DNA hypomethylation reduces homologous pairing of inserted tandem repeat arrays in somatic nuclei of Arabidopsis thaliana

*† These authors contributed equally to the paper. Summary Fluorescent chromatin tagging makes possible tracking of specific loci in vivo and in situ. Loci tagged by the lac operator (lacO)/GFP-LacI/Nuclear Localization Signal (NLS) system show rapid motility and constrained chromatin dynamics in somatic nuclei of a transgenic line, designated EL702C, in Arabidopsis thaliana. The tagged loci associated with each other significantly more often than expected at random, due to homologous pairing of the lacO tandem repeat arrays. Furthermore, these arrays associated significantly more often than average euchromatic regions with heterochromatic chromocenters (CCs). We show now that the inserted lacO array in this transgenic line became strongly methylated at CG sites in the T3 generation, which can be reversed upon transfer into the mutant backgrounds of decrease in DNA methylation 1 (ddm1) and methyltransferase 1 (met1). Concomitantly, the tagged loci showed lower association frequencies as compared with the transgenics in wild-type background, which is correlated with a significant decrease in allelic and ectopic pairing of the lacO repeat arrays as visualized by fluorescence in situ hybridization. In contrast, the preferential association of the lacO arrays with heterochromatin, locus mobility in somatic nuclei and transcription of neighboring transgenes were not altered by reduced DNA methylation in ddm1 and met1 backgrounds. Our results show that repeat arrays can activate hypermethylation of the inserted locus that correlates with high frequencies of homologous pairing in somatic cells. In contrast, the preferential association of these inserted arrays with CCs in plant cells occurs through another mechanism.