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: 10706598
• 负责人: MARK M EMERSON
• 金额: $ 23.55万
• 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/10706598
• 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; 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.

项目总结/摘要 基因的精确时空转录是大多数发育过程的基本组成部分， 多细胞生物中的过程。尽管其重要性，关键的细节，如蛋白质的身份， 在其天然细胞环境中占据特定顺式调节元件的基因通常是未知的。此外还有 需要一种方法来稳健地评估顺式调节元件内的特定序列要求 DNA-蛋白质相互作用所必需的。因此，在确定 基因调控网络的蛋白质和DNA成分。该项目的长期目标是开发一个 用于鉴定在其天然的顺式调节元件中占据特定顺式调节元件的蛋白质的稳健的、无偏倚的方法 细胞背景该建议的中心假设是，用于DNA导入的电穿孔方法 将允许相关转录因子的高灵敏度检测，也是一种实验途径， 评估顺式调控元件的序列要求。进行这项研究的理由是， 在发育的背景下，缺乏关于调控转录的基本信息， 和疾病这里描述的方法将建立一个平台，可以用来克服这一关键问题。 障碍，并可以适应不同的范式。为了实现这一目标，以下两个目标是 提出：1）用共价结合的生物素转移剂创建修饰的顺式调节元件质粒 和2）用修饰的顺式调节元件质粒评价蛋白质的生物素标记。到 提供了一个基础，以评估这种技术，这些目标将使用先前建立的电穿孔 在发育中的脊椎动物视网膜和顺式调节元件与已知的 转录因子第一个目标将使用标准的DNA和蛋白质修饰程序来产生顺式- 调节元件报告质粒，其具有生物素化紧邻顺式- 调节元件。第二个目标将确定这种方法标记已知的 转录因子相互作用使用技术来直接评估这些特定的蛋白质以及 无偏质谱法，以确定这种无偏技术的灵敏度，以确定这些 已知的DNA-蛋白质相互作用。这种方法是创新的，因为它将使用顺式调节元件靶点 与共价结合的生物素化试剂，其可能增加检测蛋白质的灵敏度 互动者此外，与当前技术相比，拟议的系统将允许强大且 一种简便的方法来检查转录因子募集中特定DNA序列的要求， 设计严格的控制以确认相互作用。在项目完成后，一部小说 可用于鉴定天然细胞环境中DNA-蛋白质相互作用的方法将是 开发和验证。该方法的应用有望提供对关键参数的了解 顺式调节的影响，为我们理解的发展机制和疾病的病因。