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Epigenetic analysis of perinatal maturation of supporting cells in the organ of Corti

ABSTRACT: Sensory hair cells cannot be regenerated through transdifferentiation of neighboring supporting cells in the organ of Corti in mature mammalian animals, but limited regeneration capacity exists in supporting cells at neonatal stage in mouse and this transdifferentiation potential is rapidly lost during the first week of postnatal maturtion. We hypothesized that epigenetic decommissioning of hair cell gene enhancers in supporting cells during postnatal maturation leads the permanent silencing of hair cell genes and the loss of transdifferentiation potential. To test this hypothesis, we FACS purified hair cells and supporting cells from cochleae at different developmental stages for transcritomic analysis (RNAseq), chromatin accessibility assay (ATACseq) and histone modification profiling (ChIPseq or CUT&RUN). We first defined hair cell genes and predicted their potential active enhancers. We found that hair cell gene promoters and enhancers were kept in a primed-but-silenced status (H3K4me1/3+, low H3K27ac but high H3K27me3) in supporting cells at neonatal stage. During postnatal maturation, hair cell gene enhancers are decommissioned through H3K4me1 removal, leading to the permanent silencing of hair cells genes. We also found that hair cell gene enhancer decommissioning process correlated with the base-to-apex wave of transdifferentiation potential loss. In addition, hair cell gene enhancer commissioning status is preserved in mature utricular supporting cells, which can regenerate hair cells through transdifferentiation even at adult stage. Those data together suggest that decommissioning of hair cell gene enhancers in supporting cells during postnatal maturation is the epigenetic mechanism underlying the loss of regeneration capacity in the organ of Corti.

ORGANISM(S): Mus musculus

PROVIDER: GSE150000 | GEO | 2021/07/09

REPOSITORIES: GEO

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Project description:Purpose - To profile epigenetic changes in cochlear progenitor cells, hair cells, and supporting cells across postnatal development. Methods- Cochlear cell types were obtained from fluorescently labeled transgenic mice. E13.5 cochlear prosensory progenitor cells (E13.5_PG, p27Kip1-GFP+), P1 hair cells (P1_HC, Atoh1-GFP+), P1 supporting cells (P1_SC, Lfng-GFP+), and P6, P8, P21 supporting cells (P6_SC, P8_SC, P21_SC, Lfng-creER, NuTRAP lineage-traced) were FACS purified for epigenetic profiling using WGBS (DNA methylation), CUT&Tag (H3K4me1, H3K4me3, H3K27me3, CUTAC H3K4me2). DNA demethylation of hair cell-specific promoters was interrogated by comparing % mCpG profiles of transdifferentiating supporting cells (P1_SC_tdt_gfp, Lfng-creER/TdTomato+, Atoh1-GFP+) against non-transdifferentiating supporting cells (P1_SC_tdt, P6_SC_tdt, Lfng-creER/TdTomato+) purified via FACS. P1 and P8 wildtype cochleas were enzymatically dissociated and used directly for scMultiome simultaneous profiling of RNA and ATAC at the single cell level (P1_scMultiome, P8_scMultiome, 10x Genomics). For scMultiome of P70 wildtype (P70_wt_scMultiome, Lfng-CreERT2, NuTRAP) and P70 deafened (P70_deaf_scMultiome, Lfng-CreERT2, NuTRAP, Pou4f3DTR) supporting cells, mice received 100 mg/kg tamoxifen (Sigma-Aldrich T5648) at P21, 0.01 mg/kg Diptheria toxin (Sigma-Aldrich D0564) at P28, and allowed to mature to P70, at which point cochlear tissues were harvested, FACS purified for NuTRAP+ supporting cells, and inputted into a scMultiome reaction. Results- WGBS highlighted differentially methylated regions (DMR) across E13.5_PG, P1_HC, P1_SC, P6_SC, and P21_SC. Pre-established DMRs become hypermethylated in supporting cells between P1 and P21. This corresponds with increasing heterochromatin characteristics, such as loss of accessibility (CUTAC), loss of H3K4me1, and switch between H3K27me3-mediated repression to DNA methylation-mediated silencing. WGBS of P1_SC_tdt_gfp compared to P1_SC_tdt and P6_SC_tdt showed DNA demethylation of hair cell-specific DMRs, suggesting that DNA demethylation is required for transdifferentiation of supporting cells into hair cells. P1_scMultiome, P8_scMultiome, P70_wt_scMultiome, and P70_deaf_scMultiome revealed changes in chromatin accessibility associated with age, as well as from long-term deafening at the single cell level. scMultiome datasets also highlight cell type-specific enhancers active during different stages of postnatal development.

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Epigenetic analysis of perinatal maturation of supporting cells in the organ of Corti

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