Chromosome termini form a specialized type of heterochromatin that is important for chromosome stability. The recent discovery of telomeric RNA transcripts in yeast and vertebrates raised the question of whether RNA–based mechanisms are involved in the formation of telomeric heterochromatin. In this study, we performed detailed analysis of chromatin structure and RNA transcription at chromosome termini in Arabidopsis. Arabidopsis telomeres display features of intermediate heterochromatin that does not extensively spread to subtelomeric regions which encode transcriptionally active genes. We also found telomeric repeat–containing transcripts arising from telomeres and centromeric loci, a portion of which are processed into small interfering RNAs. These telomeric siRNAs contribute to the maintenance of telomeric chromatin through promoting methylation of asymmetric cytosines in telomeric (CCCTAAA)n repeats. The formation of telomeric siRNAs and methylation of telomeres relies on the RNA–dependent DNA methylation pathway. The loss of telomeric DNA methylation in rdr2 mutants is accompanied by only a modest effect on histone heterochromatic marks, indicating that maintenance of telomeric heterochromatin in Arabidopsis is reinforced by several independent mechanisms. In conclusion, this study provides evidence for an siRNA–directed mechanism of chromatin maintenance at telomeres in Arabidopsis.

Highlights d A database combining genomic information and chromatin profiles for Marchantia d Correlations between chromatin marks and transcription are conserved in land plants d A significant portion of constitutive heterochromatin is marked by H3K27me3 d Insights into the evolution of TAD organization in plants

Telomeres in mammals and plants are protected by the terminal t loop structure, the formation of which parallels the first steps of intrachromatid homologous recombination (HR). Under some circumstances, cells can also utilize an HR-based mechanism (alternative lengthening of telomeres [ALT]) as a back-up pathway for telomere maintenance. We have found that the Ku70/80 heterodimer, a central nonhomologous end-joining DNA repair factor, inhibits engagement of ALT in Arabidopsis telomerase-negative cells. To further assess HR activities at telomeres, we have developed a sensitive assay for detecting extrachromosomal telomeric circles (t circles) that may arise from t loop resolution and aberrant HR. We show that Ku70/80 specifically inhibits circle formation at telomeres, but not at centromeric and rDNA repeats. Ku inactivation results in increased formation of t circles that represent approximately 4% of total telomeric DNA. However, telomeres in ku mutants are fully functional, indicating that telomerase efficiently heals ongoing terminal deletions arising from excision of the t circles.