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. 2003 Sep;23(17):5989-99.
doi: 10.1128/MCB.23.17.5989-5999.2003.

Functional dissection of eyes absent reveals new modes of regulation within the retinal determination gene network

Serena J Silver  1 , Erin L Davies, Laura Doyon, Ilaria Rebay
Affiliations

Functional dissection of eyes absent reveals new modes of regulation within the retinal determination gene network

Serena J Silver et al. Mol Cell Biol. 2003 Sep.

Abstract

The retinal determination (RD) gene network encodes a group of transcription factors and cofactors necessary for eye development. Transcriptional and posttranslational regulation of RD family members is achieved through interactions within the network and with extracellular signaling pathways, including epidermal growth factor receptor/RAS/mitogen-activated protein kinase (MAPK), transforming growth factor beta/DPP, Wingless, Hedgehog, and Notch. Here we present the results of structure-function analyses that reveal novel aspects of Eyes absent (EYA) function and regulation. We find that the conserved C-terminal EYA domain negatively regulates EYA transactivation potential, and that GROUCHO-SINE OCULIS (SO) interactions provide another mechanism for negative regulation of EYA-SO target genes. We have mapped the transactivation potential of EYA to an internal proline-, serine-, and threonine-rich region that includes the EYA domain 2 (ED2) and two MAPK phosphorylation consensus sites and demonstrate that activation of the RAS/MAPK pathway potentiates transcriptional output of EYA and the EYA-SO complex in certain contexts. Drosophila S2 cell two-hybrid assays were used to describe a novel homotypic interaction that is mediated by EYA's N terminus. Our data suggest that EYA requires homo- and heterotypic interactions and RAS/MAPK signaling responsiveness to ensure context-appropriate RD gene network activity.

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Figures

FIG. 1.
FIG. 1.
The N-terminus of EYA is a potent transactivator. (A) Drosophila EYA contains two conserved regions, the ED2 and ED. The P/S/T-rich region includes both the ED2 and the two MAPK phosphoacceptor sites. (B) Gal4DBD-EYA fusions were used to assay the ability of EYA to activate transcription from a UAS-luciferase reporter gene. Transactivation potentials were calculated by taking the luciferase/β-galactosidase activity ratio for each construct and were plotted relative to the activity of the Gal4DBD vector alone. As shown in panel B, for construct 2, full-length EYA can activate transcription 3.5-fold above Gal4DBD alone. The N terminus of EYA (construct 3), a construct that lacks the ED, is a potent transactivator, activating transcription over 250-fold (note scale change on axis). Gal4DBD-EYA 1-353 (construct 4), a truncation that contains ED2 but removes part of the P/S/T-rich region, reduces transactivation potential to only fivefold. ED2 plus part of the P/S/T-rich region is able to activate transcription at low levels (construct 5), indicating that the entire N terminus is necessary for full transactivation potential. Deletion of the conserved ED2 within the N terminus of EYA (construct 6) sharply reduces transactivation to 41-fold above background, one-fifth the activity of the intact N terminus. Strikingly, deletion of the entire P/S/T-rich region (construct 7) results in complete loss of transactivation potential. (C) Deletion of the EYA domain does not affect protein expression levels. WT, wild type.
FIG. 2.
FIG. 2.
RAS/MAPK signaling activates EYA transactivation. Transactivation via Gal4DBD-EYA ΔED is increased significantly but variably upon the addition of RASV12. Mutations of the MAPK phosphoacceptors to alanine (EYAS-A) to prevent phosphorylation, or to aspartic or glutamic acid (EYAS-D/E) to mimic phosphorylation, both increase Gal4DBD-EYA ΔED transactivation activity, suggesting that these sites are important for regulation of EYA transactivation potential. Taken with the increase seen upon addition of RASV12, we conclude that RAS signaling can increase EYA transactivation potential.
FIG. 3.
FIG. 3.
ARE-luciferase is responsive to the EYA-SO transcription factor. (A) EYA and SO alone do not affect transcription of the ARE-luciferase reporter gene, but together can activate transcription 27-fold. Full-length EYA ΔED2 is unable to fully activate transcription of this gene, and neither is a construct missing the entire P/S/T-rich region, EYA Δ223-438. This construct was not expressed at the same level as wild-type EYA (EYAWT), so we transfected two (++) and three (+++) times the amount of plasmid to raise protein levels to and above the levels of EYA. These levels still do not activate ARE-luciferase. (B) Quantitative Western blotting shows that EYA (lane 1) and EYA ΔED2 (lane 2) are expressed at similar levels, while the same amount of EYA Δ223-438 plasmid (lane 3) is not. However, transfection of two and three times more EYA Δ223-438 plasmid results in robust expression, as shown in lanes 4 and 5. (C) EYA and SO show a strong interaction in an S2-2H assay. The deletions in EYA do not affect interactions with SO, and in fact, EYA Δ223-438 appears to have a stronger interaction with SO. DAC does not interact with EYA in S2-2H assays. (D) Overexpression of EYA using the 57A1dpp-Gal4 driver causes ectopic eye induction in over 98% of animals (n = 413). Overexpression of EYA ΔED2 results in ectopic eyes in only 35% of animals (n = 506). Deletion of the entire P/S/T-rich region, EYA Δ223-438, results in a protein that can only rarely induce ectopic eyes, seen in only 1.5% of animals examined (n = 204).
FIG. 4.
FIG. 4.
EYA-EYA interactions are mediated by the EYA N terminus. Using the S2-2H system, Gal4DBD-EYA fusions shown on the left were coexpressed with (+) and without (−) full-length Gal4AD-EYA. Full-length EYA interacts with itself sevenfold above the background level. This interaction is mediated by aa 223 to 317, because all constructs that contain this minimal region can interact with Gal4AD-EYA. Strikingly, Gal4DBD-EYA 2-353 and Gal4DBD-EYA ΔED2 (constructs 4 and 6) show a more than threefold-stronger interaction than that of full-length EYA. The Gal4DBD-ED fusion (construct 7) does not interact with Gal4AD-EYA, consistent with our finding that the EYA N terminus mediates this interaction.
FIG. 5.
FIG. 5.
The EYA-SO transcription factor is regulated by phosphorylation. (A) As shown in Fig. 3, the EYA-SO transcription factor can activate expression of ARE-luciferase. This expression is not affected by addition of RASV12 nor upon mutation of the MAPK phosphoacceptor sites to alanine (EYAS-A). A striking increase in activation is seen when the EYAS-D/E phosphomimetic mutant is used, showing that phosphorylation acts to increase EYA transactivation potential. That RASV12 itself does not produce the same increase on wild-type EYA (WT) in this assay suggests that RAS signaling may have multiple effects on the RD gene network and in particular may negatively regulate SO. (B) EYA phosphoacceptor mutations do not affect protein expression levels.
FIG. 6.
FIG. 6.
The EYA-SO transcription factor is negatively regulated by interactions with GROUCHO. (A) When coexpressed with EYA and SO, GRO is a repressor of the EYA-SO transcription factor. (B) Lanes 1 and 2 show that MYC-EYA and GRO are not pulled down by anti-Flag beads. In lane 3, IP of SO can co-IP EYA. Lane 4 shows that IP of SO can co-IP EYA but not GRO; however, in lane 5, we see that without EYA, GRO can associate with SO. Thus, co-IP of GRO with SO is disrupted by cotransfection of EYA. This result does not seem to be due to direct competition between EYA and SO for GRO, because IP of EYA cannot co-IP GRO. All proteins were expressed at similar levels in crude cell lysates (data not shown).
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