Transcriptional networks establishing the precise gene expression states that define neurosensory cell identity in the inner ear

The goal of this project is to characterize how a network of transcription factors, chromatin remodeling factors and gene interactions establishes and maintains the precise gene expression states that drive otic epithelial cell development toward sensory or neuronal cells for hearing. The precursor cells for sensory hair cells (HCs) and neurons are specified in the otocyst, which develops from the otic placode, an ectoderm thickening that differentiates to form all inner ear structures. However, we know little about the regulatory circuits involved in initial specification of pro-neurosensory progenitors and their subsequent neuronal versus sensory cell commitment, thus presenting a major challenge for regenerating these cells to restore hearing loss. We have shown that the Eya1 forms a key transcriptional complex with Six1 and Sox2 to induce HC fate by activating Atoh1 in cochlear explant. Through mass spectrometry analyses, we identified components of the SWI/SNF chromatin remodeling complex as Eya1’s interacting proteins. We demonstrated that forced expression of Eya1-Six1 with Brg1-BAFs in cochlear explant induces nonsensory epithelial cells to differentiate into Neurog1+Neurod1+Tuj1+ neurons. Our recent data uncover an essential role of Eya1-Six1 in regulating the functional specificity of Brg1-BAFs to initiate pro-neurosensory fate in otic ectoderm by activating Sox2 expression through co-binding to multiple distal 3' Sox2 enhancers. The Brg1-BAFs are known to play a vital role in promoting depletion of nucleosomes to allow binding of TFs to enhancers. As chromatin regulation is critical for defining cellular identity, this application proposes to systematically characterize chromatin regulation by Brg1-BAF complex and its interaction with TFs Eya1, Six1 and Sox2 in establishing the precise gene expression states that define neurosensory cell identity by testing the central hypotheses: 1) depletion of Brg1 in Eya1+ otic progenitors at different stages reduces the accessibility of enhancers with regulatory roles in neurosensory cell lineage commitment and 2) many of these enhancer sites are co-regulated by Brg1- BAFs/Eya1-Six1 or Sox2. This study provides a rich resource of genomic sites with regulatory potential and also a ‘temporal’ clue for their activities critical to establishing neurosensory cell identity for hearing.