Dissecting the sensory hair cell nucleus: Development of a novel method for investigating chromatin interactions (Tn5-Capture) in small cell numbers

项目总结： 染色质以远端增强子与靶基因启动子相互作用的方式堆积在微小的细胞核中 在3D空间中以细胞类型特异的方式促进靶基因的表达，尽管增强剂 启动子以染色质的线性形式被几千碱基或有时百万碱基分开。 因此，了解染色质的相互作用对于我们解释转录调控是很重要的 网络支配细胞的识别，并通过转录操作逆转或改变细胞的命运。 然而，数以百万计的细胞需要通过传统的接近法来描述全基因组染色质的相互作用 基于连接的染色质构象捕获方法，使得询问染色质相互作用变得困难 在科尔蒂器官的感觉毛细胞中。头发中缺乏增强子-启动子相互作用信息 细胞限制了我们对毛细胞特异性基因表达和调控网络的理解 阻碍通过表达操作识别毛细胞再生的潜在靶点。至 为了满足这种需要，我们建议研究感觉毛细胞中增强子-启动子的相互作用。 利用Tn5转座酶的“剪切粘贴”转座活性(Tn5捕获)。TN5的可行性-- 猫纯检测增强子-启动子的相互作用已经被我们的初步研究证明，我们将 利用Tn5-Capture启动几个必需转录因子基因的增强子-启动子相互作用分析 然后通过结合Tn5-Capture和Next将分析扩展到全基因组范围 世代排序。最后，通过CRISPRi(CRISPR-dCAS9-EZH2)系统，我们将验证 已知和新发现的特定增强子-启动子相互作用的调控功能 增强子-启动子相互作用。通过这个项目，我们将收集增强子-启动子的数据集 毛细胞中的相互作用，可以与现有的转录和表观遗传学数据相结合，以建立 调控毛细胞特异性基因表达的毛细胞调控网络和识别潜在靶点 刺激毛细胞再生。CRISPRi技术在内耳系统中的实现 在耳聋的治疗方面提供了一个强大的工具。此外，Tn5-Capture 方法是一种新的染色质相互作用询问技术，在国外的研究中具有广泛的应用前景 内耳系统的。

Project Summary: Chromatins are packed into the tiny nucleus in a way that distal enhancers interact with target gene promoters in 3D space to facilitate the expression of the target genes in a cell type-specific manner, though enhancers and promoters are separated by several kilobases or sometimes megabases in the linear forms of chromatins. Thus, understanding of the chromatin interactions is important for us to interpret the transcription regulatory networks dictating cell identifies and to reverse or convert cell fate through transcription manipulations. However, millions of cells are required to profile genome-wide chromatin interactions by conventional proximity ligation-based chromatin conformation capture methods, making it difficult to interrogate chromatin interactions in sensory hair cells of the organ of Corti. The lack of enhancer-promoter interaction information in the hair cells restrains our understanding of the regulatory networks governing hair cell-specific gene expression and hinders the identification of potential targets for hair cell regeneration through expression manipulation. To address this need, we are proposing to investigate enhancer-promoter interactions in sensory hair cells utilizing the “cut-and-paste” transposition activity of Tn5 transposase (Tn5-Capture). The feasibility of Tn5- Catpure to detect enhancer-promoter interactions has been demonstrated by our preliminary study, we will start enhancer-promoter interaction analysis for a few essential transcription factor genes using Tn5-Capture followed by qPCR, and then expand the analysis to genome-wide scale by combining Tn5-Capture and Next Generation Sequencing. Finally, with the CRISPRi (CRISPR-dCAS9-EZH2) system, we will validate the regulatory functions of selected enhancer-promoter interactions for already known and newly discovered enhancer-promoter interactions. Through this project, we will collect datasets of enhancer-promoter interactions in hair cells, which can be integrated with available transcriptomic and epigenetic data to establish hair cell regulatory networks governing hair cell-specific gene expression and to identify potential targets to stimulate hair cell regeneration. Implementation of the CRISPRi technology in the inner ear system also provide a powerful tool in the context of therapeutical treatment of deafness. Besides, the Tn5-Capture method, a new chromatin interaction interrogating technique, will have broad applications in research outside of the inner ear system.