Developing a robust method for analyzing transcription factor mediated chromatin interactions

开发一种稳健的方法来分析转录因子介导的染色质相互作用

• 批准号: 10667811
• 负责人: LIN CHEN
• 金额: $ 24.81万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10667811
• 关键词: 3-Dimensional; Adopted; Alkynes; Antibodies; Azides; Binding; Binding Sites; Bioinformatics; Biological Models; Biotin; Biotinylation; Cell Line; Cells; ChIP-seq; Chemistry; Chromatin; Chromatin Interaction Analysis by Paired-End Tag Sequencing; Communities; Complex; Computer Analysis; DNA; DNA Binding; DNA Ligation; DNA Modification Process; Data; Disease; Distal; Epigenetic Process; FOXP3 gene; Feedback; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Genome; Genome Mappings; Hi-C; Histones; Hybridomas; Immunoprecipitation; In Situ; In Vitro; Joints; Knock-in; Label; Ligase; Ligation; Link; Locus Control Region; Maps; Mediating; Methods; Modification; Mutation; Names; Nucleotides; Open Reading Frames; Pathologic; Physiological; Process; Proteins; Protocols documentation; Publishing; Reaction; Regulator Genes; Regulatory Element; Regulatory T-Lymphocyte; Reproducibility; Research; Resources; Role; Running; Series; Signal Transduction; Streptavidin; T-Lymphocyte; Techniques; Technology; Testing; Transcriptional Regulation; Transgenic Model; Untranslated RNA; Work; base; biochip; chromatin immunoprecipitation; cofactor; design; differential expression; experimental study; genome-wide; genome-wide analysis; genomic tools; histone modification; human disease; improved; in vivo; interest; model organism; novel; promoter; restriction enzyme; success; three dimensional structure; tool; transcription factor; transcriptome; transcriptome sequencing

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.

开发一种分析转录因子介导的染色质相互作用的稳健方法 转录因子介导远端调控元件与基因启动子之间的相互作用 控制特定的基因表达;通过蛋白质编码区的突变破坏这一过程，或 TF的非编码DNA结合位点可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）已被开发用于绘制长距离染色质图谱 交互.在这些以DNA为中心的方法中缺少的是TF及其辅因子，尽管 已经开发了生物信息学方法来推断TF在染色质相互作用中的作用。电流 用于绘制蛋白质介导的染色质相互作用的技术（如HiChIP）已经与 高丰度蛋白质，但不是大多数TF。这在很大程度上是由于高可变性和低效率， 用于免疫沉淀与目标TF相关的染色质复合物的抗体。的 引入原位生物素化的TF大大提高了ChIP-seq的灵敏度和重现性 （a.k.a biochip-seq），但这种策略不能扩展到TF介导的染色质的定位 交互.这是因为所有基于Hi-C的定位技术，包括HiChIP，都使用生物素化的 核苷酸以标记连接点以富集接触信号。因此，链霉亲和素结合将 不加区别地拉下所有的染色质接触。为了克服这个问题，拟议的研究旨在 开发一种新的方法，通过调整HiChIP协议来映射TF介导的染色质相互作用， 生物素化TF（称为bioHiChIP）（目的1），并测试bioHiChIP在作图中的应用 生物素化FOXP 3介导的染色质相互作用（Aim 2）。bioHiChIP被设计成一个简单的 和易于操作的协议，用于蛋白质介导的染色质相互作用的稳健映射。它将作为一个 强大的工具，为广大的研究界利用不断增加的研究资源， 用生物素标记的转基因生物研究转录的详细机制 调节生理功能和病理疾病。