Heterochromatin is a quantitative trait associated with spontaneous epiallele formation.

Epialleles are meiotically heritable variations in expression states that are independent from changes in DNA sequence. Although they are common in plant genomes, their molecular origins are unknown. Here we show, using mutant and experimental populations, that epialleles in Arabidopsis thaliana that result from ectopic hypermethylation are due to feedback regulation of pathways that primarily function to maintain DNA methylation at heterochromatin. Perturbations to maintenance of heterochromatin methylation leads to feedback regulation of DNA methylation in genes. Using single base resolution methylomes from epigenetic recombinant inbred lines (epiRIL), we show that epiallelic variation is abundant in euchromatin, yet, associates with QTL primarily in heterochromatin regions. Mapping three-dimensional chromatin contacts shows that genes that are hotspots for ectopic hypermethylation have increases in contact frequencies with regions possessing H3K9me2. Altogether, these data show that feedback regulation of pathways that have evolved to maintain heterochromatin silencing leads to the origins of spontaneous hypermethylated epialleles.