Species-Specific Epigenetic Basis of Zebrafish Inner Ear Hair Cell Regeneration

斑马鱼内耳毛细胞再生的物种特异性表观遗传基础

• 批准号: 10732761
• 负责人: Tuo Shi
• 金额: $ 4.77万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10732761
• 关键词: ATAC-seq; Ablation; Address; Adult; Affect; Age Months; American; Animal Model; Automobile Driving; Bacterial Artificial Chromosomes; Birth; CRISPR/Cas technology; Cell Nucleus; Chromatin; Clinic; Cochlea; Code; Conserved Sequence; DNA Sequence; Data; Elements; Enhancers; Enzymes; Epigenetic Process; Fishes; Future; Generations; Genes; Hair Cells; Histones; Homeostasis; Human; Injury; Labyrinth; Learning; Life; Maintenance; Mammals; Modeling; Mus; Natural regeneration; Organ; Patients; Production; Proliferating; Property; Regenerative capacity; Regulation; Regulatory Element; Repression; Research; Rest; Role; Sensory Hair; Supporting Cell; Testing; Up-Regulation; Vertebrates; Work; Zebrafish; combinatorial; design; experimental study; hair cell regeneration; hearing impairment; hearing restoration; novel; novel therapeutic intervention; postnatal; prevent; public health relevance; regeneration following injury; regeneration potential; regenerative; regenerative approach; response to injury; restraint; transcription factor; transdifferentiation; zebrafish genome

PROJECT SUMMARY/ABSTRACT The major cause of hearing loss is damage to the inner ear cochlear hair cells. One potential strategy to regenerate hair cells and restore hearing is to induce the transdifferentiation of surrounding supporting cells into new functional hair cells. This is based on the observation that non-mammalian vertebrates such as zebrafish can replenish sensory hair cells throughout life from these neighboring supporting cells. However, the fact that mature mammalian supporting cells lose the ability to transdifferentiate into hair cells poses a great challenge to implement this strategy in the clinics to restore hearing in patients. Our lab recently identified an epigenetic mechanism that restricts mouse supporting cell plasticity by permanent closing of chromatin around enhancers driving expression of hair cell genes, including critical transcription factors such as ATOH1 and POU4F3. ATOH1 is a master regulator that drives the differentiation of hair cells across vertebrates, with the inability to upregulate Atoh1 expression in postnatal mouse supporting cells being a key barrier to hair cell regeneration following injury. In this proposal, I investigate how the highly regenerative zebrafish may escape such epigenetic repression by examining chromatin accessibility near zebrafish hair cell genes. My preliminary single-nuclei ATAC sequencing data reveal that several potential regulatory elements of zebrafish atoh1a remain accessible in supporting cells. This is in contrast with what we observe at the mouse Atoh1 locus. Interestingly, this maintenance of chromatin accessibility is specific to the atoh1a locus, as pou4f3 and other hair cell genes do not retain chromatin accessibility in zebrafish supporting cells. This suggests that sequence-specific features of the atoh1a locus, rather than general chromatin modifying enzymes, may account for this maintenance of accessibility in zebrafish supporting cells. I hypothesize that intrinsic properties of the zebrafish atoh1a locus maintain accessibility of its cis-regulatory elements, which facilitate its upregulation during hair cell regeneration. In Aim 1, I test whether sequence differences between zebrafish and mouse atoh1a/Atoh1 loci account for the fish-specific ability to maintain open chromatin. In Aim 2, I test a class of atoh1a enhancers that remain accessible in supporting cells for their requirement for continued hair cell generation. I also test the role of a second class of supporting cell-specific atoh1a elements in preventing ectopic hair cell formation from supporting cells in the absence of injury. By learning how zebrafish maintain atoh1a locus in a poised state in adult supporting cells, results from these aims will guide future endeavors to regenerate hair cells and restore hearing.

项目概要/摘要 听力损失的主要原因是内耳耳蜗毛细胞受损。一项潜在的策略 毛细胞再生、听力恢复是诱导周围支持细胞转分化为 新的功能性毛细胞。这是基于以下观察：斑马鱼等非哺乳动物脊椎动物 可以在整个生命过程中从这些邻近的支持细胞补充感觉毛细胞。然而，事实是 成熟的哺乳动物支持细胞失去转分化为毛细胞的能力，对 在诊所实施这一策略以恢复患者的听力。 我们的实验室最近发现了一种表观遗传机制，该机制通过永久限制小鼠支持细胞的可塑性 关闭驱动毛细胞基因（包括关键转录因子）表达的增强子周围的染色质 例如 ATOH1 和 POU4F3。 ATOH1 是驱动毛细胞分化的主要调节因子 脊椎动物，出生后小鼠支持细胞中 Atoh1 表达无法上调是关键 损伤后毛细胞再生的障碍。 在这个提案中，我研究了高度再生的斑马鱼如何通过以下方式逃避这种表观遗传抑制： 检查斑马鱼毛细胞基因附近染色质的可及性。我的初步单核 ATAC 测序 数据显示，斑马鱼 atoh1a 的几个潜在调控元件在支持细胞中仍然可以访问。 这与我们在小鼠 Atoh1 基因座上观察到的结果形成对比。有趣的是，染色质的这种维持 可及性是 atoh1a 基因座特有的，因为 pou4f3 和其他毛细胞基因不保留染色质 斑马鱼支持细胞的可及性。这表明 atoh1a 基因座的序列特异性特征， 而不是一般的染色质修饰酶，可能是斑马鱼可及性维持的原因 支持细胞。我假设斑马鱼 atoh1a 基因座的内在特性维持了其可接近性 顺式调节元件，促进其在毛细胞再生过程中的上调。 在目标 1 中，我测试斑马鱼和小鼠 atoh1a/Atoh1 位点之间的序列差异是否解释了 鱼类特有的保持染色质开放的能力。在目标 2 中，我测试了一类 atoh1a 增强子，它们仍然存在 可用于支持细胞以满足其持续毛细胞生成的需要。我也测试了一个角色 第二类支持细胞特异性 atoh1a 元件，可防止异位毛细胞形成 细胞在没有受到损伤的情况下。通过了解成年斑马鱼如何保持 atoh1a 基因座处于平衡状态 支持细胞，这些目标的结果将指导未来再生毛细胞和恢复听力的努力。