Widespread adenine N6-methylation of active genes in fungi

Stephen J. Mondo sjmondo@lbl.gov ¹ Richard O. Dannebaum ¹ Rita C. Kuo ¹ Katherine B. Louie ¹ Adam J. Bewick ² Kurt LaButti ¹ Sajeet Haridas ¹ Alan Kuo ¹ Asaf Salamov ¹ Steven R. Ahrendt ^1,3 Rebecca Lau ¹ Benjamin P. Bowen ¹ Anna Lipzen ¹ William Sullivan ¹ Bill B. Andreopoulos ¹ Alicia Clum ¹ Erika Lindquist ¹ Christopher Daum ¹ Trent R. Northen ¹ Govindarajan Kunde-Ramamoorthy ¹ Robert J. Schmitz ² Andrii Gryganskyi ⁴ David Culley ⁵ Jon Magnuson ⁵ Timothy Y. James ⁶ Michelle A. O'Malley ⁷ Jason E. Stajich ⁸ Joseph W. Spatafora ⁹ Axel Visel ^1,10 Igor V. Grigoriev ^1,3

¹US Department of Energy, Joint Genome Institute, Walnut Creek, California, USA
²Department of Genetics, University of Georgia, Athens, Georgia, USA
³Department of Plant and Microbial Biology, University of California, Berkeley, California, USA
⁴NA, L. F. Lambert Spawn Co, Coatesville, Pennsylvania, USA
⁵US Department of Energy, Pacific Northwest National Laboratory, Richland, Washington, USA
⁶Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan, USA
⁷Department of Chemical Engineering, University of California, Santa Barbara, California, USA
⁸Department of Plant Pathology and Microbiology, University of California, Riverside, California, USA
⁹Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon, USA
¹⁰School of Natural Sciences, University of California, Merced, California, USA

N6-methyldeoxyadenine (6mA) is a noncanonical DNA base modification present at low levels in plant and animal genomes, but its prevalence and association with genome function in other eukaryotic lineages remains poorly understood. Here we report that abundant 6mA is associated with transcriptionally active genes in early-diverging fungal lineages. Using single-molecule long-read sequencing of 16 diverse fungal genomes, we observed that up to 2.8% of all adenines were methylated in early-diverging fungi, far exceeding levels observed in other eukaryotes and more derived fungi. 6mA occurred symmetrically at ApT dinucleotides and was concentrated in dense methylated adenine clusters surrounding the transcriptional start sites of expressed genes; its distribution was inversely correlated with that of 5-methylcytosine. Our results show a striking contrast in the genomic distributions of 6mA and 5-methylcytosine and reinforce a distinct role for 6mA as a gene-expression-associated epigenomic mark in eukaryotes.