A novel approach combining immunodetection of target proteins, freshly replicated DNA (BrdU-labelled), nascent RNA molecules (BrUTP-labelled), and fluorescent in situ hybridisation (FISH) in plant cell nuclei flow-sorted based on their DNA content allowed to follow the cell cycle dynamics of chromatin modifications. A pronounced modulation of histone acetylation was detected at the level of large chromatin domains (euchromatin, heterochromatin, nucleoli) in plants. Heterochromatin, being hypoacetylated during the most of interphase became strongly acetylated during its replication time in late S and deacetylated again towards mitosis. Also euchromatin showed increased levels of histone acetylation during S-phase. This evolutionarily conserved global replication-dependent acetylation, masking fine-tuned acetylation of transcriptionally promoters, is probably required for post-replicative DNA repair and DNA maintenance methylation. Using Arabidopsis mutants with defects in DNA methylation (met1, ddm1) and H3K9 methylation (kyp) allowed to elucidate the relationship between DNA maintenance (CpG) methylation and H3K9 methylation during post-replicative heterochromatin re-assembly. Heterochromatic chromocenters, smaller in both DNA hypomethylation mutants due to the dispersion of pericentromeric sequences, revealed decreased levels of DNA and H3K9 dimethylation. In kyp, chromocenters lacking high levels of H3 dimethylK9, were of wild-type appearance and contained high levels of DNA methylation. These data suggest that the absence of KYP histone methyltransferase does not affect CpG-methylation, and in contrast to CpG-methylated repeats, dimethylated H3K9 is dispensable for proper formation and maintenance of constitutive heterochromatin in Arabidopsis. |