Heat-responsive ONSEN long terminal repeats integrate heat shock factor motifs, DNA methylation and natural sequence variation in Arabidopsis

ONSEN is a heat-activated Ty1/copia retrotransposon in Arabidopsis thaliana controlled by heat shock factors (HSFs) and epigenetic silencing. Heat shock element (HSE)-like sequences in ONSEN long terminal repeats (LTRs) contribute to heat responsiveness, but relationships among sequence architecture, basal DNA methylation and natural variation remain unclear. We combined transcription-factor motif prediction, transposable-element comparisons, methylome and RNA-seq data, and Arabidopsis genome assemblies. In silico disruption of five HSE cores eliminated HSF-family motif compatibility in the selected design and all 5,119 exact-GC alternatives. Across 16 curated Col-0 terminal windows, ONSEN contained 33-49 non-redundant HSF motif-coordinate placements per 800-bp window and was strongly enriched relative to 1,930 non-ONSEN transposable elements across score thresholds and continuous metrics. Direct comparison with 779 non-ONSEN LTR retrotransposons showed selectively elevated basal CHH methylation at ONSEN termini. Genome-wide RNA-seq analysis revealed broad heat-responsive gene and transposable-element changes, including strong ONSEN induction, whereas candidate-window analysis distinguished ONSEN from most HSF-rich non-ONSEN outliers. ONSEN-like variants across eight accessions generally retained HSF-compatible motifs while altering predicted DNA binding with one finger (DOF)-family motif composition. Together, these findings define ONSEN terminal regions as HSF-rich regulatory sequences that retain heat-responsive potential within a methylated chromatin context and identify candidates for functional analysis.