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.

概括 听力损失会影响500名新生儿中的1个，在65岁以上的3个人中有〜1个。这种疾病通常是 由机械学毛细胞（HCS）的丧失引起，这种缺陷在成熟中是永久的 哺乳动物耳蜗。因此，已经开发了人工方法来通过“转换”来再生HCS 非HCS进入HCS；在小鼠中，它们已被证明成功地产生了HC样细胞。但是，这些细胞 无法充分成熟，其寿命很短，表明并非所有主要HC再生的主要障碍 克服。 HC再生方法基于对物理HC发育的研究。 这些表明转录因子“ Atonal同源1”（ATOH1）对于HC是基础 发展，但其对基因表达的影响取决于上下文。这些效果在HCS与 其他表达ATOH1的细胞。那就是ATOH1功能由其他转录因子微调。利用 ATOH1对HC的再生的全部潜力将需要确定新颖的修饰符 HCS中的ATOH1活性。我们的初步数据表明ATOH1活性和HC成熟受 转录因子“胸腺细胞选择相关的HMG盒蛋白”（TOX）。托克斯基因的敲除 小鼠（toxΔ/∆）引起了HC丧失和耳聋，以及这些小鼠中皮尔蒂器官的RNA-seq分析 表明各种基因在异常水平上表达。 “绝对低表达”组 包括ATOH1靶基因，“ RE1沉默转录因子”（REST）靶基因和转录 阻遏物编码基因蓖麻锌指1（CASZ1）。 CASZ1在Corti器官中的靶向诱变 培养表明，Casz1是需要在异常表达的几种基因表达的表达 高水平的Corti的毒/∆器官。我们有条件的CASZ1敲除小鼠的初步表征 发现CASZ1的HC特异性缺失会导致外部HC（OHC）变性和听力损失。此外， 我们先前对REST功能的分析表明，需要对REST活动的围产期下调 HC成熟。拟议的研究的目的是定义毒物在成熟中的作用 HCS。我们的中心假设是，托克斯通过调节ATOH1，REST和 CASZ1开发HCS的活动。我们建议通过两个特定目的检验这一假设：目标1） 确定毒物对人工耳蜗各个阶段中HC成熟的影响，以及 它支持ATOH1活性，REST调节和人为地诱导HC样细胞的产生；目的 2）确定casz1对HC形态和人工耳蜗表达的影响，并鉴定基因 介导某些间接靶基因的CASZ1依赖表示的阻遏物复合物 HCS中的毒物。这些目标将使用从RNA-seq到体细胞的多种方法来实现 基因组编辑。拟议的研究很重要，因为鉴定了ATOH1的新修饰符 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