Three distinct Atoh1 enhancers cooperate for sound receptor hair cell development [ATAC-seq]

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. Atoh1 expression is dynamic and tightly regulated during development, but the cis-regulatory elements mediating this regulation remain unresolved. Unexpectedly, we found that deleting the only recognized Atoh1 enhancer, defined here as Eh1, failed to impair HC development. By using ATAC-seq (assay for transposase-accessible chromatin with high-throughput sequencing), we discovered two additional Atoh1 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 Atoh1 regulation during HC development, indicating potential therapeutic approaches for HC regeneration. Overall design: Examination of genome wide chromatin accessibility in mouse P1 cochlear hair cells and P7 cerebellar granule neuron progenitors and Atoh1 binding sites in mouse P1 cochlear hair cells. For ATAC-seq, 2 replicates are involved. Each replicate contain 500 FACS purified cochlear hair cells or cerebellar granule neuron progenitors from Atoh1-3×V5-P2A-tdTomato/+ mouse strain. For CUT&RUN, Atoh1-GFP mouse strain was used and each replicate contain 2-3 fresh cochlear tissue. rabbit anti-GFP and rabbit anti-mouse IgG antibody was used for experimental and control group, respectively.