High-throughput methods to analyze protein-DNA and protein-RNA interactions
Abstract
The genome of a cell contains the information to make thousands of RNA and protein molecules. However, a cell typically expresses only a fraction of its genes, with different phenotypes arising because of differentially expressed genes. A cell controls its gene expression at different levels. For example, it can control when and how often a gene is transcribed, or it can selectively destabilize mRNA molecules. These biological processes are controlled by DNA- and RNAbinding proteins. In this dissertation, I first discuss the current state of the field of transcription factor (TF) variation, and I describe in vitro and in vivo technologies for mapping TF-DNA interactions. I then describe studies that apply the “calling card” assay to analyze interactions between chromatin and TF variants. In Chapter 2, I use this assay to characterize the interactions with chromatin of six single amino acid variants of Ste12, a yeast transcription factor regulating mating and invasion. My study showed that while subtle changes in the coding region of this transcription factor can result in large regulatory rewiring, the major determinants of organismal phenotype are the changes in the expression of a small, related set of genes. In Chapter 3, I use next generation sequencing technology to score the RNA-binding activity of variants in each of eight repeats present within a PUF domain. In this assay, in addition to the PUF variants, I generated a library of RNA variants in which each possible RNA base was present at the cognate position recognized by one of the PUF domain repeats. I identified many PUF domain variants with highly specific interactions by comparing their binding across the four RNA bases. This approach allows us to propose a complete code for RNA recognition by this PUF domain. In Chapter 4, I discuss a new method to concurrently profile the whole and newly synthesized transcriptome in each of many single cells, and use the data to quantify the dynamics of the cell cycle and glucocorticoid receptor activation. Finally, in Chapter 5, I discuss some of the 2 outstanding questions for mapping protein-DNA and protein-RNA interactions, including research directions that I believe will be important in the future.
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