Yongqiang Liu1,2,7, Junhua Niu1,2,7, Fei Ye1,2,7, Therese Solberg3,4,5, Borong Lu1,2, Chundi Wang1,6, Mariusz Nowacki3 and Shan Gao1,2 1MOE Key Laboratory of Evolution and Marine Biodiversity and Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China; 2Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, Qingdao 266237, China; 3Institute of Cell Biology, University of Bern, 3012 Bern, Switzerland; 4Department of Molecular Biology, Keio University School of Medicine, 160-8582 Tokyo, Japan; 5Human Biology Microbiome Quantum Research Center (WPI-Bio2Q), Keio University, 108-8345 Tokyo, Japan; 6Laboratory of Marine Protozoan Biodiversity and Evolution, Marine College, Shandong University, Weihai 264209, China

↵7 These authors contributed equally to this work.

Corresponding author: shangaoouc.edu.cn Abstract

The formation of resting cysts commonly found in unicellular eukaryotes is a complex and highly regulated survival strategy against environmental stress that involves drastic physiological and biochemical changes. Although most studies have focused on the morphology and structure of cysts, little is known about the molecular mechanisms that control this process. Recent studies indicate that DNA N6-adenine methylation (6mA) could be dynamically changing in response to external stimuli; however, its potential role in the regulation of cyst formation remains unknown. We used the ciliate Pseudocohnilembus persalinus, which can be easily induced to form cysts to investigate the dynamic pattern of 6mA in trophonts and cysts. Single-molecule real-time (SMRT) sequencing reveals high levels of 6mA in trophonts that decrease in cysts, along with a conversion of symmetric 6mA to asymmetric 6mA. Further analysis shows that 6mA, a mark of active transcription, is involved in altering the expression of encystment-related genes through changes in 6mA levels and 6mA symmetric-to-asymmetric conversion. Most importantly, we show that reducing 6mA levels by knocking down the DNA 6mA methyltransferase PpAMT1 accelerates cyst formation. Taken together, we characterize the genome-wide 6mA landscape in P. persalinus and provide insights into the role of 6mA in gene regulation under environmental stress in eukaryotes. We propose that 6mA acts as a mark of active transcription to regulate the encystment process along with symmetric-to-asymmetric conversion, providing important information for understanding the molecular response to environmental cues from the perspective of 6mA modification.

Received December 1, 2023. Accepted February 14, 2024.