Figure 3. A genome-wide analyses of endo-siRNAs that depend on SET-25 or SET-32.

(A) An expression analysis of endo-siRNAs targeting transposons and repetitive elements classes (left panel) or protein-coding genes (right panel). Shown are the expression values as log2 of number of Reads per Million (RPM) in set-25 mutants (y-axis) compared to wild type animals (x-axis). Gene targets of endo-siRNAs, which display significant differential expression (analyzed with Deseq2, adjusted p-value<0.1) are marked in Red (B) An analysis of H3K9me3 signals (based on published data from McMurchy et al., 2017) on differet sets of gens: highly expressed genes (top 10%, Blue), lowly expressed genes (top 10%, Yellow) and gene targets of endo-siRNAs that depend on SET-25 (based on Lev et al., 2017, Red), SET-32 (based on Kalinava et al., 2018), Dark Red) or endo-siRNAs associated with WAGOs (HRDE-1,WAGO-1, and ERGO-1, Light Red). H3K9me3 signal is aligned according to gene's Transcription Start Sites (TSS), and the regions of 1000 base pairs upstream and downstream of the TSS are shown on the x axis. The y axis shows the averaged signal of the H3K9me3 modification as a function of distance from the TSS. For statistical analysis, control data sets (shown in Gray) were created by sampling the H3K9me3 levels of randomly selected gene sets of the same size as the examined gene list. (C and D) An enrichment analysis of genes with significantly lowered levels of endo-siRNAs targeting them in set-25 and set-32 mutants compared to wild type. Fold enrichment values (log2 scale) are color coded. (C) An enrichment analysis for expression in specific tissues. (D) An enrichment analysis for different small RNA pathways. The p-values were calculated using 10,000 random gene sets identical in their size to the examined endo-siRNA-target gene list. Asterisk denotes statistically significant enrichment values (p-value<0.05).

Figure 3—figure supplement 1. An analysis of genes targeted by endo-siRNAs upregulated in set-25 and set-32 mutants.

(A) An analysis of H3K9me3 signals (based on published data from McMurchy et al., 2017) on gene targets of endo-siRNAs up-regulated in set-25 mutants (based on Lev et al., 2017, left panel) and in set-32 mutants (based on Kalinava et al., 2018). All genes are aligned according to their Transcription Start Sites (TSS), and the regions of 1000 base pairs upstream and downstream of the TSS are shown on the x axis. The y axis shows the averaged signal of the H3K9me3 modification as a function of distance from the TSS. (B) An analysis of H3K9me3 signals (based on Kalinava et al., 2018) on gene targets of SET-32 dependent endo-siRNAs. Genes are aligned according to their TSS, and the regions of 1000 base pairs upstream and downstream of the TSS are shown on the x axis. The y axis shows the averaged signal of the H3K9me3 modification in wild type (Blue) and set-32 mutants (Red). For statistical analysis control data sets (Gray) were created by sampling the H3K9me3 levels of randomly selected gene sets of the same size as the examined gene list. (C) An enrichment analysis for expression in specific tissues or association with different small RNA pathways. Fold enrichment values are color coded. For statistical analysis, p-values were calculated using 10,000 random gene sets identical in their size to the examined gene set. Asterisk denotes statistically significant enrichment values (p-value<0.05).

Figure 3—figure supplement 2. An analysis of overlap between different endo-siRNA target gene sets.

(A) A Venn diagram analysis of the overlap between gene targets of HRDE-1-bound endo-siRNAs (Buckley et al., 2012) and germline expressed genes (Ortiz et al., 2014). (B) A Venn diagram comparison of gene targets of endo-siRNAs that depend on either SET-25 or SET-32 and genes targeted by known endo-siRNA pathways: HRDE-1 (Buckley et al., 2012), Mutator pathway (Phillips et al., 2014), ERGO-1 dependent small RNA targets (Vasale et al., 2010) and endo-siRNAs dependent on either SET-25 (Lev et al., 2017) or SET-32 (Kalinava et al., 2018).