The DFNA15 Deafness Mutation Affects POU4F3 Protein Stability, Localization, and Transcriptional Activity

Abstract

A mutation in the POU4F3 gene (BRN-3.1, BRN3C) is responsible for DFNA15 (MIM 602459), autosomaldominant nonsyndromic hearing loss. POU4F3 is a member of the POU family of transcription factors and is essential for inner-ear hair cell maintenance. To test the potential effects of the human POU4F3 mutation, we performed a series of experiments in cell culture to mimic the human mutation. Mutant POU4F3 loses most of its transcriptional activity and most of its ability to bind to DNA and does not function in a dominantnegative manner. Moreover, whereas wild-type POU4F3 is found exclusively in the nucleus, our studies demonstrate that the mutant protein is localized both to the nucleus and the cytoplasm. Two nuclear localization signals were identified; both are essential for proper nuclear entry of POU4F3 protein. We found that the mutant protein half-life is longer than that of the wild type. We propose that the combination of defects caused by the mutation on the function of the POU4F3 transcription factor eventually leads to hair cell morbidity in affected family H members.The POU domain transcription factors play an important role in tissue-specific gene regulation. They were originally defined by sequence homology between four transcription factors: mammalian Pit-1, Oct-1, and Oct-2 and nematode Caenorhabditis elegans 7,15,16). The region of homology, referred to as the POU domain, is a bipartite DNAbinding domain that contains a POU-specific (POU S ) domain and the POU-homeodomain (POU HD ) joined by a variable linker. High-affinity DNA binding of POU transcription factors requires both domains (1, 32). Based on sequence homology in the POU domain, the POU transcription factors were divided into six subclasses from I to VI (37). The class IV POU subfamily includes POU4F1 (Brn3a/Brn-3.0), POU4F2 (Brn3b/ Brn-3.2), and POU4F3 (Brn3c/Brn-3.1) in mammals (11,14,21,25,35,44), C. elegans Unc-86, and Drosophila factors I-POU and tI-POU (37).The three mammalian members of the POU IV subfamily are expressed in distinct but overlapping populations of neuronal cells during development and in the adult organism (11,25,35). Gene-targeted mutagenesis in the mouse of each of these class IV transcription factors demonstrates that the cell type in which it is first expressed is the system that is most affected (6,10,23,39,40). In the inner ear, Pou4f3 is uniquely and strongly expressed in cochlear and vestibular hair cells. Targeted deletion of Pou4f3 results in profound deafness and impaired balance due to complete loss of auditory and vestibular hair cells. This is followed by a partial secondary loss of spiral and vestibular ganglion neurons (6,39,42). Pou4f3 is required for the final differentiation and survival of hair cells (41).A mutation in the POU4F3 gene is associated with hearing loss in a large Israeli Jewish family, family H (36). Affected members of family H suffer from progressive autosomal-dominant sensorineural hearing loss (9). In hearing impaired members of family H, an 8-bp deletion was identified in exon 2...

Clinical and molecular characterizations of novelPOU3F4mutations reveal that DFN3 is due to null function of POU3F4 protein

“…Similar to our POU3F4 results, molecular analyses of POU4F3 mutations identified in DFNA15 patients showed that the mutant POU4F3 proteins lack transcriptional activity due to the loss of nuclear localization and DNAbinding ability (6,35). Thus, the two POU family transcription factors share a common molecular pathological mechanism, whereby transcription of target genes essential for inner ear development is affected.…”

Caprin-1 is a target of the deafness genePou4f3and is recruited to stress granules in cochlear hair cells in response to ototoxic damage

“…In Pou4f3-null mice, hair cells fail to mature and undergo apoptosis during early postnatal development, resulting in mice with auditory or vestibular dysfunction (Erkman et al, 1996;Xiang et al, 1997;Xiang et al, 1998). In addition, mutations in human POU4F3 result in adultonset non-syndromic hearing loss (DFNA15) Pauw et al, 2008;Vahava et al, 1998;Weiss et al, 2003).…”

Specific Amino Acid Residues in the Basic Helix-Loop-Helix Domain of SRC-3 Are Essential for Its Nuclear Localization and Proteasome-Dependent Turnover

“…It is known that proteasomes are localized in both the nucleus and the cytosol, where intracellular proteins can be degraded by them (16). A number of transcription factors have previously been reported to be degraded in the nucleus (4,9,11,13,28,46,49,63).…”