Transcriptional regulation of hair cell development in the hearing organ

听觉器官毛细胞发育的转录调控

• 批准号: 10659051
• 负责人: Botond Banfi
• 金额: $ 38.63万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-04 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10659051
• 关键词: Affect; Age; Alternative Splicing; Audiology; Cell Maturation; Cell Survival; Cells; Cellular Morphology; Cochlea; Data; Development; Disease; Down-Regulation; Epithelium; Event; Exons; Functional disorder; Funding; Gene Expression; Gene Silencing; Genes; Genetic Transcription; Goals; HMGB Family Gene; Hair Cells; Hearing; Homologous Gene; Individual; Knock-out; Knockout Mice; Knowledge; Longevity; Measures; Mediating; Methods; Mus; Mutagenesis; Mutation; Natural regeneration; Neurons; Newborn Infant; Organ; Organ of Corti; Outcome Study; Outer Hair Cells; Perinatal; Phase; Physiological; Production; Public Health; RE1-silencing transcription factor; RNA Splicing; Regulation; Repression; Repressor Proteins; Research; Role; Sensory; Testing; Thymocyte Selection; Transcription Repressor; Transcriptional Regulation; Transgenes; Viral Vector; Zinc Fingers; cell type; cochlear development; deaf; deafness; design; gene interaction; hair cell regeneration; hearing impairment; human old age (65+); improved; knockout gene; novel; prenatal; prevent; programs; rational design; somatic cell gene editing; transcription factor; transcriptome sequencing

SUMMARY Hearing loss affects 1 in 500 newborns and ~1 in 3 individuals over the age of 65. This disorder is often caused by the loss of mechanosensory hair cells (HCs), a deficiency that is permanent in the mature mammalian cochlea. Therefore, artificial approaches have been developed to regenerate HCs by `converting' non-HCs into HCs; and in mice they have proven successful in generating HC-like cells. However, these cells fail to mature fully and their lifespan is short, indicating that not all major barriers to HC regeneration have been overcome. The HC-regenerating approaches have been based on studies of physiological HC development. These have shown that the transcription factor `Atonal homolog 1' (ATOH1) is fundamental for HC development but that its effects on gene expression are context dependent. These effects differ in HCs versus other ATOH1-expressing cells. Thus, ATOH1 function is fine-tuned by other transcription factors. Harnessing the full potential of ATOH1 for the regeneration of HCs will require the identification of novel modifiers of ATOH1 activity in HCs. Our preliminary data suggest that ATOH1 activity and HC maturation are regulated by the transcription factor `thymocyte selection‐associated HMG box protein' (TOX). Knockout of the Tox gene in mice (Tox∆/∆) caused HC loss and deafness, and RNA-seq analysis of the organ of Corti in these mice revealed that a variety of genes are expressed at abnormal levels. The `abnormally low expression' group includes ATOH1 target genes, `RE1-silencing transcription factor' (REST) target genes, and the transcriptional repressor-encoding gene castor zinc finger 1 (Casz1). Targeted mutagenesis of Casz1 in organ of Corti cultures revealed that CASZ1 is needed for the repression of several genes that are expressed at abnormally high levels in the Tox∆/∆ organ of Corti. Our preliminary characterization of conditional Casz1 knock-out mice revealed that HC-specific deletion of Casz1 causes outer HC (OHC) degeneration and hearing loss. In addition, our previous analyses of REST function showed that perinatal downregulation of REST activity is needed for HC maturation. The objective of the proposed research is to define the role of TOX in the maturation of HCs. Our central hypothesis is that TOX supports cochlear HC maturation by modulating ATOH1, REST, and CASZ1 activities in developing HCs. We propose to test this hypothesis through 2 specific aims: Aim 1) determine the effects of TOX on HC maturation during various phases of cochlear development, and the extent to which it supports ATOH1 activity, REST regulation, and artificially induced production of HC-like cells; Aim 2) determine the effects of CASZ1 on HC morphology and cochlear gene expression, and identify gene repressor complexes that mediate the CASZ1-dependent repression of some of the indirect target genes of TOX in HCs. These aims will be achieved using a variety of methods ranging from RNA-seq to somatic cell genome-editing. The proposed studies are significant because identification of novel modifiers of ATOH1 activity will be necessary for improving the rational design of HC-regenerating approaches.

总结 听力损失影响500名新生儿中的1名和65岁以上的3名个体中的1名。这种疾病往往是 由机械感觉毛细胞（HC）的损失引起，这种缺陷在成熟的 哺乳动物耳蜗因此，开发了通过“转化”来再生碳氢化合物的人工方法。 在小鼠中，它们已被证明成功地产生了HC样细胞。然而，这些细胞 不能完全成熟，寿命短，这表明并非所有HC再生的主要障碍都已消除。 克服HC再生方法基于对生理HC发育的研究。 这些结果表明，转录因子“无调同源物1”（ATOH 1）是HC的基础 但它对基因表达的影响是依赖于环境的。这些效应在HC中与 其他ATOH 1表达细胞。因此，ATOH 1的功能是由其他转录因子微调。利用 ATOH 1再生HC的全部潜力将需要鉴定新的修饰剂， HC中的ATOH 1活性。我们的初步数据表明，ATOH 1活性和HC成熟是受 转录因子“胸腺细胞选择相关HMG盒蛋白”（TOX）。Tox基因的敲除 小鼠（Tox）引起HC丧失和耳聋，并对这些小鼠中的Corti器官进行RNA-seq分析 揭示了多种基因以异常水平表达。“异常低表达”组 包括ATOH 1靶基因、“RE 1沉默转录因子”（REST）靶基因，以及转录抑制因子。 阻遏物编码基因蓖麻锌指1（Casz 1）。Corti器中Casz 1的定点突变 培养物显示，CASZ 1是抑制几个基因所必需的，这些基因在细胞中异常表达。 高浓度的毒素我们对条件性Casz 1基因敲除小鼠的初步表征 HC特异性Casz 1缺失导致外HC（OHC）变性和听力损失。此外，本发明还提供了一种方法， 我们先前对REST功能的分析表明， HC成熟。拟议研究的目的是确定TOX在 HC。我们的中心假设是TOX通过调节ATOH 1、REST和 CASZ 1在开发碳氢化合物方面的活动。我们建议通过两个具体目标来检验这一假设：目标1） 确定TOX在耳蜗发育的各个阶段对HC成熟的影响，以及 支持ATOH 1活性、REST调节和人工诱导的HC样细胞产生;目的 2)测定CASZ 1对HC形态和耳蜗基因表达的影响，并鉴定基因 阻遏物复合物介导CASZ 1依赖性阻遏某些间接靶基因， 碳氢化合物中的毒性。这些目标将使用从RNA-seq到体细胞的各种方法来实现 基因组编辑所提出的研究是有意义的，因为ATOH 1的新修饰剂的鉴定 活动对于改善HC再生方法的合理设计是必要的。

项目成果

Defects in the Alternative Splicing-Dependent Regulation of REST Cause Deafness.

• DOI: 10.1016/j.cell.2018.06.004
• 发表时间: 2018-07-26
• 期刊: Cell
• 影响因子: 64.5
• 作者: Nakano Y;Kelly MC;Rehman AU;Boger ET;Morell RJ;Kelley MW;Friedman TB;Bánfi B
• 通讯作者: Bánfi B