Mapping DNA structure & protein-DNA interactions using hydroxyl radical footprinting & high-throughput sequencing
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Development of biochemical techniques to examine chromatin structure and protein-DNA interactions on a global scale has allowed for extensive characterization of functional and regulatory elements essential to cellular biological processes. In particular, chromatin accessibility and susceptibility to damage, coupled with high-throughput sequencing, have served as means for characterizing these elements. To better understand protein occupancy in relation to chromatin architecture, a technique that can impartially probe DNA structure at high resolution is required. The hydroxyl radical, generated from a modified Fenton reaction or ɣ-irradiation of water molecules, is a chemical tool used for probing nucleic acid structure, and capable of mapping protein-DNA binding sites at single-nucleotide resolution. Adapting hydroxyl radical footprinting for analysis by high-throughput sequencing (OH-seq) aims to provide a detailed profile of the chromatin landscape in whole genomes. Initial development of OH-seq was carried out on a model system using synthetic oligonucleotides to mimic a hydroxyl radical damage site. The single-strand break was enzymatically converted to a double-strand break to allow for end-repair and ligation to a sequencing adapter. This dissertation describes the further development of OH-seq in vitro, and the optimization of this technique for application to whole genomes in vivo. To show that OH-seq can successfully map protein-DNA interactions, the technique was tested on the well characterized λ repressor-operator complex. Analyses for sequencing libraries, tagging single- and double-strand breaks created from hydroxyl radical cleavage of plasmid DNA in the absence and presence of λ repressor, show footprints similar to those from previous studies. Application of OH-seq to human and S. cerevisiae genomes captured double-strand breaks in genomic DNA following ɣ-irradiation of cells. Analyses examining the damage profile across aggregated transcription start sites and nucleosome positions in the human genome reveal high damage at promoters, and highly periodic nucleosomal footprints. OH-seq profiles for select transcription factors in yeast show distinct footprints comparable to those from other genome-wide studies. These preliminary results show the potential OH-seq has for characterizing chromatin structure and protein-DNA interactions. Further optimization will make the technique a useful addition to the current repertoire of tools for studying genome structure and function.