Fig. 5: CLSY3 and CLSY4 play a disproportionately strong role in shaping the epigenome.

a and b Heatmaps showing the WT_av levels of 24nt-siRNAs and DNA methylation for each tissue at the combined set of 24nt-siRNA clusters reduced in either the clsy1,2 (a) or clsy3,4 (b) mutants. The heatmaps were clustered (grey scale) into 8 groups based on their 24nt-siRNA levels and the CHH methylation levels at these regions are shown in the same order to the right. c Boxplots showing the coefficient of variation (CV) between the 24nt-siRNA expression levels (log₂ fpkm+1) or percent CHH methylation (% mCHH) at the regions shown in a and b. Comparisons were made including all four tissues (Fl, Rs, Lv, and Ov) or just three tissues (Ov excluded). P-values based on two-sided student’s t-tests are indicated for each clsy mutant pair below each graph. d Bar graphs showing the number of 24nt-siRNA clusters (1k = 1000) dependent on clsy1,2 or clsy3,4 that reside in the chromosome arms versus pericentromeric heterochromatin, as designated in Yelina et al.⁶⁹. The percent of clusters in each category is indicated above. Violin plots showing the distribution for 1000 permutations of randomly selected 24nt-siRNA clusters from the master set of clusters (12,939). For the clsy1,2 and clsy3,4 loci each permutation included 8629 or 2739 clusters, respectively. For each tissue, the observed percentage of 24nt-siRNA clusters located in the chromosome arms are shown as triangles, with an asterisks indicating significant deviations from the random distributions (* for P-values < 0.001 and ns for P-values of 0.695 and 0.106 for the ovule and leaf data at clsy3,4 loci, respectively). e Distributions of reduced 24nt-siRNA clusters in the clsy1,2 or clsy3,4 mutants along the 5 chromosomes. The pericentromeric heterochromatin is marked in red and denoted by vertical dashed lines. Scale bars for x-axis (Mb) and y-axis (clusters/100 kb bin) are indicated in the lower left corner and the tissue types are indicated in the upper left corner of each track. f Plot showing the expression levels [log₂ FC (mut/WT)] of the 326 pol-iv-upregulated transcripts represented as horizontal lines (log₂ FC ≥ 1.5 and FDR ≤ 0.05) or as dots (not significantly upregulated; n.s.) in the four tissues. The horizontal lines are colored based on the expression level in pol-iv from flower tissue and the number of DE loci are indicated above each column.