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

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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.