Sequence and epigenetic landscapes of active and silent nucleolus organizer regions in Arabidopsis

Arabidopsis thaliana has two ribosomal RNA (rRNA) gene loci, nucleolus organizer regions NOR2 and NOR4, whose complete sequences are missing in current genome assemblies. Ultralong DNA sequences assembled using an unconventional approach yielded ~5.5- and 3.9-Mbp sequences for NOR2 and NOR4 in the reference strain, Col-0. The distinct rRNA gene subtype compositions of the NORs enabled the positional mapping of their active and inactive regions, using RNA sequencing to identify subtype-specific transcripts and DNA sequencing to identify subtypes associated with flow-sorted nucleoli. Comparisons of wild-type and silencing-defective plants revealed that most rRNA gene activity occurs in the central region of NOR4, whereas most, but not all, genes of NOR2 are epigenetically silenced. Intervals of low CG and CHG methylation overlap regions where gene activity and gene subtype homogenization are high. Collectively, the data reveal the genetic and epigenetic landscapes underlying nucleolar dominance (differential NOR activity) and implicate transcription as a driver of rRNA gene concerted evolution.