Long-range Atoh1 enhancers maintain competency for hair cell regeneration in the inner ear
During tissue regeneration, lineage-related cells can switch their fate to replace missing cells. This cell plasticity is particularly prominent in more regenerative vertebrates such as zebrafish, yet the molecular basis by which cells transdifferentiate into another cell type upon injury remains unclear. Here we investigate the epigenetic basis of regenerative transdifferentiation in the inner ear, where supporting cells (SCs) generate mechanosensory hair cells (HCs) upon damage. By comparing the chromatin landscapes in regenerative zebrafish and green anole lizards versus non-regenerative mice, we identified a class of enhancers that function in progenitors to generate HCs and then are selectively maintained in SCs of regenerative vertebrates to regenerate HCs. In particular, we uncovered a syntenic class of long-range enhancers for Atoh1, a master transcription factor for HC differentiation. In the absence of injury, these enhancers maintain accessibility in SCs through adulthood but are prevented from driving zebrafish atoh1a expression through Notch repression. Deletion of these enhancers not only impaired atoh1a expression and HC formation during development but also blocked the ability of SCs to transdifferentiate into HCs during regeneration. Moreover, defects were specific to the inner ear versus the lateral line, revealing distinct mechanisms of regeneration in these mechanosensory organs. These findings reveal a class of regenerative enhancer that maintains competency of inner ear SCs to upregulate atoh1a and transdifferentiate into HCs upon damage. We propose that the continued accessibility of developmental enhancers for one cell fate in lineage-related cells may be a common theme underlying adult cell plasticity in regenerative vertebrates. Overall design: Inner ears were dissected from the Sox10Cre;UbiSwitch zebrafish at 14dpf (n=40) and 12mpf (n=6) zebrafish. Converted cells were isolated by Fluorescence-activated cell sorting (FACS) according to the presence or absence of mCherry signal and analyzed using 10x genomics based snATACseq or multimome ATAC+Gene Expression kits. scRNA/snRNA associated with these two timepoints were previous deposted at GSE211728. Reads were aligned to danRer11 using the Cellranger pipeline. Inner ear saccules were dissected from adult green anole lizards heads (n=7, heads were 2.5-3cm) and FACS sorted for live cells by DAPI exlucsion. 10x Genomics multiome ATAC+gene expression library was constructed and reads were aligned to AnoCar2.0v2 using the cellranger pipline