Methylcytosine oxidation derivatives are preferentially associated with repetitive chromatin and mark active rDNA in Silene latifolia.

INTRODUCTION

DNA methylation and its oxidative derivatives are key components of epigenetic regulation, yet their roles in plant repetitive DNA remain poorly understood. Here, we investigate the genomic distribution of oxidized cytosine modifications: 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) within the repeat-rich genome of Silene latifolia, a dioecious plant with heteromorphic sex chromosomes.

MATERIALS AND

METHODS

Using low-coverage oxi-mC sequencing combined with RepeatExplorer2 clustering, ChIP-seq Mapper analysis, genome mapping, and immunoFISH validation, we identify widespread association of oxi-mCs across major repeat classes, including LTR retrotransposons, DNA transposons, satellite repeats, and ribosomal DNA (rDNA).

RESULTS

While transposable elements largely reflect genomic abundance, satellite DNA shows selective and context-dependent overrepresentation, particularly within the major centromeric repeat clusters (STAR-C), which exhibits association with 5caC in specific chromosome-biased clusters rather than across centromeres globally. In contrast, 45S rDNA arrays display strong and consistent accumulation of oxi-mCs, especially 5fC and 5caC, in transcriptionally active nucleolar organizing regions, supported by both sequencing and cytological evidence.

DISCUSSION: These findings suggest that oxidative cytosine modifications are not randomly distributed but are linked to chromatin state and transcriptional activity, particularly in rDNA loci. Overall, our results reveal cytosine oxidation as a potential additional layer of epigenetic regulation in plant repetitive DNA.