Developing a robust method for analyzing transcription factor mediated chromatin interactions

建立一种分析转录因子介导的染色质相互作用的可靠方法 转录因子(TF)介导末端调控元件与基因启动子的相互作用 控制特定基因的表达；通过蛋白质编码区的突变或 转录因子的非编码DNA结合位点可能导致转录失调和人类疾病。一个 越来越多的基因组学工具(如Hi-C)已被开发用于定位长范围染色质 互动。这些以DNA为中心的方法缺少的是转录因子及其辅助因子，尽管 生物信息学方法已经被用来推断转录因子在染色质相互作用中的作用。当前 绘制蛋白质介导的染色质相互作用图的技术(如HiChIP)与 高丰度蛋白质，但不适用于大多数转录因子。这在很大程度上是由于 用于免疫沉淀与目标转移因子相关的染色质复合体的抗体。这个 原位生物素化转移因子的引入大大提高了芯片序列的灵敏度和重复性 (也称为生物芯片-SEQ)，但这一策略不能扩展到TF介导的染色质的定位 互动。这是因为所有基于Hi-C的映射技术，包括HiChIP，都使用生物素化 核苷酸标记连接连接，以丰富接触信号。因此，链霉亲和素结合将 不分青红皂白地拔掉所有染色质接触。为解决这一问题，拟议的研究旨在 建立一种新的方法，通过调整HiChIP协议来定位TF介导的染色质相互作用 Biotinylated TFS(简称BioHiChIP)(目标1)，并测试BioHiChIP在地图绘制中的应用 生物素化FOXP3介导的染色质相互作用(目标2)。BioHiChIP被设计成一个简单的 以及易于操作的方案，用于蛋白质介导的染色质相互作用的稳健图谱。它将作为一种 为广大研究界利用日益增长的研究资源提供了强大的工具 用生物素化的转录因子转基因模型生物研究转录的详细机制 对生理功能和病理疾病的调节。

Developing a robust method for analyzing transcription factor mediated chromatin interactions Transcription factors (TFs) mediate the interactions between distal regulatory elements and gene promoters to control specific gene expression; disruption of this process by mutations in the protein coding regions or the non-coding DNA binding sites of TFs could lead to dysregulation of transcription and human diseases. An increasing number of genomics tools (such as Hi-C) have been developed for mapping long range chromatin interactions. What is missing in these DNA-centric approaches are TFs and their cofactors, although bioinformatics approaches have been developed to infer the roles of TFs in chromatin interactions. Current technologies (such as HiChIP) for mapping protein-mediated chromatin interactions have worked well with high abundance proteins but not for most TFs. This is largely due to the high variability and low efficiency of antibodies used in the immunoprecipitation of chromatin complexes associated with the TF of interest. The introduction of in situ biotinylated TFs has greatly improved the sensitivity and reproducibility of ChIP-seq (a.k.a biochip-seq), but this strategy cannot be extended to the mapping of TF-mediated chromatin interactions. This is because all Hi-C based mapping techniques, including HiChIP, use biotinylated nucleotide to label the ligation junction to enrich contact signals. Consequently, streptavidin binding would pull down all chromatin contacts indiscriminately. To overcome this problem, the proposed studies aim to develop a new method for mapping TF-mediated chromatin interaction by adapting the HiChIP protocol to biotinylated TFs (referred to as bioHiChIP) (Aim 1) and test the application of bioHiChIP in mapping chromatin interactions mediated by biotinylated FOXP3 (Aim 2). The bioHiChIP is designed to be a simple and easy-to-operate protocol for robust mapping of protein-mediated chromatin interactions. It will serve as a powerful tool for the broad research community to take advantage of the increasing research resources of transgenic model organisms with biotinylated TFs to study the detailed mechanisms of transcription regulation in physiological functions and pathological diseases.