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

这个项目的目标是描述一个转录因子网络，染色质重塑因子 和基因相互作用建立并维持精确的基因表达状态， 细胞向感觉细胞或神经元细胞的发展以获得听觉。感觉毛细胞（HCs） 神经元在耳囊中被指定，耳囊从耳基板发育而来，耳基板是一种外胚层增厚， 分化形成所有内耳结构。然而，我们对参与的调节电路知之甚少， 前感觉神经祖细胞及其随后神经元与感觉细胞的初始特化 因此，再生这些细胞以恢复听力损失是一个重大挑战。我们有 显示Eya 1与Six 1和Sox 2形成关键的转录复合物，通过激活 耳蜗外植体中的Atoh 1。通过质谱分析，我们鉴定了SWI/SNF的组分 染色质重塑复合物作为Eya 1的相互作用蛋白。我们证明了强迫表达 Eya 1-Six 1与Brg 1-BAFs在耳蜗外植块中诱导非感觉上皮细胞分化为 Neurog 1 + Neurod 1 + Tuj 1+神经元。我们最近的数据揭示了Eya 1-Six 1在调节 Brg 1-BAF通过激活Sox 2启动耳外胚层中前神经感觉命运的功能特异性 通过与多个远端3'Sox 2增强子共结合来表达。已知Brg 1-BAF在生物体中起着至关重要的作用， 在促进核小体消耗以允许TF与增强子结合中的作用。由于染色质调节是 对于定义细胞身份至关重要，本申请提出系统地表征染色质 Brg 1-BAF复合物的调控及其与转录因子Eya 1、Six 1和Sox 2的相互作用， 基因表达状态，通过测试中心假设来定义神经感觉细胞身份：1） 不同阶段Eya 1+耳祖细胞中的Brg 1降低了具有调节作用的增强子的可及性， 神经感觉细胞谱系定型和2）这些增强子位点中的许多受Brg 1- BAF/Eya 1-Six 1或Sox 2。这项研究提供了丰富的具有调控潜力的基因组位点资源， 也是它们活动的“时间”线索，这些活动对建立听觉神经感觉细胞身份至关重要。

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.