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

项目总结/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， 再生毛细胞和恢复听力是诱导周围的支持细胞转分化为 新的功能性毛细胞这是基于观察到非哺乳类脊椎动物如斑马鱼 可以在整个生命过程中从这些邻近的支持细胞中补充感觉毛细胞。然而，事实上， 成熟的哺乳动物支持细胞失去转分化成毛细胞的能力， 在诊所实施这一战略，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，通过永久性的细胞毒性来限制小鼠支持细胞的可塑性。 封闭毛细胞基因（包括关键转录因子）表达增强子周围的染色质 例如ATOH 1和POU4F3。ATOH1是一个主要的调节器，驱动毛细胞的分化， 脊椎动物，不能上调Atoh1在出生后小鼠支持细胞中的表达是一个关键 损伤后毛细胞再生障碍。 在这个提议中，我调查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： 研究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 数据显示，斑马鱼atoh 1a的几个潜在调节元件在支持细胞中仍然可以接近。 这与我们在小鼠Atoh1位点观察到的相反。有趣的是，这种染色质的维持 可接近性是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，可能解释了斑马鱼这种可接近性的维持 支持细胞。我假设，斑马鱼atoh1a基因座的内在特性保持了其 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，我测试了斑马鱼和小鼠atoh1a/Atoh1基因座之间的序列差异是否解释了 鱼类特有的保持开放染色质的能力。在目标2中，我测试了一类atoh1a增强剂， 可在支持细胞中获得，因为它们需要持续的毛细胞生成。我还测试了一个 防止异位毛细胞形成的第二类支持细胞特异性ATOH 1A元件 细胞在没有损伤的情况下。通过研究成年斑马鱼如何保持atoh1a位点处于平衡状态， 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。

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.