Chromatin remodelers and lineage specific factors interact to target enhancers to establish pro-neurosensory fate within otic ectoderm

Specification of Sox2+ pro-neurosensory progenitors within otic ectoderm is a prerequisite for the production of sensory cells and neurons for hearing. However, the underlying molecular mechanisms driving this lineage specification remain unknown. Here, we show that Brg1-based SWI/SNF chromatin-remodeling complex interacts with the neurosensory-specific transcriptional regulators Eya1/Six1 to induce Sox2 expression and promote pro-neurosensory-lineage specification. Ablation of the ATPase-subunit Brg1 or both Eya1/Six1 results in loss of Sox2 expression and lack of neurosensory identity, leading to abnormal apoptosis within the otic ectoderm. Brg1 binds to two of three distal 3' Sox2 enhancers occupied by Six1, and Brg1-binding to these regions depends on Eya1-Six1 activity. We demonstrate that the activity of these Sox2 enhancers in otic neurosensory cells specifically depends on binding to Six1. Furthermore, genome-wide and transcriptome profiling indicate that Brg1 may suppress apoptotic factor Map3k5 to inhibit apoptosis. Together, our findings reveal an essential role for Brg1, its downstream pathways, and their interactions with Six1/Eya1, in promoting pro-neurosensory fate induction in the otic ectoderm and subsequent neuronal lineage commitment and survival of otic cells. Overall design: Genome-wide Brg1/Six1/Histone modification binding feature was characterized in whole embryo and cochlea using ChIP-seq.

耳外胚层（otic ectoderm）内Sox2阳性（Sox2+）前神经感觉祖细胞（pro-neurosensory progenitors）的特化，是听觉相关感觉细胞与神经元生成的必要前提。然而，驱动该谱系特化的潜在分子机制仍未阐明。本研究发现，基于Brg1的SWI/SNF染色质重塑复合物（SWI/SNF chromatin-remodeling complex）可与神经感觉特异性转录调控因子Eya1/Six1相互作用，诱导Sox2基因表达并促进前神经感觉谱系的特化。敲除ATP酶亚基（ATPase-subunit）Brg1，或同时敲除Eya1与Six1，会导致Sox2表达缺失及神经感觉特性丧失，进而引发耳外胚层内的异常细胞凋亡（apoptosis）。Brg1可结合被Six1占据的3个Sox2基因3'端远端增强子（distal 3' Sox2 enhancers）中的2个，且Brg1对这些区域的结合依赖于Eya1-Six1复合物的活性。本研究证实，上述Sox2增强子在耳神经感觉细胞中的活性，特异性依赖于其与Six1的结合。此外，全基因组（genome-wide）与转录组分析（transcriptome profiling）结果显示，Brg1可通过抑制凋亡因子Map3k5的表达以阻断细胞凋亡。综上，本研究结果揭示了Brg1及其下游通路，以及它们与Six1/Eya1的相互作用，在促进耳外胚层前神经感觉命运特化、后续神经元谱系定型以及耳细胞存活过程中发挥的关键作用。实验设计概述：本研究通过染色质免疫共沉淀测序（ChIP-seq）技术，在全胚胎与耳蜗中解析了Brg1、Six1及组蛋白修饰（Histone modification）的全基因组结合特征。