| Summary: | <p><em>cello</em> is a recessive mouse model of early-onset, sensorineural hearing loss that was identified from a large-scale <em>N</em>-ethyl-<em>N</em>-nitrosourea (ENU) mutagenesis screen undertaken at MRC Harwell. Linkage analysis and whole-genome sequencing identified that <em>cello</em> mice have a non-synonymous point mutation in <em>Ikaros family zinc finger</em> 2 (<em>Ikzf2</em>), which encodes a zinc finger transcription factor called helios.</p> <p>To investigate the role of helios in the auditory system, <em>cello</em> mice underwent comprehensive molecular, phenotypic and functional characterisation. The <em>cello</em> mutation was found to disrupt a highly conserved, zinc-coordinating residue in the final C-terminal zinc finger domain of helios. <em>In vitro</em> analyses demonstrated that mutant helios can still localise to the nucleus, but has an impaired ability to homodimerise – a prerequisite for DNA binding. In both <em>Ikzf2<sup>+/+</sup></em> and <em>Ikzf2<sup>cello/cello</sup></em> cochleae, helios is expressed in the nuclei of the outer hair cells (OHCs) from the first postnatal week, coinciding with a critical period of OHC functional maturation. Although Ikzf2<sup>cello/cello</sup> exhibit severely impaired hearing at postnatal day (P) 16, cochlear morphology appears grossly normal up to one month of age, after which the OHCs and inner hair cells begin to progressively degenerate. Transcriptome profiling was subsequently undertaken to identify potential target genes of helios in the auditory system. Results suggested that loss of helios function leads to dysregulation of over 450 genes in <em>Ikzf2<sup>cello/cello</sup></em> cochleae at P8. This included the OHC-specific genes Slc26a5 (which encodes the OHC motor protein, prestin) and Ocm (oncomodulin), both of which were significantly decreased in <em>Ikzf2<sup>cello/cello</sup></em> mutants compared to <em>Ikzf2<sup>+/+</sup></em> controls. Electrophysiological studies demonstrated that <em>Ikzf2<sup>cello/cello</sup></em> OHCs fail to acquire full electromotility, a property that is fundamental to their function, but otherwise mature normally. Using luciferase reporter assays, wild-type helios was found to induce transactivation of the <em>Slc26a5</em> promoter, whilst this ability was lost in the mutant protein. Additional <em>in silico</em> network analyses identified helios as a putative direct regulator of 40 genes that are enriched in the OHCs and upregulated during development, which suggests that helios has a key role in regulating the maturing OHC transcriptome.</p> <p>Overall, the work presented in this thesis identifies <em>Ikzf2</em> as a novel hearing loss-causing gene and establishes that the helios transcription factor is essential for OHC functional maturation in the mammalian auditory system.</p>
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