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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.

概括成年哺乳动物中存在一种未知机制阻碍毛细胞再生的现象，但我们发现最近表明，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 信号传导刺激祖细胞生成毛细胞在新生儿耳蜗中，我们假设 Wnt 通路的下游靶点变得更少随耳蜗年龄的增长而容易接近。我们的初步数据表明，组蛋白脱乙酰酶抑制剂，保留组蛋白上的乙酰基，增加新生儿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。我们寻求学习两者染色质变化的本质以及使用表观遗传药物和 CRISPR dCas9 逆转这些变化的潜力介导的表观遗传修饰。尽管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，再生受到限制。在这里，我们评估了对表观遗传修饰剂的抑制的反应新生儿和成人耳蜗，重点关注转录因子 Atoh1 及其表达的控制下游目标。我们评估了 3 个表观遗传修饰因子的影响，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， Tcf4、Setd7 和 Lsd1，关于耳蜗类器官的表观遗传标记和染色质可及性，我们询问操纵其表达水平是否可以逆转染色质不可接近性并增加毛细胞的分化。在目标 2 中，我们评估 Lsd1 和 HDAC 抑制介导的染色质修饰对 Atoh1 激活和毛细胞分化的影响。在目标 3 中，我们检验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的分化受损的耳蜗。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，我们提出了一系列关键的问题毛细胞再生和功能恢复的表观遗传机制。