Mapping Genomic Binding Sites of Low Abundance Transcription Factors

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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.