Differential Chromatin Accessibility, Gene Expression, and mRNA Splicing Between Developing Cochlear Inner and Outer Hair Cells

Abstract

Purpose: The mammalian cochlea has two types of low abundance and highly specialized inner (IHC) and outer (OHC) mechanosensory hair cells. Their malfunction or death is a common cause of congenital and acquired deafness. IHCs and OHCs exhibit different transcriptomes during development. We wondered how differences in gene expression are regulated at the chromatin level in developing IHCs and OHCs, and whether there were also differences in mRNA splicing between IHCs and OHCs. Methods: We separately collected developing mouse IHCs and OHCs to identify their mRNAs and chromatin states. We examined their transcriptomes by bulk (full coverage) RNA-seq from six biological replicates each to reveal differences in gene expression and in alternative mRNA splicing. We also examined their chromatin conformation by bulk ATAC-seq from two biological replicates each to reveal open vs. closed promoter and enhancer elements. We then compared ATAC-seq with RNA-seq datasets to determine if differential chromatin accessibility can account for differential gene expression. Each biological replicate consists of hair cells pooled from multiple neonatal mice of both sexes. Results: We found that developing IHCs and OHCs have differentially accessible promoters in many differentially expressed genes. This includes functional genes whose expression is incipient in neonatal hair cells but will be maintained throughout life, and developmental genes which are only expressed transiently. We also found that different mRNA isoforms result from alternative mRNA splicing and transcription start sites. Finally, our data reveals that cochlear hair cells utilize unique promoters and mRNA isoforms absent in other cell types. Conclusion: Differential transcriptomes between developing hair cell types result from pre- and post-transcriptional mechanisms. The unique promoters and mRNA isoforms in cochlear HCs highlight the importance of elucidating transcriptomes and epigenomes of rare cell types. We provide a comprehensive resource for the identification of promoters and mRNA isoforms of genes expressed by neonatal IHCs or OHCs, which is publicly-accessible for visualization of any gene of interest at https://igvviewer.s3.us-east-2.amazonaws.com/index.html.