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

项目概要： 染色质以远端增强子与靶基因启动子相互作用的方式被包装到微小的细胞核中 以促进靶基因以细胞类型特异性方式表达，尽管增强子 并且启动子被几种酶或有时是染色质线性形式的大酶分开。 因此，了解染色质相互作用对于我们解释转录调控机制是非常重要的。 控制细胞身份的网络，并通过转录操作逆转或转换细胞命运。 然而，需要数百万个细胞才能通过常规的邻近分析来分析全基因组染色质相互作用。 基于连接的染色质构象捕获方法，使得难以询问染色质相互作用 在Corti器的感觉毛细胞中。毛发中缺乏增强子-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调控网络的理解， 阻碍了通过表达操作鉴定毛细胞再生的潜在靶标。到 为了满足这一需求，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Tn 5转座酶（Tn 5-Capture）的“剪切-粘贴”转座活性。Tn 5的可行性 Catpure检测增强子-启动子相互作用已被我们的初步研究所证明，我们将 应用Tn 5-Capture分析几种重要转录因子基因启动子-增强子相互作用 随后进行qPCR，然后通过结合Tn 5-Capture和Next将分析扩展到全基因组范围 世代排序。最后，利用CRISPRi（CRISPR-dCAS 9-EZH 2）系统，我们将验证 选择的增强子-启动子相互作用对已知和新发现的 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，这可以与可用的转录组学和表观遗传学数据相结合，以建立 毛细胞调控网络管理毛细胞特异性基因表达，并确定潜在的目标， 刺激毛细胞再生。在内耳系统中实施CRISPRi技术还 为耳聋的治疗提供了有力的工具。此外，Tn 5-Capture 方法作为一种新的染色质相互作用的检测技术，在国外的研究中有着广泛的应用前景 内耳系统。

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.