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Recording Transcription Factor Binding During Development
Date of Award
Doctor of Philosophy (PhD)
This dissertation addresses the development and application of a novel method for mapping the binding sites of transcription factors using transposons to insert into the genome near to where the factors are bound. The method, named "Calling Cards", offers an alternative to chromatin immunoprecipitation (ChIP) methods and furthermore has the ability to record transcription factor binding through cell division and development. I first demonstrated the method using the Ty5 transposon in the budding yeast Saccharomyces cerevisiae. The method was benchmarked against existing ChIP methods and demonstrated to be capable of multiplexing several transcription factors in a single experiment. I applied the method to map the transcriptional network of 28 multiplexed transcription factors during the yeast developmental process of pseudohyphal growth. The method was next re-engineered to use a different transposon, piggyBac, making the technique applicable to mammalian systems. Using human cell culture, I mapped the binding sites of the transcription factor SP1 and benchmarked them against ChIP data. Finally, I employed this system to record the early binding events of the transcription factor Olig2 during neural differentiation. In summary, this dissertation presents a useful method for recording transcription factor binding during development.
Donald Conrad, Shelly Sakiyama-Elbert, Gary Stormo