Identifying the Atoh1 targetome in hair cells with deep sequencing

听力和平衡障碍是美国最常见的残疾，0.1%的新生儿患有某种形式的遗传性听力损失，大约一半的成年人在达到退休年龄时患有某种程度的听力损失。年龄依赖性听力损失的原因很复杂，遗传和环境因素都起着作用。听力损失最常见的原因是内耳感觉毛细胞的死亡。除了遗传因素外，噪音暴露或某些抗生素和化疗药物的损害也会杀死毛细胞。一旦哺乳动物的毛细胞死亡，它们就永远不会被取代，因此听力损失是永久性的。过去15年的许多实验表明，转录因子Atoh 1在感觉毛细胞的发育中起着核心作用。首先，它是 在分化的毛细胞中最早表达的基因。第二，Atoh 1在小鼠中的失活 导致耳蜗和前庭感觉器官中毛细胞分化的完全失败。第三，Atoh 1是在非哺乳类脊椎动物毛细胞再生过程中最先表达的基因之一。最后，Atoh 1的异位表达足以在体外和体内内耳的某些区域产生新的毛细胞。由于这些原因，Atoh 1已被提议作为基因治疗的工具，以取代丢失的听觉或前庭毛细胞。尽管人们对Atoh 1在发育中的功能及其在再生疗法中的潜在用途非常感兴趣，但对Atoh 1在毛细胞中调控的基因知之甚少。在一个极端，Atoh 1可能调节相对少量的其他转录因子，这些转录因子各自调节毛细胞发育，成熟和存活的不同方面。或者，Atoh 1可能在直接调节毛细胞结构和功能的许多方面具有更广泛的作用。此外，尚不清楚Atoh 1转录靶点是否不同 听觉和前庭毛细胞之间的差异，或者这些差异是否是由其他因素决定的，Atoh 1只是调节一般的毛细胞特性。在这个试点项目中，我们提议使用尖端的深度测序和生物信息学方法来识别毛细胞中Atoh 1的直接靶点-所谓的Atoh 1“targetome”。我们将与贝勒医学院的Huda Zoghbi博士合作，他的实验室最近使用类似的技术成功地表征了小脑颗粒细胞中的Atoh 1 targetome。由Atoh 1直接调控的基因应该具有三个属性-它应该以Atoh 1依赖的方式表达，基因的基因组位点应该具有转录活性，Atoh 1蛋白应该与基因附近的调控区域结合。Zoghbi实验室的成功策略涉及对这三种特性的全基因组分析，再加上统计排名，以生成Atoh 1直接靶点的列表。我们现在建议应用这些相同的方法来识别毛细胞中的Atoh 1靶组。

DESCRIPTION (provided by applicant): Hearing and balance disorders are some of the most common disabilities in the United States, with 0.1% of newborns having some form of hereditary hearing loss and roughly half of all adults suffering from some degree of hearing loss by the time they reach retirement age. The causes of age-dependent hearing loss are complex, with both genetic and environmental factors playing a role. The most common reason for hearing loss is the death of sensory hair cells in the inner ear. In addition to genetic factors, damage from noise exposure or from certain kinds of antibiotics and chemotherapy drugs can kill hair cells. Once hair cells die in mammals, they are never replaced and hearing loss is therefore permanent. Many experiments over the last 15 years have suggested that the transcription factor Atoh1 plays a central role in the development of sensory hair cells. First, it is one of the earliest genes to be expressed in differentiating hair cells. Second, inactivation of Atoh1 in mice causes a complete failure of hair cell differentiation in both the cochlear and vestibular sensory organs. Third, Atoh1 is one of the first genes to be expressed during hair cell regeneration in non-mammalian vertebrates. Finally, ectopic expression of Atoh1 is sufficient to generate new hair cells in some regions of the inner ear in vitro and in vivo. For these reasons, Atoh1 has been proposed as tool for gene therapy to replace lost auditory or vestibular hair cells. Despite the great interest in Atoh1's function in development and its potential use in regenerative therapies, very little is known about the genes that are regulated by Atoh1 in hair cells. At one extreme, Atoh1 might regulate a relatively small number of other transcription factors, which would each regulate different aspects of hair cell development, maturation and survival. Alternatively, Atoh1 might have a much broader role in directly regulating many aspects of hair cell structure and function. Moreover, it is not clear whether Atoh1 transcriptional targets differ between auditory and vestibular hair cells, or whether these differences are determined by other factors, with Atoh1 simply regulating generic hair cell properties. In this pilot project, we propoe to use cutting-edge deep sequencing and bioinformatic approaches to identify the direct targets of Atoh1 in hair cells - the so-called Atoh1 "targetome". We will work in collaboration with Dr. Huda Zoghbi at Baylor College of Medicine, whose lab recently used similar technology to successfully characterize the Atoh1 targetome in cerebellar granule cells. A gene that is directly regulated by Atoh1 should possess three attributes - it should be expressed in an Atoh1-dependent manner, the genomic locus of the gene should be transcriptionally active, and Atoh1 protein should bind to regulatory regions adjacent to the gene. The successful strategy of the Zoghbi lab involved a genome-wide analysis of these three properties, coupled with statistical ranking to generate a list of direct Atoh1 targets. We now propose to apply these same methods to identify the Atoh1 targetome in hair cells.