Project Summary A major focus of biomedical research that involves sequencing of the genome and transcriptome is to understand how genes are regulated and also dysregulated in disease. Integrated analyses of epigenome and epitranscriptome changes are urgently needed to have a complete molecular profile of cellular changes and to understand the molecular mechanisms of gene regulation. Yet, standard sequencing methods are unable to capture the full complexity of the epigenome (DNA modifications and chromatin accessibility) or the epitranscriptome (RNA modification). Problems inherent to current Illumina-based sequencing include: introduction of PCR bias, short-length of the reads, and the inability to directly sequence RNA molecules. To address these needs, we are developing experimental and computational approaches that allow us to (a) simultaneously detect, in vivo, DNA modifications and DNA accessibility on long stretches of single DNA molecule sequences and correlate these changes with effects on the epitranscriptome by (b) directly profiling full-length alternative RNA isoforms, RNA edits, and RNA modifications from single RNA molecule sequences. Our combined approach will rely on Oxford Nanopore long-read technologies which is capable of distinguishing modified bases in DNA and RNA, and on in vivo methods of marking accessible regions of chromatin. To demonstrate the applicability and relevance of our methods, we will perform these experiments under biological conditions known to impact both chromatin structure and the epitranscriptome. We also plan to (c) profile epigenomic and epitranscriptomic changes in response to knockdown of key chromatin remodeling genes and RNA binding proteins to test if, and how broadly, these regulatory factors affect the epigenome and epitranscriptome. Our combined approach of long-range detection of modified and accessible regions of DNA with detection of isoform-specific RNA processing events will provide a much-needed broadly applicable tool to elucidate the mechanisms governing cell signaling responses involving chromatin and transcriptome alterations.

Project Narrative Comprehensive profiling of DNA and RNA molecules, including nucleotide modifications, is important for a complete understanding of how genes are normally regulated or dysregulated in disease; however, this requires methods for higher resolution profiling than what is currently available by standard sequencing techniques. To this end, we will develop approaches using long-read technology to simultaneously detect in vivo genome-wide DNA modification and accessibility and correlate changes with complete RNA sequences, along with RNA modifications. We will apply our approaches to the study of well-characterized biological responses known to impact chromatin in established mammalian cell lines and probe the inter-dependence of the epigenome and epitranscriptome by suppressing expression of key protein modulators.