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: 10535065
• 负责人: Tuo Shi
• 金额: $ 4.68万
• 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/10535065
• 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; Property; Regenerative capacity; Regulation; Regulatory Element; Repression; Research; Rest; Role; Sensory Hair; Supporting Cell; Testing; Up-Regulation; Vertebrates; Work; XCL1 gene; Zebrafish; base; 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; transcription factor; transdifferentiation; zebrafish genome; 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; Property; Regenerative capacity; Regulation; Regulatory Element; Repression; Research; Rest; Role; Sensory Hair; Supporting Cell; Testing; Up-Regulation; Vertebrates; Work; XCL1 gene; Zebrafish; base; 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; 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.

项目摘要/摘要 听力损失的主要原因是对内耳人耳蜗细胞的损害。一个潜在的策略 再生毛细胞和恢复听力是为了诱导周围支撑细胞的转变 新的功能性毛细胞。这是基于这样的观察到的，即非哺乳动物脊椎动物（例如斑马鱼） 可以通过这些相邻的支持细胞来补充一生中的感觉毛细胞。但是，事实是 成熟的哺乳动物辅助细胞失去转变成毛细胞的能力，对 在诊所实施此策略以恢复患者的听力。 我们的实验室最近确定了一种表观遗传机制，该机制限制了通过永久支撑细胞可塑性的小鼠 围绕增强子驱动毛细胞基因表达的染色质关闭，包括关键转录因子 例如ATOH1和POU4F3。 ATOH1是一个主调节器，可驱动毛细胞的分化 脊椎动物，无法上调产后小鼠的ATOH1表达，作为关键 受伤后毛细胞再生的障碍。 在此提案中，我调查了高度再生斑马鱼如何通过 检查斑马鱼毛细胞基因附近的染色质可及性。我的初步单核ATAC测序 数据表明，在支持细胞中，斑马鱼ATOH1A的几个潜在调节元件仍然可以访问。 这与我们在鼠标ATOH1基因座上观察到的相反。有趣的是，这种染色质的维护 可访问性特定于AtOH1a基因座，因为POU4F3和其他毛细胞基因不保留染色质 斑马鱼辅助细胞中的可及性。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，可以解释斑马鱼中可及性的这种维护 支持细胞。我假设斑马鱼Atoh1a基因座的内在特性保持其可访问性 顺式调节元素，促进其在毛细胞再生过程中的上调。 在AIM 1中，我测试斑马鱼和鼠标ATOH1A/ATOH1 Loci之间的序列差异是否为 鱼类特定的维持开放染色质的能力。在AIM 2中，我测试了一类ATOH1A增强器 可以在支持细胞中访问其持续毛细胞产生的要求。我还测试了一个 第二类支持细胞特异性ATOH1A元素，以防止异位毛细胞形成支持 在没有伤害的情况下细胞。通过学习斑马鱼如何在成人中保持现状的状态保持ATOH1A基因座 这些目标的支持细胞将指导将来的努力再生毛细胞并恢复听力。