PNAS:耳聋研究新突破:刘志勇/陆发隆合作揭示耳蜗中三个Atoh1增强子协同调控听觉毛细胞发育

2022-08-06 “生物世界”公众号 “生物世界”公众号

解析了新生小鼠耳蜗毛细胞的全基因组开放染色质图谱,发现和验证了两个之前未知的 Atoh1 增强子。研究利用 CRISPR-Cas9 基因编辑技术敲除单个或同时敲除多个 Atoh1 增强子。

耳蜗毛细胞是声音的感受器,转录因子 Atoh1 突变小鼠完全丧失毛细胞,相反,过表达 Atoh1 能够额外产生更多的毛细胞。Atoh1 的重要生物学功能使之成为毛细胞发育再生领域的一个重要靶点。因此,探索 Atoh1 表达的调控机制具有重要的科学意义。

Atoh1 在毛细胞发育过程中高度动态变化,但其精准调控机制知之甚少。过去20年,Atoh1 增强子1(Eh1)被认为对 Atoh1 的表达至关重要。然而,这一假说一直未得到体内实验的证实,且毛细胞中是否存在尚未发现的其他 Atoh1 增强子也未知。

2022年8月4日,中国科学院脑科学与智能技术卓越创新中心(神经科学研究所)刘志勇研究组与中科院遗传与发育生物学研究所陆发隆研究组合作,在《美国国家科学院院刊》(PNAS)在线发表了题为:Three distinct Atoh1 enhancers cooperate for sound receptor hair cell development 的研究论文。

该研究解析了新生小鼠耳蜗毛细胞的全基因组开放染色质图谱,发现和验证了两个之前未知的 Atoh1 增强子。研究利用 CRISPR-Cas9 基因编辑技术敲除单个或同时敲除多个 Atoh1 增强子,揭示了 Atoh1 的三个增强子以剂量依赖性协同调控毛细胞发育。

该研究为 Atoh1 表达的精准表观遗传学调控机制提供了更深入的理论依据,并为耳蜗毛细胞再生提供了新的潜在靶点。

该研究利用 CRISPR-Cas9 基因编辑技术敲除 Eh1、Eh1 -/- 小鼠耳蜗毛细胞发育完全正常,这表明 Eh1 对于毛细胞的发育不是必需的。该结果和预期完全相反,提示毛细胞存在尚未发现的其他 Atoh1 增强子。

研究流式分选获得新生小鼠耳蜗毛细胞(图1A),并利用染色质开放性测序技术(ATAC-seq),建立了毛细胞的全基因组染色质开放图谱,发现了两个位于 Atoh1 远端的新增强子(Eh2和Eh3)。Eh2 和 Eh3 只在毛细胞中开放,而在对照组(小脑颗粒细胞前体细胞,同样表达 Atoh1)中处于关闭状态,提示 Eh2 和 Eh3 是毛细胞特异的调控元件(图1B)。

科研团队构建了转基因小鼠,验证了 Eh2 和 Eh3 是特异在毛细胞中活跃的增强子(图1C)。为了进一步探究 Eh2 和 Eh3 在毛细胞发育中的功能,研究在小鼠中分别敲除 Eh2 和 Eh3,毛细胞发育完全正常或者仅有弱的发育异常。而同时敲除 Eh2 和 Eh3,毛细胞数量明显减少;进一步发现,同时敲除三个 Atoh1 增强子使毛细胞数量进一步下降到最低点。正常的耳蜗毛细胞数量约2800个(图1D),敲除 Atoh1 的三个增强子后,毛细胞数量急剧减少到约86个(图1E),上述实验结果表明 Atoh1 的三个增强子协同调控毛细胞的形成。

图1.耳蜗毛细胞ATAC-seq,增强子验证和功能分析。A、耳蜗毛细胞流式分选和ATAC-seq示意图;B、Eh2和Eh3在毛细胞中保持开放,而在对照组中处于关闭状态;C、转基因小鼠耳蜗染色分析发现Eh3特异驱动毛细胞中EGFP报告基因的表达;D、正常野生型耳蜗毛细胞;E、同时敲除Atoh1的三个增强子引起毛细胞数量急剧减少。

研究人员利用 CUT&RUN 技术,揭示了 Atoh1 蛋白在耳蜗毛细胞中的全基因组结合位点,发现 Atoh1 可以直接结合到 Eh1、Eh2 和 Eh3 这三个增强子上,从而调控 Atoh1 自身的表达(图2)。该结果提供 Atoh1 的三个增强子被敲除后,Atoh1 表达水平显着下降,引起毛细胞数量急剧减少的分子机制。

图2.Atoh1和三个增强子的调控示意图。在正常毛细胞中,Atoh1蛋白可以直接结合到三个增强子(Eh1、Eh2和Eh3)上,促进Atoh1自身的表达。三个增强子被同时敲除,失去了自主调控作用,引起Atoh1表达下降,毛细胞数量减少。

该研究为在成年耳蜗支持细胞内如何重新启动 Atoh1 的表达提供了理论指导,有助于促进未来成年耳蜗毛细胞再生恢复研究。

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    2022-10-15 tulenzi
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    2023-06-27 drwjr
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    2023-03-10 zxxiang
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    2022-08-08 neurowu

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复旦附属眼耳鼻喉科医院新成果:小巧“基因魔剪”精准治疗遗传性耳聋

这是国际上首个基于RNA单碱基编辑技术成功精准治疗显性遗传性耳聋的研究。

Braz J Otorhinolaryngol:突发感音神经性耳聋中不良预后的预测参数研究

突发感音神经性耳聋的预后可以通过使用实验室血液分析的一些参数来进行预测。最近,有研究人员鉴定和调查了突发感音神经性耳聋不良预后的最显著的指示参数。

Otol Neurotol:耳蜗死区对突发感音神经性耳聋听力结果的影响研究

最近,有研究人员在多中心研究中前瞻性的评估了死区(DRs)在突发感音神经性耳聋(SSNHL)中的患病率,并根据DRs的有无比较了SSNHL的临床特征和听力结果。