Functional Dissection of Eyes absent Reveals New Modes of Regulation within the Retinal Determination Gene Network

Whitehead Institute for Biomedical Research. MIT Department of Biology, Cambridge, Massachusetts 02142, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 10/2003; 23(17):5989-99. DOI: 10.1128/MCB.23.17.5989-5999.2003
Source: PubMed


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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    • "cDNA-based studies have also suggested that an internal proline-serine-threonine-rich (PST) domain in Eya is required for transcriptional activation in cell culture reporter assays and for efficient induction of ectopic eyes in vivo [36]. Consistent with these results, our genomic rescue assays also show that the PST domain is required for Drosophila survival as well as normal eye development (data not shown; to be reported elsewhere). "
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    ABSTRACT: Eyes absent (Eya) is an evolutionarily conserved transcriptional coactivator and protein phosphatase that regulates multiple developmental processes throughout the metazoans. Drosophila eya is necessary for survival as well as for the formation of the adult eye. Eya contains a tyrosine phosphatase domain, and mutations altering presumptive active-site residues lead to strongly reduced activities in ectopic eye induction, in vivo genetic rescue using the Gal4-UAS system, and in vitro phosphatase assays. However, these mutations have not been analyzed during normal development with the correct levels, timing, and patterns of endogenous eya expression. To investigate whether the tyrosine phosphatase activity of Eya plays a role in Drosophila survival or normal eye formation, we generated three eya genomic rescue (eyaGR) constructs that alter key active-site residues and tested them in vivo. In striking contrast to previous studies, all eyaGR constructs fully restore eye formation as well as viability in an eya null mutant background. We conclude that the tyrosine phosphatase activity of Eya is not required for normal eye development or survival in Drosophila. Our study suggests the need for a re-evaluation of the mechanism of Eya action and underscores the importance of studying genes in their native context.
    PLoS ONE 03/2013; 8(3):e58818. DOI:10.1371/journal.pone.0058818 · 3.23 Impact Factor
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    • "eyes absent (eya) encodes a highly conserved transcriptional coactivator and protein phosphatase whose homologs play vital roles in human development [1], [2], [3], [4]. Mutations in human EYA1 lead to the autosomal dominant disorder known as branchio-oto-renal (BOR) syndrome, characterized by craniofacial anomalies, hearing loss, and kidney defects [5]. "
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    ABSTRACT: Eyes absent (Eya) is a highly conserved transcription cofactor and protein phosphatase that plays an essential role in eye development and survival in Drosophila. Ectopic eye induction assays using cDNA transgenes have suggested that mitogen activated protein kinase (MAPK) activates Eya by phosphorylating it on two consensus target sites, S402 and S407, and that this activation potentiates the ability of Eya to drive eye formation. However, this mechanism has never been tested in normal eye development. In the current study, we generated a series of genomic rescue transgenes to investigate how loss- and gain-of-function mutations at these two MAPK target sites within Eya affect Drosophila survival and normal eye formation: eya(+)GR, the wild-type control; eya(SA)GR, which lacks phosphorylation at the two target residues; and eya(SDE)GR, which contains phosphomimetic amino acids at the same two residues. Contrary to the previous studies in ectopic eye development, all eya genomic transgenes tested rescue both eye formation and survival equally effectively. We conclude that, in contrast to ectopic eye formation, MAPK-mediated phosphorylation of Eya on S402 and S407 does not play a role in normal development. This is the first study in Drosophila to evaluate the difference in outcomes between genomic rescue and ectopic cDNA-based overexpression of the same gene. These findings indicate similar genomic rescue strategies may prove useful for re-evaluating other long-standing Drosophila developmental models.
    PLoS ONE 12/2012; 7(12):e50776. DOI:10.1371/journal.pone.0050776 · 3.23 Impact Factor
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    • "Biol. (2012), doi:10.1016/j.ydbio.2012.02.030 2003; Silver et al., 2003; Tootle et al., 2003). Taking advantage of our recent finding that cytoplasmic Eya phosphatase function contributes to photoreceptor axon targeting in the larval brain (Xiong et al., 2009), we first asked whether Nmo might interact with Eya in this context. "
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    ABSTRACT: The retinal determination gene network comprises a collection of transcription factors that respond to multiple signaling inputs to direct Drosophila eye development. Previous genetic studies have shown that nemo (nmo), a gene encoding a proline-directed serine/threonine kinase, can promote retinal specification through interactions with the retinal determination gene network, although the molecular point of cross-talk was not defined. Here, we report that the Nemo kinase positively and directly regulates Eyes absent (Eya). Genetic assays show that Nmo catalytic activity enhances Eya-mediated ectopic eye formation and potentiates induction of the Eya-Sine oculis (So) transcriptional targets dachshund and lozenge. Biochemical analyses demonstrate that Nmo forms a complex with and phosphorylates Eya at two consensus mitogen-activated protein kinase (MAPK) phosphorylation sites. These same sites appear crucial for Nmo-mediated activation of Eya function in vivo. Thus, we propose that Nmo phosphorylation of Eya potentiates its transactivation function to enhance transcription of Eya-So target genes during eye specification and development.
    Developmental Biology 02/2012; 365(1):267-76. DOI:10.1016/j.ydbio.2012.02.030 · 3.55 Impact Factor
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