Fig. 5

PKL is required for RNA Pol V-dependent noncoding RNA accumulation and nucleosome occupancy. a Non-coding RNA levels at six IGN loci were examined by real-time PCR. No RT (reverse transcriptase) samples serve as controls for genomic DNA contamination. All the transcript levels are shown on a relative scale with the level in WT (Col-0) plants being set to one. Error bars represent standard deviations calculated from three biological replicates. b Diagram showing the IGN5 locus on chromosome 4. Arrows above and below the coordinates indicate the position and direction where Pol V-dependent transcripts start. Positions of amplicons used for assaying nucleosome density in (d) were indicated by black lines labeled as A1 through A11. c A screen shot of IGV (Integrative Genomics Viewer) showing DNA methylation levels at the IGN5 locus. The colored bars (red, blue, green) represent the methylation levels of specific cytosines on the DNA double strands on a scale from –1 to 1; minus values indicate the methylated cytosine is on the reverse strand. d Nucleosome densities at the IGN5 locus assayed by anti-histone H3 ChIP. Error bars indicate standard deviations calculated from three biological replicates. All the signals are normalized to the ACT2 + 1 nucleosome; stars indicate p < 0.05 between the mutant and WT (Col-0) based on two-tailed t-tests. e PKL affects the positioning of Pol V-stabilized nucleosomes (PVS). Nucleosome positioning was examined by histone H3 ChIP following micrococcal nuclease digestion of the chromatin. The +1 nucleosome at HSP70 served as a negative control. Error bars represent standard deviation calculated from three biological replicates