Mapping Genomic Binding Sites of Low Abundance Transcription Factors

绘制低丰度转录因子的基因组结合位点图谱

• 批准号: 1144407
• 负责人: Patricia Springer
• 金额: $ 19.99万
• 依托单位: University of California-Riverside
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1144407&HistoricalAwards=false
• 关键词: Mapping; Genomic; Binding; Sites; Low

Transcription factors, proteins that regulate gene expression, play critical roles in all aspects of growth and development in higher organisms. In order to understand how an individual transcription factor exerts its function, it is necessary to identify the genes that it regulates. One way to do this is to identify DNA sequences that a transcription factor binds to in a live cell. However, many transcription factors are present in very low abundance and/or are expressed in a small number of cells, making the identification of target genes technically challenging. The goal of this proposal is to develop a method for purifying transcription factor-DNA complexes from the model plant Arabidopsis thaliana. A genetic approach will be used to tag individual Arabidopsis transcription factors with the small molecule biotin. Biochemical methods will be used to purify the biotin-tagged transcription factor and associated DNA from plant cells, taking advantage of the strong interaction between biotin and the protein streptavidin. Following purification, the sequence of DNA fragments that co-purify with the transcription factor will be determined. This project will determine the genomic binding sites of SHOOT MERISTEMLESS and LATERAL ORGAN BOUNDARIES, two developmentally important Arabidopsis transcription factors. Identification of downstream targets of these transcription factors will further our understanding of genetic networks that regulate organ formation and stem cell maintenance, critical processes in plants. More generally, this project will develop methods for tagging and purifying any plant transcription factor and associated DNA, which will be a major scientific advance. The project will contribute to training of graduate and undergraduate students, who will be directly involved in the research.

转录因子是调节基因表达的蛋白质，在高等生物生长发育的各个方面起着关键作用。为了了解单个转录因子如何发挥其功能，有必要确定它调节的基因。一种方法是鉴定活细胞中转录因子结合的DNA序列。 然而，许多转录因子以非常低的丰度存在和/或在少量细胞中表达，使得靶基因的鉴定在技术上具有挑战性。本研究的目的是建立一种从模式植物拟南芥中纯化转录因子-DNA复合物的方法。将使用遗传方法用小分子生物素标记单个拟南芥转录因子。利用生物素和蛋白质链霉亲和素之间的强相互作用，生物化学方法将用于从植物细胞中纯化生物素标记的转录因子和相关DNA。 纯化后，将确定与转录因子共纯化的DNA片段的序列。本计画将探讨拟南芥中两个重要的转录因子SHOOT MERISTEMLESS和LATERAL ORGAN BOUNDARIES的基因组结合位置。这些转录因子的下游靶点的鉴定将进一步加深我们对调节植物器官形成和干细胞维持的遗传网络的理解，这些是植物中的关键过程。更广泛地说，该项目将开发标记和纯化任何植物转录因子和相关DNA的方法，这将是一个重大的科学进步。该项目将有助于培训研究生和本科生，他们将直接参与研究。