Project Summary: Though a reference human genome and even excellent characterization of the epigenome (ENCODE, Epigenetics Roadmap, 4D Nucleome Project) has been generated, we still lack a clear understanding of how the different facets of the epigenome collaborate to control gene expression. With the advent of affordable DNA-sequencing technologies, methods have been developed for examining nuclear organization, protein-DNA interaction, chromatin accessibility, and methylation state. But these methods generally interrogate only one aspect of the epigenome at a time and reads are typically too short to provide critical correlative information. We propose the development of a novel epigenetic characterization methodology, in this round of funding focusing on protein- DNA interaction through leveraging the long reads and modified bases detectable with a nanopore sequencing platform. We will enhance the signal from subtle modifications being used to profile protein-DNA interactions. We are developing a method to insert sequence tags (nanoTUBI) near the sites of protein-DNA binding. And we will implement these methods focusing on long read sequencing, gaining insights into long correlative measurements and heretofore unexplored repetitive areas, phasing the entire epigenome. These tools and analysis pipelines will grant new insights into mechanisms of gene regulation, as well as their implications for human disease.

Project Narrative: Gene regulation is controlled in part through the binding of different proteins to DNA, proteins which control how DNA is organized and how it can be transcribed to RNA and from there to protein. Specifically, sequencing of protein-DNA interactions is logistically difficult and limited with current 2nd generation technologies: only short-range interactions are studied, extensive chemical or enzymatic treatments are needed, and heterogeneity is hard to probe. In this funding period, we are seeking to add to our toolbox to explore the epigenetic state of the human genome via measurement of modifications and insertions to the DNA sequence, with a focus on protein-DNA interactions.