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

基本信息

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
项目类别：
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

项目摘要

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.

开发一种稳健的方法来分析转录因子介导的染色质相互作用转录因子（TF）介导远端调控元件和基因启动子之间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破坏这一过程或 TF 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（例如 Hi-C）被开发用于绘制长距离染色质图谱互动。这些以 DNA 为中心的方法中缺少的是 TF 及其辅助因子，尽管已经开发出生物信息学方法来推断转录因子在染色质相互作用中的作用。当前的用于绘制蛋白质介导的染色质相互作用的技术（例如 HiChIP）与高丰度蛋白质，但不适用于大多数 TF。这主要是由于高变异性和低效率造成的。用于免疫沉淀与目标 TF 相关的染色质复合物的抗体。这原位生物素化 TF 的引入大大提高了 ChIP-seq 的灵敏度和重现性（又名生物芯片测序），但该策略不能扩展到 TF 介导的染色质作图互动。这是因为所有基于 Hi-C 的作图技术（包括 HiChIP）都使用生物素化核苷酸来标记连接点以丰富接触信号。因此，链霉亲和素结合将不加区别地拉下所有染色质接触点。为了克服这个问题，拟议的研究旨在通过采用 HiChIP 协议来开发一种绘制 TF 介导的染色质相互作用的新方法生物素化转录因子（简称bioHiChIP）（目标1）并测试bioHiChIP在作图方面的应用由生物素化 FOXP3 介导的染色质相互作用（目标 2）。 bioHiChIP 被设计为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图谱。它将作为为广大研究界利用不断增加的研究资源的强大工具具有生物素化转录因子的转基因模型生物，用于研究转录的详细机制生理功能和病理疾病的调节。