Summary Blocks to regeneration of hair cells by an unknown mechanism exist in the adult mammal, but we have recently shown that cochlear hair cells have a capacity for spontaneous regeneration in the first few postnatal days. We have recently discovered that Wnt signaling stimulates generation of hair cells from progenitor cells in the newborn cochlea, and we hypothesize that downstream targets of the Wnt pathway become less accessible with age of the cochlea. Our preliminary data show that histone deacetylase inhibitors, which preserve acetyl groups on histones, increase the proliferation of newborn cochlear supporting cells and promote hair cell differentiation in the newborn and to a lesser extent the adult inner ear. We seek to learn both the nature of chromatin changes and the potential to reverse them with epigenetic drugs and CRISPR dCas9 mediated epigenetic modifications. Although our recent work has provided an important proof-of-principle for hair cell replacement in the adult, regeneration was limited. Here, we assess the response to inhibition of epigenetic modifiers of both the newborn and adult cochlea with a focus on the control of expression of transcription factor Atoh1 and its downstream targets. We assess the effects of 3 epigenetic modifiers that we propose to be key to enhancer- based activation of genes required for HC differentiation. In Aim 1, we assess the effects of these modifiers, Tcf4, Setd7 and Lsd1, on epigenetic marks and chromatin accessibility in cochlear organoids and we ask whether manipulation of their level of expression can reverse chromatin inaccessibility and increase differentiation of hair cells. In Aim 2 we assess chromatin modification mediated by Lsd1 and HDAC inhibition for effects on Atoh1 activation and differentiation of hair cells. In Aim 3, we test our hypothesis that manipulation of epigenetic changes through these modifiers of chromatin will increase hair cell differentiation in the damaged cochlea. We test the epigenetic modifiers and inhibitors for their effect on hair cell regeneration in a noise damage model of mouse deafness. Through these experiments, we ask a crucial series of questions on epigenetic mechanisms in hair cell regeneration and recovery of function.