Identification and characterisation of gene regulation by the POU4F3 (BRN-3C) transcription factor in the inner ear

Hair cells are the mechanosensory cells of the inner ear whose loss causes irreversible hearing loss in mammals. POU4F3, a POU-domain transcription factor, is only expressed in hair cells in the inner ear and is essential for hair cell terminal maturation in mice. In humans, it is essential for hair cell survival as all three identified families with a POU4F3 mutation show autosomal dominant adult-onset progressive hearing loss. The pathways by which POU4F3 has this effect are unknown as little is known about its target genes (only four such genes are known: Bdnf, Ntf3, Gfi1 and Lhx3). Therefore, identification of unknown POU4F3 target genes would aid the understanding of hair cell maturation and survival. Prior to starting my doctoral work, a subtractive hybridization was carried out in an inner ear cell line (UB/OC-2) which was manipulated to either over- or under-express POU4F3 to identify unknown POU4F3 target genes. The clones produced by this screen were first matched to their corresponding gene; the presence of these genes in POU4F3-expressing UB/OC-2 cells was then confirmed by western blot and/or reverse transcriptase PCR. Where possible, expression was also confirmed in cochlear hair cells by immunofluorescence microscopy. Subsequently, the interaction of POU4F3 with these genes was characterised. Bioinformatics software was used to identify putative POU4F3 recognition elements in target gene promoters, and these sequences were then synthesised and used in a DNA binding assay (EMSA) to assess POU4F3 binding. The ability of POU4F3 to regulate putative gene promoters was also tested using luciferase assays. By these methods, one gene (Nr2f2) was shown to be a direct target of POU4F3 in vitro and two other genes were shown to be likely POU4F3 targets. The identification of these genes improves our understanding of the mechanism of POU4F3 function and further investigation of these networks may yield therapies for deafness caused by hair cell loss

Characterization of the Usher Syndrome gene CDH23: implications for mechanosensation in the vertebrate inner ear

Deafness is the most common form of sensory impairment afflicting the human population. Approximately one in eight hundred children is born with serious hearing impairment and more than half of these cases are likely due to single gene defects. In addition to hearing loss, mutations in some genes cause Usher Syndrome, not only affecting the auditory apparatus but also causing visual impairment eventually leading to blindness. Several genetic loci have been linked to Usher Syndrome Type I, the most severe form of the disease, and so far five of the relevant genes have been identified. Understanding their molecular role in the context of ear and retina physiology will be invaluable to the design of effective therapies against this devastating disease. Some forms of Usher Syndrome as well as other hearing disorders are caused by defects in the inner ear that contains the end organs for the perception of sound waves, the cochlea, and for the detection of gravity and acceleration, the vestibule. Both end organs contain mechanosensory hair cells that are named after actin rich stereocilia projecting from their apical surface. The stereocilia contain mechanically gated ion channels that open or close upon deflection of the stereocilia. This in turn triggers ion influx into the hair cells, causing changes in cell polarization and alterations in the rate of neurotransmitter release from the hair cells onto innervating neurons. The mechanically gated transduction channel implicated in this event has remained elusive. Mechanical gating of the transduction channel is believed to be triggered by thin filaments, the tip links, connecting adjacent stereocilia into a bundle. It has been suggested that these connector molecules are being stretched during hair cell stimulation, thereby actively pulling open the transduction channel. Although these filaments are clearly detectable on the ultrastructural level, their molecular nature has remained elusive. One molecule that might participate in mechanoelectrical transduction is the transmembrane protein cadherin 23 (CDH23). Mutations in its gene can cause Usher Syndrome, non-syndromic forms deafness and age-related hearing loss in human patients. Mice and zebrafish that carry mutations in the orthologous genes show splayed stereocilia bundle morphology, arguing for a function of the protein product in the cell compartment harboring the transduction channel. Furthermore, CDH23 is large enough to be the tip link, the extracellular filament proposed to gate the mechanotransduction channel. Here we show that antibodies against CDH23 label the entire stereocilia bundle during hair cell morphogenesis. In mature hair cells CDH23 labelling is confined to the tip links. Further, CDH23 has biochemical properties similar to those of the tip link. In cell-aggregation experiments CDH23 displays Ca2+-dependent, homophilic adhesion potential, an attribute typically observed for members of the cadherin superfamily, which may explain how adjacent stereocilia are linked together. Moreover, CDH23 forms a complex with myosin 1c (MYO1C), the only known component of the mechanotransduction apparatus, suggesting that CDH23 and MYO1C cooperate to regulate the activity of mechanically gated ion channels in hair cells. Computer assisted alignments with sequences encoding the cytosolic domain of CDH23 reveal two putative PDZ-binding motifs. Others and we can show that CDH23 interacts with the product of a second Usher Syndrome gene, harmonin. Two PDZ domains within harmonin interact with two complementary binding surfaces in the CDH23 cytoplasmic domain. One of the binding surfaces is disrupted by sequences encoded by an alternatively spliced CDH23 exon that is expressed in hair cells, but not in any other tissue analyzed so far. In the ear, harmonin is expressed in the stereocilia of developing hair cells. Since mice with a targeted deletion of the harmonin gene have been reported to phenocopy the splayed stereocilia bundle morphology observed in CDH23 deficient mice, the complex of the two Usher Syndrome proteins is predicted to be important for the stereocilia bundle. Whether the harmonin–CDH23 complex might be involved in mechanotransduction is unclear, since harmonin´s presence in mature stereocilia has not been reported yet. We concluded that CDH23 may serve a dual function in auditory hair cells: together with harmonin the molecule is important to shape the hair bundle during hair bundle morphogenesis and in mature stereocilia the molecule is part of the tip link complex

Molecular modelling insights into DFNA15 mediated enhancement of POU4F3 stability.

peer reviewedThe POU4F3 transcription factor is expressed in the cochlear and vestibular hair cells of the inner ear and its targeted deletion results in a loss of inner ear hair cells. The DFNA15 truncation mutation has been demonstrated to result in a loss of transcriptional activity, but an increase in the stability of the protein. Molecular modelling is utilised to propose a mechanism of stability enhancement, via an interaction between the truncated POU(HD) domain and the POU(S) domain of the transcription factor

Hearing Loss

Authored by 17 international researchers and research teams, the book provides up-to-date insights on topics in five different research areas related to normal hearing and deafness. Techniques for assessment of hearing and the appropriateness of the Mongolian gerbil as a model for age-dependent hearing loss in humans are presented. Parental attitudes to childhood deafness and role of early intervention for better treatment of hearing loss are also discussed. Comprehensive details are provided on the role of different environmental insults including injuries in causing deafness. Additionally, many genes involved in hearing loss are reviewed and the genetics of recessively inherited moderate to severe and progressive deafness is covered for the first time. The book also details established and evolving therapies for treatment of deafness