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

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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 Our unpublished data revealed dynamic changes in transcription and chromatin accessibility during HC regeneration.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.