RNA-directed DNA methylation mutants reduce histone methylation at the paramutated maize booster1 enhancer

Abstract

Abstract Paramutation is the transfer of mitotically and meiotically heritable silencing information between two alleles. With paramutation at the maize (Zea mays) 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 pathway, including the RNA-dependent RNA polymerase (encoded by mediator of paramutation1, mop1), the second-largest subunit of RNA polymerase IV and V (NRP(D/E)2a, encoded by mop2), and the largest subunit of RNA Polymerase IV (NRPD1, encoded by 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. Mutations in mop1 and mop3 resulted in decreased repressive histone modifications H3K9me2 and H3K27me2 at the hepta-repeat. Associated with this decrease were 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, H3K27me2 and a single-loop structure like in wild-type B′ were retained. Surprisingly, high CG and CHG methylation levels at the B′ hepta-repeat remained in all three mutants, and CHH methylation was low in both wild type and mutants. Our results raise the possibility of MOP factors mediating RNA-directed histone methylation rather than RNA-directed DNA methylation at the b1 locus.