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