Figure 3.

TEs Are Repressed by a DICER Mechanism

(A) Knockdown (KD) of RNAi players. Left: schematic of siRNA KD in Dnmt1 cKO ESCs. The genome gets demethylated (5mC, orange) and IAPs get transcriptionally activated and resilenced (red) if small RNAs are present (gray); however, KD of the RNAi pathway will deplete small RNAs. Lower right: quantitative real-time PCR analysis showing KD efficiencies of Dicer, Ago2, and Dgcr8 upon treatment with siRNAs after Dnmt1 deletion. Upper right: expression of IAPs upon Dicer, Ago2, Dgcr8, or non-targeting siRNA transfection. The data are normalized to non-targeting control. Bars represent mean ± SD, n = 3. ^∗p < 0.05, ^∗∗p < 0.005, two-tailed Student’s t test.

(B) Small RNA-seq of Dicer/Dnmt1 cDKO and Dnmt1 cKO ESCs. Sense (orange) and antisense (blue) small RNAs are separated by size and were mapped to all TEs. Reads were normalized to non-induced WT (Dicer^fl/fl/Dnmt1^fl/fl) ESCs.

(C) Quantitative real-time PCR analysis of TE classes in ESCs following conditional Dnmt1 cKO or Dnmt1/Dicer cDKO by treatment with 4OHT or Dicer KO. Bars represent mean of two biological replicates with two technical replicates. Values were normalized to Atp5b and Hspcb, and major satellites were normalized to U1. ^∗p < 0.05, ^∗∗p < 0.005, two-tailed Student’s t test.

(D) Quantitative real-time PCR analysis of IAPEz in the indicated ESC lines. Conditional deletions were induced by treatment with 4OHT for the indicated days. Values were normalized to Atp5b and Hspcb and are relative to the respective WT sample for each KO line, indicated by dashed line. Error bars represent mean ± SD; n = 3 for Dnmt1 cKO, Dicer KO/Dnmt1 cKO, and Ago2 KO/Dnmt1 cKO; n = 2 for Dicer/Dnmt1 cDKO and Ago2/Dnmt1 cDKO. Ago2 KO/Dnmt1 cKO time points days 9 and 11 were not collected.

(E) Heatmap of unbiased hierarchical clustering of all TE classes responsive to Dicer KO. Heatmap depicts relative expression (Z score) of TEs upon Dicer KO.

See also Figures S3 and S4I and Tables S2 and S3.