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

项目摘要/摘要 听力损失的主要原因是内耳耳蜗毛细胞受损。一个潜在的战略是 再生毛细胞和恢复听力是诱导周围支持细胞转分化为 新的功能毛细胞。这是基于对斑马鱼等非哺乳类脊椎动物的观察 可以从这些邻近的支持细胞中补充一生中的感觉毛细胞。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 在临床上实施这一策略，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学机制，它通过永久限制小鼠的支持细胞可塑性 染色质封闭在驱动毛细胞基因表达的增强子周围，包括关键转录因子 例如Atoh1和POU4F3。Atoh1是一种主控调节因子，可以驱动毛细胞的分化。 脊椎动物，无法上调出生后小鼠支持细胞中Atoh1的表达是关键 损伤后毛细胞再生的障碍。 在这项建议中，我研究了高度再生的斑马鱼如何通过 研究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Toh1a的几个潜在调控元件仍然可以在支持细胞中获得。 这与我们在小鼠Atoh1基因座观察到的情况形成了鲜明对比。有趣的是，染色质的这种维持 由于pou4f3和其他毛细胞基因不保留染色质，所以可及性是toh1a基因特有的。 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，这可能是斑马鱼保持可获得性的原因 支持细胞。我假设斑马鱼toh1a基因座的固有属性维持其 顺式调节元件，在毛细胞再生过程中促进其上调。 在目标1中，我测试了斑马鱼和小鼠Toh1a/Atoh1基因座之间的序列差异是否可以解释 鱼特有的保持染色质开放的能力。在目标2中，我测试了一类仍然存在的toh1a增强剂 可在支持细胞中访问，以满足其对持续毛细胞生成的需求。我还测试了一个 第二类支持细胞特异性toh1a元件在防止异位毛细胞形成中的支持作用 细胞在没有损伤的情况下。通过了解斑马鱼如何在成年时保持ath1a基因的稳定状态 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。

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.