Overlapping RdDM and non-RdDM mechanisms work together to maintain somatic repression of a paramutagenic epiallele of maize pericarp color1

Allelic variation at the Zea mays (maize) pericarp color1 ( p1 ) gene has been attributed to epigenetic gene regulation. A p1 distal enhancer, 5.2 kb upstream of the transcriptional start site, has demonstrated variation in DNA methylation in different p1 alleles/epialleles. In addition, DNA methylation of sequences within the 3’ end of intron 2 also plays a role in tissue-specific expression of p1 alleles. We show here a direct evidence for small RNAs’ involvement in regulating p1 that has not been demonstrated previously. The role of mediator of paramutation1 ( mop1 ) was tested in the maintenance of somatic silencing at distinct p1 alleles: the non-paramutagenic P1-wr allele and paramutagenic P1-rr ’ epiallele. The mop1-1 mutation gradually relieves the silenced phenotype after multiple generations of exposure; P1-wr ; mop1-1 plants display a loss of 24-nt small RNAs and DNA methylation in the 3’ end of the intron 2, a region close to a Stowaway transposon. In addition, a MULE sequence within the proximal promoter of P1-wr shows depletion of 24nt siRNAs in mop1-1 plants. Release of silencing was not correlated with small RNAs at the distal enhancer region of the P1-wr allele. We found that the somatic silencing of the paramutagenic P1-rr ’ is correlated with significantly reduced H3K9me2 in the distal enhancer of P1-rr ’; mop1-1 plants, while symmetric DNA methylation is not significantly different. This study highlights that the epigenetic regulation of p1 alleles is controlled both via RdDM as well as non-RdDM mechanisms.