A Comparative Study of Eya1 and Eya4 Protein Function and Its Implication in Branchio-oto-renal Syndrome and DFNA10

Abstract

Allele variants of EYA1 and EYA4, two members of the vertebrate Eya gene family, underlie two types of inherited human deafness, branchio-oto-renal (BOR) syndrome and DFNA10, respectively. To clarify how mutations in these two genes and their encoded proteins impact the normal biology of hearing, we completed a number of functional studies using the yeast-two-hybrid system. We verified that bait constructs of the homologous region (Eya1HR and Eya4HR) interact with Six1 prey constructs, although no interaction with Dach1 prey was demonstrable. To compare interaction affinities, we evaluated a-galactosidase activity after cotransformation of Eya1HR/ Six1 and Eya4HR/Six1 and found that the latter interaction was weaker. By immunofluorescence staining, we showed Eya4HR localization to the cytoplasm. After coexpression of Six1, Eya4HR was translocated to the nucleus. Results with Eya1HR were similar. Translation of mutant constructs (Eya4HR R564X and Eya1HR R539X ) could not be demonstrated. Using dual Eya-containing constructs (with two wild-type alleles or wild-type and mutant alleles), we confirmed no translation of the mutant allele, even if the mutation was nontruncating. These results are consistent with clinical data and implicate haploinsufficiency as the cause of BOR syndrome and DFNA10.

The preplacodal region: an ectodermal domain with multipotential progenitors that contribute to sense organs and cranial sensory ganglia

“…Eya1 and Six1 have been studied extensively and play a role in the formation of most placode derivatives (see below), reflecting their widespread expression in the preplacodal region. Likewise, mutations in Eya4 and Six5 are associated with defects in placode derivatives (Klesert, et al, 2000;Wayne, et al, 2001;Zhang, et al, 2004), while information about Six2 and Eya2 is very sparse. Mice lacking Eya2 function have been generated, however, their placodal phenotype has not been described in detail (Grifone, et al, 2007).…”

“…Eya1 mutations in humans are also associated with congenital eye defects (Azuma, et al, 2000), although these have not been described in mice. Finally, mutations in the eya-homologous region of Eya4 lead to late-onset deafness in humans (Wayne, et al, 2001;Zhang, et al, 2004;Schonberger, et al, 2005).…”

The Eyes Absent Family of Phosphotyrosine Phosphatases: Properties and Roles in Developmental Regulation of Transcription

“…However, studies of vertebrate Eya1-4 in cultured cells have revealed that these proteins localize to the cytoplasm and require cotransfection with Six1/2 or Six4/5 subfamilies to accumulate stably in the nucleus (24,119). Intracellular localization of the vertebrate Eya homologs in the presence and absence of Six family proteins in whole animals has not been reported.…”

Genome-wide discovery of Pax7 target genes during development

“…Likewise, Pax7 and Eya4 share significantly similar developmental expression patterns; both play critical roles in neurogenesis and myogenesis and are highly expressed in developing brain, craniofacial mesenchyme, dermomyotome and limb during embryogenesis (6,25,49), and both Eya4 and Pax7 are robustly expressed in brain and skeletal muscle in adult mouse tissues (65,70,82). Eya family members function as transcriptional coactivators controlling cell fate specification, cell survival and apoptosis, proliferation, differentiation, and morphogenesis (34,49,57).…”