Fig. 4.

Full-length DNMT3L is required and sufficient to establish long-term repression facilitated by KRAB-dCas9 and Ezh2-dCas9. Long-term repression was evaluated using three different approaches for targeted DNA methylation: a D3A-based recruitment (D3A-dCas9 + overexpressed D3L), b Co-targeting of D3A + D3L (D3A-dCas9 + dCas9-D3L) and c D3L-based recruitment (dCas9-D3L). a Comparison of the D3A-based recruitment approach with the targetable D3a3L fusion (dCas9-D3a3L), HER2 mRNA levels in HCT116 cells and Snurf mRNA levels in Neuro2A cells were determined by RT-qPCR 14 days after co-transfection of plasmids expressing the indicated epi-dCas9 fusions with the three gRNAs targeted to the HER2 or Snurf promoter (Dunnett’s test, *P < 0.05, **P < 0.01, ***P < 0.001; n = 3; mean ± SEM). b Long-term repression by co-targeting D3A and D3L fusions. Performance of D3L fusions to the N or C terminus of dCas9 was compared. HER2 mRNA levels in HCT116 cells and Snurf mRNA levels in Neuro2A cells were determined by RT-qPCR 14 days after co-transfection of plasmids expressing the indicated epi-dCas9 fusions with the three gRNAs targeted to the Snurf or HER2 promoter (Dunnett’s test, *P < 0.05, **P < 0.01, ***P < 0.001; n = 3; mean ± SEM). c Long-term repression was evaluated by RT-qPCR 14 days after co-transfection of dCas9-DNMT3L with KRAB-dCas9 or Ezh2-dCas9 with gRNAs targeted to the SNURF and HER2 promoter in LNCaP cells. Expression levels were compared to dCas9 with no ED (Dunnett’s test, *P < 0.05, **P < 0.01, ***P < 0.001; n = 3; mean ± SEM). d Schematic of overexpressed full-length DNMT3L and design of dCas9 fusions. DNMT3A or DNMT3L were fused to the N-terminus of dCas9 (D3A-dCas9 and D3L-dCas9, respectively) or the C terminus of dCas9 (dCas9-D3L). dCas9-D3a3L contains D3a and the C-terminal portion of D3L (D3a3L) fused to the N-terminus of dCas9. e Average methylation levels were determined by bisulfite cloning (left panel) and high-throughput bisulfite amplicon sequencing (right panel) at the HER2 promoter 10 days after transfection with the indicated Ezh2-dCas9 and KRAB-dCas9 cocktails or dCas9 (no effector domain). Methylation after treatment with only dCas9-D3L and three HER2 gRNAs was also evaluated. Bisulfite cloning results are displayed as lollipop plots. Each line represents a single clone, filled and empty circles represent a single methylated or unmethylated CpG, respectively. Analysis of bisulfite amplicons sequencing was performed from two biological replicates. Average % methylation is plotted for each indicated epi-dCas9 treatment. Methylation obtained with targetable dCas9-D3L (orange bars) was compared with methylation status after combinatorial KRAB-dCas9 (K) and Ezh2-dCas9 (E) with D3A-dCas9 (D3A) and overexpressed D3L (gray bars). All treatments resulted in significant HER2 target methylation when compared to cells treated with dCas9 or cells that were not transfetcted (NT) (Dunnett’s test, ***P < 0.001; n = 2; mean ± SEM)