RdDM mutants reduce histone methylation rather than DNA methylation at the paramutated maize b1 enhancer

Paramutation is the transfer of mitotically and meiotically heritable silencing information between two alleles. With paramutation at the maize booster1 (b1) locus, the low expressed B epiallele heritably changes the high expressed B-I epiallele into B with 100% frequency. This requires specific tandem repeats and multiple components of the RNA-directed DNA methylation (RdDM) pathway, including the RNA-dependent RNA polymerase (RdRP, mediator of paramutation1, MOP1), the second-largest subunit of RNA polymerase IV and V (NRP(D/E)2a, mediator of paramutation2, MOP2), and the largest subunit of RNA Polymerase IV (NRPD1, mediator of paramutation3, MOP3). Mutations in mop genes prevent paramutation and release silencing at the B epiallele. In this study we investigated the effect of mutations in mop1, mop2 and mop3 on chromatin structure and DNA methylation at the B epiallele, and especially the regulatory hepta-repeat 100 kb upstream of the b1 gene. We show that mutations in mop1 and mop3 result in decreased repressive histone modifications H3K9me2 and H3K27me2 at the hepta-repeat. Associated with this decrease are partial activation of the hepta-repeat enhancer function, formation of a multi-loop structure, and elevated b1 expression. In mop2 mutants, which do not show elevated b1 expression, H3K9me2 and H3K27me2 and a single-loop structure like in wild type B are retained. Surprisingly, high DNA methylation levels at the B hepta-repeat remains in all three mutants. Our results raise the possibility of MOP factors mediating RNA-directed histone methylation rather than RNA-directed DNA methylation at the b1 locus.