Transposon-Induced Paramutation in Maize

Paramutation is an interaction of alleles resulting in heritable gene silencing, but the molecular events that initiate paramutation are largely unknown. We observed unexpected silencing of the maize P1-rr gene by p1 alleles containing two nearby Ac/fAc transposable elements. A panel of derivative alleles containing these paired Ac/fAc elements with different Inter Transposon Segments (ITS) were tested for their silencing ability; two cases exhibit strong and frequent silencing. Both alleles retain small overlapping segments (3748 and 3815 bp) of the p1 5'regulatory region, including sequences of a transcribed ∼600 bp p1 enhancer region. Previous studies have shown that this enhancer sequence is sufficient to induce paramutagenic silencing in transgenic maize plants. Here, we show that the inducing and silenced alleles are enriched for small RNAs and cytosine methylation in the ITS, relative to the normal progenitor allele. Interestingly, unique DNA junctions corresponding to circular ITS are found in plants carrying the inducing alleles, as evidenced by genomic PCR and sRNA reads crossing the presumed circle junction. We hypothesize that Reversed-Ends Transposition (RET) generates extrachromosomal circular DNA (eccDNA) containing the p1 enhancer, whose transcription leads to trans silencing via production of small RNA and DNA methylation. The silenced P1-rr* alleles have severely reduced p1 transcript levels, are highly heritable, and are themselves paramutagenic. These results show a new origin of paramutation, and a novel genetic function of eccDNA.