LAUSR

Significance The expression of Atoh1, a gene essential for cochlear hair cell (HC) development, is tightly regulated. A previously identified Atoh1 enhancer is considered vital for regulating Atoh1 expression, but direct evidence is lacking. It also remains unknown whether additional enhancers exist and play critical roles in controlling Atoh1 expression. This study makes four contributions: 1) genomewide open chromatin regions of neonatal cochlear HCs are revealed, 2) two previously unknown Atoh1 enhancers are identified and validated, 3) individual and synergistic functions of the three enhancers are uncovered using seven mutant models, and 4) Atoh1 protein is shown to bind to all three Atoh1 enhancers. These provide insights into the molecular mechanisms underlying Atoh1 expression and subsequent HC development.