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

项目摘要 损伤诱导细胞死亡后，人类不能更新内耳中的机械感受器(毛细胞，HCS) 到听力损失。与哺乳动物不同，斑马鱼可以再生HCS。我们的目标是揭示关键的基因开关， 最终引导我们找到可能在人类身上触发HC再生计划的方法。以避免发展 ，我们评估斑马鱼成体内耳，以真正了解基因组元素在动态平衡和 再生。HC再生是支持细胞(SCs)和HC祖细胞(Hcp)的平衡行为，这些细胞振荡 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续签 更好地理解为基因网络在动态平衡、损伤和 再生，它可以与基因组的空间组织联系在一起。超分辨率显微镜和 基于测序的基因组技术，如Hi-C，揭示了基因组是按层次组织的 真核细胞的核。这种3D组织由多层染色质架构特征表示，例如 染色体区域、A/B区、拓扑相关结构域(TADS)和长距离染色质 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 未被开发的。稳定的TADS被认为是通过CTCF介导和调节的，CTCF是一种DNA结合转录和 边界因素和粘附素复合体。有趣的是，这些在发育和分化中发挥了作用。 我们未发表的数据揭示了在HC再生过程中转录和染色质可及性的动态变化。 动态平衡过程中斑马鱼内耳的大量ATAC-SEQ结果显示CTCF是最重要的基序。 此外，scatac-seq数据显示，由于碳氢化合物的结果，四氯化碳富含在出现的碳氢化合物峰中。 再生。根据我们的发现，我假设在HC过程中TADS和染色质环的从头形成 再生将显示一个3D“动态平衡与再生”的轮廓，任何重排都将修改hcp 再生可塑性。拟议研究的目标是采用多维基因组学方法来 构建成人体内HC发育和再生调控程序的全面图景 耳朵。这一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 结构处于动态平衡和HC再生期间。我们将使用scatac-seq、scRNA-seq和schi-C检测。 为了比较野生型和甘油三酯(myo6b：hDTR)在成人内耳中的三维基因组，a 体内唯一具有条件消融能力的转基因斑马鱼。在目标2中，我们将在体内测试 利用CRISPR/CAS9和超分辨率显微镜，TADS和调控元件之间的功能。 AIMS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 并在功能上描绘了潜在的HC再生计划的调节区。这项研究是 由于结果将确定HC基因组结构变化的基础和后果，因此具有重要意义 再生可塑性作为了解伤口愈合和组织再生的更广泛领域的指标。

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.