TRIM28 recruitment on HERVK leads to repression and methylation of an adjacent promoter in hES. (A) Sequences corresponding to TRIM28-bound regions of two (R1 and R2) HERVKs only expressed when TRIM28 was depleted, or the corresponding 600-bp-long fragment of three (NR1–NR3) HERVKs expressed in WT cells were cloned in the antisense direction in depicted LV. The resulting LVs were used to transduce hESC in duplicates, and GFP expression was monitored over time by FACS. The average and SD of the duplicates are shown. (NT) Nontransduced. (B) Similar experiment in LV-shE transduced (WT) versus TRIM28 KD cells. Repression is TRIM28-dependent. The average and SD of the duplicates are shown. (C) Similar experiment with a 39-bp-long, PBS-encompassing HERVK fragment, sufficient to induce TRIM28-mediated repression. Alignment of PBS R1- and NR1-derived sequences is on top, with mismatches highlighted in yellow. (D) ChIP-qPCR (4 d post-transduction) showing that TRIM28 is recruited and H3K9me3 deposited on ERE (top panel) and PRKG1 (bottom panel) sequences of proviruses from the repressed LV-HERVK-R but not from the repression-resistant LV-HERVK-NR. Immunoprecipitates were normalized to their respective total input and enrichment on the positive control ZNF180. Results represent two independent experiments with technical replicates (n = 3) for TRIM28 ChIP and technical replicates (n = 3) for H3K9 ChIP. (**) P-value ≤ 0.01, (***) P-value ≤ 0.001. (Ctrol) LV devoid of ERE fragment. (E) Human ES or 293T cells were transduced with LV.HERVK.R or NR, and 2 wk later, percentage of de novo methylation at each tested CpG (n = 8) of the PRKG1 promoter was measured by bisulfite quantitative pyrosequencing. (***) P-value < 0.0001.
