SUMMARY Hearing loss affects 1 in 500 newborns and ~1 in 3 individuals over the age of 65. This disorder is often caused by the loss of mechanosensory hair cells (HCs), a deficiency that is permanent in the mature mammalian cochlea. Therefore, artificial approaches have been developed to regenerate HCs by `converting' non-HCs into HCs; and in mice they have proven successful in generating HC-like cells. However, these cells fail to mature fully and their lifespan is short, indicating that not all major barriers to HC regeneration have been overcome. The HC-regenerating approaches have been based on studies of physiological HC development. These have shown that the transcription factor `Atonal homolog 1' (ATOH1) is fundamental for HC development but that its effects on gene expression are context dependent. These effects differ in HCs versus other ATOH1-expressing cells. Thus, ATOH1 function is fine-tuned by other transcription factors. Harnessing the full potential of ATOH1 for the regeneration of HCs will require the identification of novel modifiers of ATOH1 activity in HCs. Our preliminary data suggest that ATOH1 activity and HC maturation are regulated by the transcription factor `thymocyte selection‐associated HMG box protein' (TOX). Knockout of the Tox gene in mice (Tox∆/∆) caused HC loss and deafness, and RNA-seq analysis of the organ of Corti in these mice revealed that a variety of genes are expressed at abnormal levels. The `abnormally low expression' group includes ATOH1 target genes, `RE1-silencing transcription factor' (REST) target genes, and the transcriptional repressor-encoding gene castor zinc finger 1 (Casz1). Targeted mutagenesis of Casz1 in organ of Corti cultures revealed that CASZ1 is needed for the repression of several genes that are expressed at abnormally high levels in the Tox∆/∆ organ of Corti. Our preliminary characterization of conditional Casz1 knock-out mice revealed that HC-specific deletion of Casz1 causes outer HC (OHC) degeneration and hearing loss. In addition, our previous analyses of REST function showed that perinatal downregulation of REST activity is needed for HC maturation. The objective of the proposed research is to define the role of TOX in the maturation of HCs. Our central hypothesis is that TOX supports cochlear HC maturation by modulating ATOH1, REST, and CASZ1 activities in developing HCs. We propose to test this hypothesis through 2 specific aims: Aim 1) determine the effects of TOX on HC maturation during various phases of cochlear development, and the extent to which it supports ATOH1 activity, REST regulation, and artificially induced production of HC-like cells; Aim 2) determine the effects of CASZ1 on HC morphology and cochlear gene expression, and identify gene repressor complexes that mediate the CASZ1-dependent repression of some of the indirect target genes of TOX in HCs. These aims will be achieved using a variety of methods ranging from RNA-seq to somatic cell genome-editing. The pr...