Transcriptional regulation of hair cell development in the hearing organ

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 proposed studies are significant because identification of novel modifiers of ATOH1
activity will be necessary for improving the rational design of HC-regenerating approaches.

Grant Number: 5R01DC014953-09
NIH Institute/Center: NIH
Principal Investigator: Botond Banfi