Three distinct <i>Atoh1</i> enhancers cooperate for sound receptor hair cell development.

Abstract

Cochlear hair cells (HCs) in the inner ear are responsible for sound detection. For HC fate specification, the master transcription factor Atoh1 is both necessary and sufficient. <i>Atoh1</i> expression is dynamic and tightly regulated during development, but the <i>cis</i>-regulatory elements mediating this regulation remain unresolved. Unexpectedly, we found that deleting the only recognized <i>Atoh1</i> enhancer, defined here as Eh1, failed to impair HC development. By using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we discovered two additional <i>Atoh1</i> enhancers: Eh2 and Eh3. Notably, Eh2 deletion was sufficient for impairing HC development, and concurrent deletion of Eh1 and Eh2 or all three enhancers resulted in nearly complete absence of HCs. Lastly, we showed that Atoh1 binds to all three enhancers, consistent with its autoregulatory function. Our findings reveal that the cooperative action of three distinct enhancers underpins effective <i>Atoh1</i> regulation during HC development, indicating potential therapeutic approaches for HC regeneration.