes identified as direct EYA–SO transcriptional targets are largely uncharacterized “CGs” whose expression patterns in the eye will have to be studied in detail to gain further insight to EYA–SO-mediated regulation, but a few have predicted or well-studied functions that may provide insight into how EYA–SO functions during normal development and how misregulation can result in disease. Most notable on this list is stg. Given that overexpression of eya and so results in overproliferation, while their loss leads to tissue reduction (Bonini et al., 1997; Pignoni et al., 1997), EYA–SO control of stg

expression provides a mechanism for how EYA–SO regulation of the cell cycle may in turn affect cell proliferation. An interesting question for future investigation is how the relatively broad expression of EYA and SO throughout the developing retina activates stg expression only in a relatively narrow stripe of cells just anterior to the morphogenetic furrow. Given the apparent complexity of stg cis-regulatory elements revealed by our and previous studies (Baonza et al., 2002; Deng et al., 2001; Johnston and Edgar, 1998), a likely explanation is that EYA–SO act combinatorially with transcriptional effectors of other signaling pathways to effect this developmental precision. Consistent with eya and so overexpression leading to increased tissue overgrowth in Drosophila (Bonini et al., 1997; Pignoni et al., 1997), elevated levels of Eya and Six family members have been observed in a variety of cancers (Coletta et al., 2004; Ford et al., 1998; Yu et al., 2006; Zhang et al., 2005). Studies of the transcriptional targets of mammalian Eya and SO/Six proteins have identified the cell cycle regulatory genes, cyclin D1 and cyclin A1 (Coletta et al., 2004; Yu et al., 2006), the proto-oncogene c-Myc (Li et al., 2003) and ezrin (Yu et al., 2004), a regulator of the cytoskeleton and contributor to metastasis, suggesting intermediates through which Eya and Six family genes regulate proliferation and contribute to cancer. Identification of stg as a transcriptional target of EYA and SO in Drosophila provides not only the first direct cell cycle target in Drosophila, but also suggests another target through which EYA and SO might regulate proliferation in other organisms. Before we can draw parallels to how EYA–SO targets important for Drosophila retinal development might be relevant to development and disease in other organisms, it will be necessary to examine the conservation of the transcriptional regulatory circuits. However, given the predicted functions of the gene products encoded by our candidate EYA–SO targets, together with knowledge of Eya–Six function in mammalian systems, it is tempting to speculate. For example, CG12030, the Drosophila homolog of the human Gale, encodes a sugar epimerase required for galactose metabolism (predicted by Interpro (Apweiler et al., 2000)). As metabolic abnormalities have been demonstrated to play a part in cataract formation, and mutations in eya have been observed in patients with congenital cataracts (Azuma et al., 2000), the identification of CG12030 as an EYA–SO target suggests that intermediates through with eya might function to maintain homeostasis in the eye. Mal, which encodes a molybdenum cofactor sulfurase important for ommochrome biosynthesis, is expressed in Drosophila pigment cells in the eye (Amrani et al., 2000) and would seem a logical target of the RDGN. Mutations of the human homolog of mal, HMCS, can result in renal failure and myositis (Ichida et al., 2001), both intriguing phenotypes given the importance of Eya–Six in vertebrate kidney and muscle development (Abdelhak et al., 1997; Grifone et al., 2005; Heanue et al., 1999; Laclef et al., 2003; Li et al., 2003; Ozaki et al., 2001; Xu et al., 1999, 2003). CG15879 encodes the Drosophila homolog of human SERHL2, a member of a serine hydrolase-like family predicted to regulate muscle growth, a developmental context in which Eya and Six family genes function in Drosophila and

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vertebrates (Clark et al., 2006; Grifone et al., 2005; Heanue et al., 1999; Laclef et al., 2003; Li et al., 2003). Lastly, CG8449 has a predicted RabGAP/TBC domain. While RabGAPs function in a variety of developmental contexts, RabGAP-like proteins have been predicted to function in phototransduction and synaptic transmission in Drosophila (Xu et al., 1998) and mutations in RabGAP genes have been isolated in cases of Warburg Micro syndrome (Aligianis et al., 2005), a severe autosomal recessive disorder characterized by abnormalities in the eye, as well as the central nervous system and genitals, all contexts in which Drosophila eya and so are expressed (Bonini et al., 1998; Boyle et al., 1997; Cheyette et al., 1994; Fabrizio et al., 2003). Identification of additional EYA–SO targets and the examination of the conservation of EYA–SO transcriptional regulation across homologous genes in different species will be necessary to determine how EYA and contribute to development and disease. Given the importance of achieving appropriate levels of gene expression during the course of development, it is not surprising that multiple signaling pathways converge to regulate common target genes at the level of transcription. For example, hh and lz are coordinately regulated by receptor tyrosine kinase (RTK) downstream effectors of the ETS family (Behan et al., 2002; Rogers et al., 2005) in conjunction with EYA and SO. Here, we have identified stg as an EYA–SO target, and work by us and others suggests that stg transcription is also regulated by Notch and Wingless (Wg) signaling (Deng et al., 2001; Johnston and Edgar, 1998) and by RTK signaling (Baonza et al., 2002). Thus, our results suggest a mechanism by which members of the RDGN are integrated with Notch and Wg signaling to coordinate cell proliferation. Identification of additional EYA–SO targets is likely to reveal new nodes for integration of the RDGN with other signaling pathways, explaining how signaling pathways cooperate to yield specific developmental outcomes. Acknowledgments Microarray experiments were performed by Jennifer Love at the Whitehead Institute Center for Microarray Technology. Thanks to J. Troy Littleton, A. Neisch and P. Vivekanand for comments on the manuscript and to members of the Fehon and Rebay labs for helpful discussions. Special thanks to the Singh, Goldstein, and Crispino labs for use of equipment. This work was supported in part by National Institutes of Health grant R01 EY12549 to I.R. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ydbio.2007.07.024. References Abdelhak, S., Kalatzis, V., Heilig, R., Compain, S., Samson, D., Vincent, C., Weil, D., Cruaud, C., Sahly, I., Leibovici, M., Bitner-Glindzicz, M., Francis, M., Lacombe, D., Vigneron, J., Charachon, R., Boven, K.,

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