Single-molecule long-read methylation profiling reveals regional DNA methylation regulated by Elongator Complex Subunit 2 in Arabidopsis roots experiencing spaceflight

AbstractThe Advanced Plant Experiment-04 - Epigenetic Expression (APEX04-EpEx) experiment onboard the International Space Station examined the spaceflight-altered cytosine methylation in two genetic lines ofArabidopsis thaliana, wild-type Col-0 and the mutantelp2-5,which is deficient in an epigenetic regulator Elongator Complex Subunit 2 (ELP2). Whole-genome bisulfite sequencing (WGBS) revealed distinct spaceflight associated methylation differences, presenting the need to examine specific space- altered methylation at single molecule resolution to associate specific changes over large regions of spaceflight related genes. Therefore flap-enabled next-generation capture (FENGC), a novel targeted multiplexed DNA capture and enrichment technique allowing cleavage at any specified sites, was applied to survey spaceflight-altered DNA methylation at the single-molecule level. The FENGC capture panel contained 108 targets ranging from 509 to 704 nt within the promoter or gene body regions of gene targets derived from spaceflight whole-genome data sets. In addition to genes with significant changes in expression and average methylation levels between spaceflight and ground control, targets without differentially methylated WGBS but with space- altered distributions of the proportion of methylated cytosines per molecule were further identified. Moreover, trends of co-methylation of different cytosine contexts were exhibited in the same DNA molecules. We further identified significant DNA methylation changes in three previously biological process-unknown genes, and two of them showed enhanced root growth rate in loss-of-function mutants (named asELP2- regulated Methylation in Orbit 1and2). FENGC makes multiplexed targeted single- molecule profiling of methylation simple and cost-effective, which provides an additional power to population-based data for methylation analysis and identification of epigenetically modified substrates in plants. This case study has revealed regional spaceflight-altered methylation in selected targets, which were not identified by short- read methods in spaceflight plants. The newly identifiedEMO1andEMO2are root growth regulators, which can be epigenetically involved in plant adaptation to spaceflight.