A novel method to characterize cis-regulatory complexes during development

一种表征开发过程中顺式调控复合物的新方法

• 批准号: 10511551
• 负责人: MARK M EMERSON
• 金额: $ 19.63万
• 依托单位: CITY COLLEGE OF NEW YORK
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-20 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10511551
• 关键词: Binding; Binding Sites; Biological Assay; Biotin; Biotinylation; Cells; Chick; Chickens; Complex; Coupled; DNA; DNA Modification Process; DNA Sequence; DNA-Protein Interaction; Development; Developmental Process; Disease; Electroporation; Elements; Engineering; Enzymes; Etiology; Evaluation; Formulation; Foundations; Future; Genetic Transcription; Genetic Variation; Goals; Immunoprecipitation; Knowledge; Label; Ligase; Location; Mass Spectrum Analysis; Methodology; Methods; Modeling; Modification; Mutation; Noise; Organism; Plasmids; Polymerase; Post-Translational Protein Processing; Procedures; Process; Proteins; Proteome; Quality Control; Reagent; Regulation; Regulatory Element; Reporter; Retina; Role; Signal Transduction; System; Techniques; Technology; Trans-Activators; Transcriptional Regulation; Ultraviolet Rays; Untranslated RNA; Western Blotting; cell type; chromatin modification; design; detection sensitivity; effectiveness evaluation; gene regulatory network; innovation; insight; next generation sequencing; novel; novel strategies; protein complex; recruit; transcription factor

PROJECT SUMMARY/ABSTRACT The precise temporal and spatial transcription of genes is a fundamental component of most developmental processes in multicellular organisms. Despite its importance, critical details such as the identity of the proteins that occupy specific cis-regulatory elements in their native cellular context are often unknown. In addition, there is a need for methods to robustly evaluate the specific sequence requirements within cis-regulatory elements that are required for DNA-protein interactions. Thus, there exist critical barriers to the identification of the protein and DNA components of gene regulatory networks. The long-term goal of this project is to develop a robust, unbiased method for identification of proteins that occupy specific cis-regulatory elements in their native cellular context. The central hypothesis of this proposal is that the electroporation method for DNA introduction will allow for high sensitivity detection of associated transcription factors and also an experimental avenue to evaluate the sequence requirements of cis-regulatory elements. The rationale to undertake this study is that there is a lack of fundamental information with regards to regulated transcription in the context of development and disease. The approach described here will establish a platform that can serve to overcome this critical barrier and can be adapted to diverse paradigms. To accomplish this goal, the following two aims are proposed: 1) Creation of modified cis-regulatory element plasmids with biotin transfer agents covalently bound to DNA and 2) Evaluation of biotin-labeling of proteins with modified cis-regulatory element plasmids. To provide a basis to evaluate this technique, these aims will use a previously established electroporation paradigm in the developing vertebrate retina and a cis-regulatory element with defined binding sites for known transcription factors. The first aim will use standard DNA and protein modification procedures to generate cis- regulatory element reporter plasmids with the ability to biotinylate proteins in close proximity to the cis- regulatory element. The second aim will determine the effectiveness of this approach to label the known transcription factor interactors using techniques to assess directly these specific proteins as well as an unbiased mass spectrometry approach to determine the sensitivity of this unbiased technique to identify these known DNA-protein interactions. This approach is innovative because it will use cis-regulatory element targets with covalently associated biotinylation agents which are likely to increase the sensitivity to detect protein interactors. In addition, in contrast to current technologies, the proposed system will allow for a robust and facile method to examine the requirements of specific DNA sequences in transcription factor recruitment and to design rigorous controls to confirm interactions. At the completion of the proposed project, a novel methodology that can be used to identify DNA-protein interactions in a native cellular context will have been developed and validated. The application of this method is expected to provide insights into critical parameters of cis-regulation with implications for our understanding of developmental mechanisms and disease etiology.

项目摘要/摘要 基因的精确时间和空间转录是大多数发育的基本组成部分 多细胞生物的过程。尽管它很重要，但诸如蛋白质的身份之类的关键细节 在本机细胞环境中占据特定的顺式调节元件通常是未知的。另外，那里 需要方法来鲁棒评估顺式调节元素中的特定序列要求 DNA蛋白相互作用所需的。因此，存在识别的关键障碍 基因调节网络的蛋白质和DNA成分。该项目的长期目标是开发 坚固的，公正的方法，用于识别占据特定顺式调节元素的蛋白质 蜂窝上下文。该提议的中心假设是DNA引入的电穿孔方法 将允许高灵敏度检测相关的转录因子，也可以进行实验性途径 评估顺式调节元件的序列要求。进行这项研究的理由是 在开发的背景下，缺乏有关调节转录的基本信息 和疾病。这里描述的方法将建立一个可以克服这一关键的平台 障碍，可以适应各种范式。为了实现这一目标，以下两个目标是 提议：1）用生物素转移剂共价结合了修饰的顺式调节元素质粒 到DNA和2）评估具有修饰的顺式调节元素质粒的蛋白质生物素标记。到 提供评估该技术的基础，这些目标将使用先前建立的电穿孔 发育中的脊椎动物视网膜和带有定义结合位点的顺式调节元件的范式 转录因子。第一个目标将使用标准DNA和蛋白质修饰程序来产生顺式 调节性元件报告基因质粒具有与顺式相近的生物素化蛋白的能力 监管元素。第二个目标将决定这种方法的有效性，以标记已知的 转录因子相互作用者使用技术直接评估这些特定蛋白质以及 公正的质谱方法来确定这种公正技术的灵敏度以识别这些方法 已知的DNA蛋白相互作用。这种方法具有创新性，因为它将使用顺式调节元件目标 共价相关的生物素化剂可能会增加检测蛋白质的敏感性 互动者。此外，与当前技术相比，拟议的系统将允许强大和 便利的方法来检查转录因子募集中特定DNA序列的要求 设计严格的控件以确认相互作用。拟议项目完成后，一本小说 可用于在天然细胞环境中鉴定DNA-蛋白质相互作用的方法学已经是 开发和验证。该方法的应用有望提供有关关键参数的见解 顺式调节，对我们对发展机制和疾病病因的理解有影响。