Novel approaches to improve comprehensive profiling of the epigenome and epitranscriptome

Project Summary In 2022, advances from long-read sequencing methods facilitated the completion of a complete human genome sequence. To interpret the genetic information from human genome sequences and understand how cells regulate which parts of the genome are transcribed, how RNA transcripts are processed, and how these processes are dysregulated in disease, it is critical to comprehensively profile the epigenome and epitranscriptome. In the last project period, our group made significant advances in multiple areas developing methods for epigenome and epitranscriptome profiling using long-read sequencing, particularly in improvements to our computational method FLAIR (Full-Length Alternative Isoform analysis of RNA). Integrating these methods in S. cerevisiae, we have uncovered novel RNA processing changes associated with chromatin remodeling mutants. In our future work, we will further optimize our method to probe chromatin accessibility with a membrane-permeable small molecule, which has the potential to facilitate epigenomic studies in low-input tissue samples. We will use our insights and expertise gained from benchmarking and evaluating long-read methods for transcript assembly to further advance the field of RNA modification detection in order to directly profile full-length alternative RNA isoforms, RNA edits, and RNA modifications from single RNA molecule sequences. By combining our approaches in long-read epigenetic and epitranscriptomic methods developed in the last project period along with future methodological advances proposed here, we will elucidate the mechanisms of RNA processing changes associated with chromatin remodeler mutants in humans, where there is additional genome and RNA processing complexities compared to yeast. Long-read epigenomic and epitranscriptomic methods developed in this work have numerous important applications to the broader scientific community and we are committed to continuing to develop and share computational tools and protocols that are well-documented and user-friendly to facilitate wide adoption.