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Determination of Hair Cell Fate from Postnatal Cochlear Supporting Cells

产后耳蜗支持细胞毛细胞命运的测定

基本信息

批准号：
10617788
负责人：
Albert Edge
金额：
$ 61.74万
依托单位：
MASSACHUSETTS EYE AND EAR INFIRMARY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-04 至 2027-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10617788
关键词：
ATAC-seq Acetylation Adult Affect Affinity Age Auditory Brainstem Responses Binding Binding Proteins Binding Sites Biological Assay Cell Differentiation process Cells Chromatin Chromatin Remodeling Factor Cochlea Complex Confocal Microscopy Cytomegalovirus DNA Binding DNA Sequence DNA-Binding Proteins DNA-Protein Interaction Data Detection Ear Enhancers Epigenetic Process Excision GFI1 gene Gene Activation Gene Expression Gene Silencing Generations Genes Genetic Transcription Hair Cells Hearing problem Histone Deacetylase Histone Deacetylase Inhibitor Histone-Lysine N-Methyltransferase Histones Inner Hair Cells KDM1A gene Laboratories Labyrinth Learning LoxP-flanked allele Mammals Measurement Measures Mediating Mediator Methylation Modeling Modification Motion Mus Natural regeneration Nature Newborn Infant Noise Organ of Corti Organoids Pathway interactions Pattern Pharmaceutical Preparations Play Proliferating Recovery of Function Regulator Genes Role Sensory Hair Series Signal Pathway Site Supporting Cell Testing Transcriptional Activation WNT Signaling Pathway Western Blotting Work age related chromatin modification deactivated CRISPR-Cas9 deafness demethylation drug testing epigenetic drug equilibration disorder experimental study genetic testing hair cell regeneration hearing impairment histone acetyltransferase histone methylation histone methyltransferase in vivo inhibitor mosaic mouse model noise exposure notch protein otoacoustic emission overexpression pharmacologic postnatal preservation prevent promoter protein protein interaction recruit response single-cell RNA sequencing sound stem cells transcription factor transcriptome sequencing transdifferentiation

项目摘要

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.

总结在成年哺乳动物中，毛细胞的再生受到一种未知机制的阻碍，最近的研究表明耳蜗毛细胞在出生后的最初几年内具有自发再生的能力，天我们最近发现Wnt信号刺激毛细胞从祖细胞生成在新生儿耳蜗中，我们假设Wnt通路的下游靶点变得更少，随着耳蜗年龄的增长，我们的初步数据显示，组蛋白去乙酰化酶抑制剂，保护组蛋白上的乙酰基，增加新生耳蜗支持细胞的增殖，促进新生儿毛细胞分化，并在较小程度上促进成人内耳毛细胞分化。我们寻求学习两者染色质变化的性质以及用表观遗传药物和CRISPR dCas 9逆转它们的潜力介导的表观遗传修饰。虽然我们最近的工作为成人毛细胞替代提供了重要的原理证明，再生是有限的。在这里，我们评估了对两种表观遗传修饰剂抑制的反应，新生儿和成人耳蜗，重点是转录因子Atoh 1的表达控制及其下游目标我们评估了3种表观遗传修饰剂的作用，我们认为它们是增强子的关键- 基于HC分化所需基因的激活。在目标1中，我们评估了这些修饰符的效果， Tcf 4，Setd 7和Lsd 1，关于耳蜗类器官的表观遗传标记和染色质可及性，我们问操纵它们的表达水平是否可以逆转染色质不可及性，毛细胞的分化。在目标2中，我们评估了Lsd 1和HDAC抑制介导的染色质修饰对Atoh 1激活和毛细胞分化的影响。在目标3中，我们检验了我们的假设，通过这些染色质修饰剂操纵表观遗传变化将增加毛细胞分化，受损的耳蜗我们测试了表观遗传修饰剂和抑制剂对毛细胞再生的影响，小鼠耳聋的噪声损伤模型。通过这些实验，我们提出了一系列至关重要的问题，毛细胞再生和功能恢复的表观遗传机制。