Defining the dynamic 3D genome during hearing regeneration in the adult zebrafish inner ear

项目摘要 在损伤诱导的细胞死亡后，人类不能更新内耳中的机械感受器（毛细胞，HC）， 听力损失与哺乳动物不同，斑马鱼可以再生HC。我们的目标是揭示关键的基因开关， 最终引导我们找到可以触发人类HC再生程序的方法。为了避免发展 变异，我们评估斑马鱼成年内耳，以真正了解基因组元素在稳态， 再生HC再生是支持细胞（SC）和HC祖细胞（HCP）的平衡行为， 在自我更新、增殖和终末分化之间，需要替换丢失的HC。我相信HC的更新 将更好地理解为在稳态，损伤和 再生，并且它可以与基因组的空间组织相关联。超分辨率显微镜和 基于测序的基因组技术，如Hi-C，已经揭示了基因组是分层组织的， 真核细胞的细胞核。这种3D组织由多级染色质结构特征表示，例如 染色体区域、A/B区室、拓扑相关结构域（TADs）和长程染色质 循环启动再生和指导HC分化的HCP的潜在基因组结构仍然存在 未开发的稳定的TADs被认为是通过CTCF介导和调节的，CTCF是一种DNA结合转录， 边界因子沿着与内聚素复合物。有趣的是，这些在发育和分化中发挥作用。 我们未发表的数据显示，在HC再生过程中转录和染色质可及性的动态变化。 来自在稳态期间获得的关于斑马鱼内耳的批量ATAC-seq的结果检测到CTCF作为顶部基序。 此外，scATAC-seq数据显示，CTCF富含由于HC而出现的HCP峰 再生基于我们的研究结果，我假设在HC过程中TADs和染色质环的重新形成 再生将显示3D“稳态与再生”曲线，并且任何重排将改变HCP 再生可塑性该研究的目的是采用多维基因组学方法， 构建一个全面的图片的调控程序HC的发展和再生在成人内部 耳朵该假设将在两个具体目标中得到解决。在目标1中，我们将识别内耳3D核 在体内平衡和HC再生过程中的结构。我们将采用scATAC-seq、scRNA-seq和scHi-C测定法， 为了比较野生型和Tg（myo 6 b：hDTR），绘制成人内耳中HCP、SC和HC的3D基因组图谱， 具有条件性体内HC消融能力的独特转基因斑马鱼。在目标2中，我们将测试体内 使用CRISPR/Cas9和超分辨率显微镜来研究TADs和调控元件之间的功能性。 目标的完成将提供现代方法的培训，以发现基因组中新位置的TADs 并在功能上描绘了HC再生程序的潜在的假定调节区。这项研究是 意义重大，因为结果将确定HC基因组结构变化的基础和后果 再生可塑性作为理解伤口愈合和组织再生的更广泛领域的代理。

Project Summary Humans cannot renew the mechanoreceptors (hair cells, HCs) in the inner ear after damage-induced cell death leading to hearing loss. Unlike mammals, zebrafish can regenerate HCs. We aim to reveal key genetic switches that will eventually lead us to approaches that could trigger HC regeneration programs in humans. To avoid developmental variations, we assess the zebrafish adult inner ear to truly understand the genomic elements during homeostasis and regeneration. HC regeneration is a balancing act of supporting cells (SCs) and HC progenitors (HCPs), that oscillate between self-renewal, proliferation, and terminal differentiation required to replace lost HCs. I believe HC renewal would be better understood as a combinatorial regulation of gene networks during homeostasis, injury, and regeneration, and that it can be linked to the spatial organization of the genome. Super-resolution microscopy and sequencing-based genomic technologies, such as Hi-C, have revealed that the genome is hierarchically organized in the nucleus of eukaryotic cells. This 3D organization is denoted by multilevel chromatin architectural features such as chromosome territories, A/B compartments, topologically associated domains (TADs), and long-range chromatin loops. The underlying genome architecture of HCPs that initiate regeneration and instruct HC differentiation remains unexplored. Stable TADs are thought to be mediated and regulated through CTCF, a DNA binding transcription and boundary factor along with the cohesin complex. Interestingly, these play a role in development and differentiation. Our unpublished data revealed dynamic changes in transcription and chromatin accessibility during HC regeneration. Results from bulk ATAC-seq on zebrafish inner ears obtained during homeostasis detected CTCF as a top motif. Moreover, scATAC-seq data showed that CTCF is enriched in HCPs emerging peaks as consequence of HC regeneration. Based on our findings, I hypothesize that de novo formation of TADs and chromatin loops during HC regeneration will reveal a 3D “homeostasis vs. regeneration” profile and any rearrangement will modify the HCP regenerative plasticity. The objective of the proposed research is to take a multi-dimensional genomics approach to construct a comprehensive picture of the regulatory program of HC development and regeneration in the adult inner ear. The hypothesis will be addressed in two specific aims. In Aim 1, we will identify the inner ear 3D nuclear architecture in homeostasis and during HC regeneration. We will employ scATAC-seq, scRNA-seq, and scHi-C assays to map the 3D genome of HCPs, SCs, and HCs in the adult inner ear comparing wild-type and Tg(myo6b:hDTR), a unique transgenic zebrafish with the capacity for conditional HC-ablation in vivo. In Aim 2, we will test the in vivo functionality between TADs and regulatory elements using CRISPR/Cas9 and super-resolution microscopy. Completion of the aims will provide training in modern approaches to uncover TADs at novel positions in the genome and functionally depict putative regulatory regions underlying HC regeneration programs. The research is significant since the results will define the foundations and consequences of genomic structural changes of HC regenerative plasticity as a proxy to comprehend the broader field of wound healing and tissue regeneration.