Mapping Genomic Binding Sites of Low Abundance Transcription Factors

转录因子是一种调节基因表达的蛋白质，在高等生物生长发育的各个方面发挥着关键作用。为了了解单个转录因子是如何发挥其功能的，有必要确定它所调控的基因。要做到这一点，一种方法是识别活细胞中转录因子与之结合的DNA序列。然而，许多转录因子以非常低的丰度存在和/或在少数细胞中表达，这使得识别目标基因在技术上具有挑战性。这项建议的目标是开发一种从模式植物拟南芥中纯化转录因子-DNA复合体的方法。一种遗传方法将被用来用小分子生物素标记个别的拟南芥转录因子。利用生物素和链霉亲和素蛋白之间的强相互作用，将使用生物化学方法从植物细胞中纯化生物素标记的转录因子和相关的DNA。纯化后，将确定与转录因子共纯化的DNA片段的序列。该项目将确定地上部无分生组织和侧器官边界的基因组结合部位，这两个转录因子是拟南芥发育中重要的转录因子。识别这些转录因子的下游靶标将进一步加深我们对调控植物器官形成和干细胞维持的遗传网络的理解。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，这将是一项重大的科学进步。该项目将有助于培养直接参与研究的研究生和本科生。

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.