Multimegabase-scale DNA hypermethylation and condensed chromatin correlate with chromosome-specific rRNA gene silencing in Arabidopsis

In eukaryotes, hundreds of ribosomal RNA (rRNA) genes are clustered at chromosomal loci called nucleolus organizer regions (NORs). Arabidopsis thaliana has two NORs, one on chromosome 2 (NOR2) and the other on chromosome 4 (NOR4). Each NOR consists of ~ 400 rRNA gene copies. We recently showed that rRNA gene subtypes that map to the NOR2 are silenced during development, whereas those that map to NOR4 are active. In several DNA methylation mutants of Arabidopsis, we show release of the NOR2 silencing to varying degrees. Significantly, the highest release of NOR2 silencing correlates with a maximum loss of cytosine methylation in the CHH context followed by CG, but independent of RNA-directed DNA methylation (RdDM). Next, we show that NOR2 is relatively hypermethylated and NOR4 is hypomethylated using methylation analysis of genomic DNA carried out with different types of methylation-sensitive restriction enzymes. We demonstrate similar differential methylation status between NOR2 and NOR4 in genotypes carrying NORs from multiple ecotypes. Lastly, using Tn5 transposon-mediated transposition assay, we show that NOR2 chromatin is in more condensed state than NOR4. ### Competing Interest Statement The authors have declared no competing interest.