Three distinct Atoh1 enhancers cooperate for sound receptor hair cell development [CUT&RUN]

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. 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.

内耳耳蜗毛细胞（Cochlear hair cells, HCs）负责感知声音信号。在毛细胞命运特化过程中，主转录因子Atoh1既是必需条件，也是充分条件。Atoh1的表达在发育进程中具有动态性且受到严格调控，但介导该调控的顺式调控元件（cis-regulatory elements）仍未明确。令人意外的是，我们发现删除本文此前唯一被鉴定的Atoh1增强子（本文命名为Eh1），并未对毛细胞发育造成损害。通过ATAC-seq（高通量测序转座酶可及性染色质实验，assay for transposase-accessible chromatin with high-throughput sequencing），我们额外发现了两个Atoh1增强子：Eh2与Eh3。值得注意的是，单独删除Eh2即可损害毛细胞发育；若同时删除Eh1与Eh2，或是删除全部三个增强子，则会导致几乎完全无毛细胞生成。最后，我们证实Atoh1可结合这三个增强子，这与其自调控功能相符。本研究结果揭示，三个不同增强子的协同作用支撑了毛细胞发育过程中Atoh1的有效调控，这为毛细胞再生的潜在治疗策略提供了理论依据。本数据集包含小鼠P1龄耳蜗毛细胞、P7龄小脑颗粒神经元前体细胞的全基因组染色质可及性检测数据，以及小鼠P1龄耳蜗毛细胞的Atoh1结合位点数据。ATAC-seq实验设置2个生物学重复，每个重复使用来自Atoh1-3×V5-P2A-tdTomato/+小鼠品系的500个经荧光激活细胞分选（FACS）纯化的耳蜗毛细胞或小脑颗粒神经元前体细胞。CUT&RUN实验使用Atoh1-GFP小鼠品系，每个重复包含2~3份新鲜耳蜗组织；实验组与对照组分别使用兔抗GFP抗体与兔抗小鼠IgG抗体。