Depositing centromere repeats induces heritable intragenic heterochromatin establishment and spreading in Arabidopsis

Stable transmission of non-DNA-sequence-based epigenetic information contributes to heritable phenotypic variants and thus to biological diversity. While studies on spontaneous natural epigenome variants have revealed an association of epialleles with a wide range of biological traits in both plants and animals, the function, transmission mechanism, and stability of an epiallele over generations in a locus-specific manner remain poorly investigated. Here, we invented a DNA sequence deposition strategy to generate a locus-specific epiallele by depositing CEN180 satellite repeats into a euchromatic target locus in Arabidopsis. Using CRISPR/Cas9-mediated knock-in system, we demonstrated that depositing CEN180 repeats can induce heterochromatin nucleation accompanied by DNA methylation, H3K9me2, and changes in the nucleosome occupancy at the insertion sites. Interestingly, both DNA methylation and H3K9me2 are restricted within the depositing sites and depletion of an H3K9me2 demethylase IBM1 enables the outward heterochromatin propagation into the neighboring regions, leading to inheritable target gene silencing to persist for at least five generations. Together, these results demonstrate the promise of employing a cis-engineering system for the creation of stable and site-specific epialleles and provide important insights into functional epigenome studies and locus-specific transgenerational epigenetic inheritance.