Molecular mechanisms for DNA N6-adenine methylation in eukaryotes

This project explores DNA N6-adenine methylation (6mA) in a unicellular eukaryotic model organism. Though known anecdotally for decades, 6mA's significance in eukaryotic cells has only recently come to light. Advanced DNA sequencing techniques will be used to map 6mA across the genome, providing insights into how this DNA modification is involved in gene regulation. The study focuses on the molecular mechanisms placing 6mA in the appropriate genomic context. Understanding 6mA could reveal new aspects of how genes are turned on and off, shedding light on fundamental processes of life. This research not only advances basic science but also has potential implications for understanding epigenetic changes in health and disease. The project also has strong educational and community outreach components. Data from the study will be shared publicly, supporting other researchers, and the project will involve training students at various levels. This ensures the next generation of scientists is equipped with the knowledge and skills needed to continue advancing this important field. This project will study DNA N6-adenine methylation (6mA) using the unicellular eukaryote Tetrahymena thermophila as a model system. 6mA’s potential as a eukaryotic epigenetic mark has attracted great interest, but its biogenesis and functional implications remain elusive. Tetrahymena is the first eukaryote with 6mA found in its nuclear DNA 50 years ago, and more recently, with a 6mA methyltransferase (MTase) identified and characterized. Using Single Molecule Real-Time, Circular Consensus Sequencing, 6mA on individual Tetrahymena genomic DNA molecules are mapped with high sensitivity and accuracy, and at single-nucleotide resolution. AMT1, a member of the MT-A70 family of MTases, is shown to be crucial for establishing the transcription-associated 6mA distribution pattern in Tetrahymena. Nonetheless, how 6mA is specifically deposited—in the context of transcription, replication, and chromatin—still needs to be elucidated. The PIs will dissect transcription-associated and replication-coupled targeting mechanisms for 6mA deposition. The PIs will also elucidate the biochemical and structural basis for AMT1-dependent, semiconservative, and chromatin-guided transmission of 6mA. This work will provide deep insight into 6mA biology in eukaryotes. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.