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theses

Spiral ganglion neurons (SGNs) of the cochlea are essential for relaying auditory signals to the brain. Loss of SGNs significantly contributes to hearing loss. Replacement of lost SGNS with otic progenitor cells is a potential strategy to alleviate hearing loss. Studies on regeneration have focused on gene expression networks that maintain self-renewal or promote differentiation, but changes in chromatin structure play an equally important role during regeneration. Understanding how the chromatin landscape regulates SGN differentiation in otic progenitors will accelerate efforts for cell replacement therapies. In the thesis, I reviewed the molecular and morphological changes during inner ear development, cells affected by hearing disorders and recent research progress in stem cell replacement therapies for hearing loss. To study inner ear regeneration, I employed an immortalized multipotent otic progenitor (iMOP) cell line, an otic fate-restricted cell type to study the chromatin changes that occur during neuronal differentiation. Under different culture conditions, iMOP cells self-renew and maintain an otic progenitor cell fate or differentiate into neurons that express neuronal markers and display SGN morphology. To study how chromatin changes affect transcription during iMOP neuronal differentiation, I studied the transcriptional activity of a pro-neural transcription factor, Neurogenin1 ( Neurog1). In iMOP cells, Neurog1 was enriched at the promoter of both cyclin-dependent kinase 2 (Cdk2) and neuronal differentiation (NeuroD1) genes. Changes in deposition of H3K9ac and H3K9me3 at the Cdk2 and NeuroD1 promoters suggested epigenetic alterations as iMOP cells transitioned between proliferation and differentiation. Chromatin changes at these promoters affected Neurog1 dependent transcriptional activity. In self-renewing iMOP cells, overexpression of Neurog1 increased Cdk2 to drive proliferation while knockdown of Neurog1 decreased Cdk2 to reduce proliferation. In differentiating cells, overexpression in Neurog1 in iMOP-derived neurons accelerated the acquisition of neuronal morphology and expression of Neurod1, while knockdown of Neurog1 prevented differentiation. My findings suggest that Neurog1 promotes either proliferation or neuronal differentiation, depending on histone modification at the promoter region of target genes. To further understand the effects of chromatin remodeling on otic neurogenesis, I studied the function of Chd7, a nucleosome repositioning protein that is upstream of Neurog1. Chd7 is associated with CHARGE syndrome, a disease that displays a constellation of symptoms including hearing loss. Chd7 mutant mice displayed defects in inner ear development and SGN formation. In iMOP cells, Chd7 expression increased as cells undergo neuronal differentiation. Knockdown of Chd7 prevented neuronal differentiation in iMOP cells. By performing Chd7 ChIP-seq in proliferating iMOP cells, Chd7 target genes were identified. Correlating the transcript levels of Chd7 target genes using RNA-seq data suggested that these genes are highly transcribed and may play a role in otic neuronal differentiation.

ETD_8726

Ph.D.

Includes bibliographical references

by Zhichao Song

doi:10.7282/T3GT5RN7

ETD doctoral

The author owns the copyright to this work.