Academic journal article Genetics

Using Drosophila to Decipher How Mutations Associated with Human Branchio-Oto-Renal Syndrome and Optical Defects Compromise the Protein Tyrosine Phosphatase and Transcriptional Functions of Eyes Absent

Academic journal article Genetics

Using Drosophila to Decipher How Mutations Associated with Human Branchio-Oto-Renal Syndrome and Optical Defects Compromise the Protein Tyrosine Phosphatase and Transcriptional Functions of Eyes Absent

Article excerpt

ABSTRACT

Eyes absent (EYA) proteins are defined by a conserved C-terminal EYA domain (ED) that both contributes to its function as a transcriptional coactivator by mediating protein-protein interactions and possesses intrinsic protein tyrosine phosphatase activity. Mutations in human EYA1 result in an autosomal dominant disorder called branchio-oto-renal (BOR) syndrome as well as congenital cataracts and ocular defects (OD). Both BOR- and OD-associated missense mutations alter residues in the conserved ED as do three missense mutations identified from Drosophila eya alleles. To investigate the molecular mechanisms whereby these mutations disrupt EYA function, we tested their activity in a series of assays that measured in vivo function, phosphatase activity, transcriptional capability, and protein-protein interactions. We find that the OD-associated mutations retain significant in vivo activity whereas those derived from BOR patients show a striking decrease or loss of in vivo functionality. Protein-protein interactions, either with its partner transcription factor Sine oculis or with EYA itself, were not significantly compromised. Finally, the results of the biochemical assays suggest that both loss of protein tyrosine phosphatase activity and reduced transcriptional capability contribute to the impaired EYA function associated with BOR/OD syndrome, thus shedding new light into the molecular mechanisms underlying this disease.

RETINAL specification and development in both vertebrates and invertebrates rely on the concerted actions of a group of evolutionarily conserved transcription factors and cofactors that include twin of eyeless ( toy), eyeless (ey), eyes absent (eya), sine oculis (so), and dachshund (dac), with the human homologs referred to as PAX6 (homolog of both toy and ey), EYA, SIX, and DACH, respectively (reviewed in WAWERSIK and MAAS 2000; PAPPU and MARDON 2002). PAX6 lies atop the hierarchy and directly activates expression of EYA and SIX family members, which operate synergistically to induce expression of DACH and other downstream genes (reviewed in PAPPU and MARDON 2002). Because of their prominent role in eye development, exemplified in Drosophila by the "eyeless" phenotype and visual system defects associated with loss-of-function mutations and the ability to induce formation of ectopic eye tissue upon overexpression, these genes have been referred to collectively as the retinal determination (RD) gene network (PAPPU and MARDON 2002). In addition to their roles in the eye, all RD genes, either individually or as a network, contribute to a diverse array of essential developmental processes in Drosophila and in vertebrates. Consequently, null mutations are lethal and exhibit complex defects in a variety of tissues and organs (reviewed in WAWERSIK and MAAS 2000; SILVER and REBAY 2005).

EYA family members encode novel nuclear proteins defined by a conserved ^275-amino-acid C-terminal motif, referred to as the EYA domain (ED), which mediates direct interactions with SO/SIX and DAC/DACH (BoNINI et al. 1993; CHEN et al. 1997; PIGNONI et al. 1997; ZIMMERMAN et al. 1997; HEANUE et al. 1999; OHTO et al. 1999). The N terminus of EYA contributes a conserved fraws-activation function to an EYA-SO bipartite transcription factor in which the homeodomain protein SO/SLX provides the DNA-binding moiety (Ïêôï et al. 1999; SILVER et al. 2003). The mechanistic implications of EYA-DACH interactions are less clear. DAC/DACH proteins function as both coactivators and corepressors and may also have DNA-binding capability, implying roles in tethering EYA to the DNA and influencing transcription of downstream genes (!KEDA et al. 2002; KIM et al. 2002; Li et al. 2003). Recent work revealed that the ED of EYA also exhibits catalytic activity, functioning as a protein tyrosine phosphatase (Li et al. 2003; RAYAPUREDDI et al. 2003; TOOTLE et al. 2003b). EYA's phosphatase activity appears critical for switching DACH between corepressor and coactivator states, suggesting an integral contribution to regulating transcriptional output (Li et al 2003). …

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