Fig. 3

Combinatorial treatment with Ezh2-dCas9, D3A-dCas9 and D3L establishes persistent DNA and histone methylation specifically at the HER2 target locus. a UCSC browser snapshot of H3K27ac ChIP-seq enrichment at the HER2 locus (human genome assembly hg19). Browser tracks are shown for biological replicates 24 days after co-transfection with indicated epi-dCas9 cocktail and 3 gRNAs targeting the HER2 promoter (dCas9 control in top two panels, Ezh2 + D3A + D3L treated cells in bottom two panels). Targeted HER2 promoter region containing H3K27ac peak is highlighted in yellow. RefSeq genes are indicated. b Scatterplot of the fold change of H3K27ac binding (-dCas9/Ezh2 + 3A + 3L) after co-targeting of Ezh2 + D3A + D3L (24 days) against peak center distance from the transcription start site (TSS). 469 H3K27ac binding peaks that are specific to the dCas9 control were plotted. Black dots represent gene promoters with a more than twofold reduction of H3K27ac binding. Reduction of H3K27ac binding at the HER2 promoter is indicated with a red dot. c RT-qPCR at six loci with reduced H3K27ac ChIP binding 23 days after treatment with Ezh2-dCas9/D3A-dCas9 plus D3L. Expression levels were calculated relative to GAPDH (Sidak’s multiple comparisons test, ***P < 0.001, n = 3; mean ± SEM). d Distribution of DNA methylation levels in promoter regions. Average DNA methylation levels of HCT116 cells co-transfected with plasmids expressing three gRNAs targeting the HER2 promoter, and epi-dCas9 cocktails expressing D3A-dCas9 and D3L with either KRAB-dCas9 (KRAB + D3A + D3L) or Ezh2-dCas9 (Ezh2 + D3A + D3L) are plotted against control cells transfected with same gRNAs and dCas9 with no ED. Hypermethylated HER2 CpG probes in promoter regions are colored in red, all other differentially methylated promoter CpG probes are colored in black. Promoter regions encompass 1500 bp upstream of the TSS as well as the 5′UTR. e Frequency of hypermethylated CpG probes per gene. Gene promoters containing more than three hypermethylated probes are considered differentially methylated promoters. After treatment with Ezh2 + D3A + D3L the HER2 target promoter shows strongest differential methylation represented by 12 hypermethylated promoter CpGs (red arrow). f Persistent hypermethylation of 25 gene promoters. Venn diagram depicts hypermethylated gene promoters with > 3 hypermethylated promoter probes 17 and 24 days after targeting the HER2 promoter with Ezh2-dCas9 + D3A-dCas9 + D3L. g RT-qPCR at potential off-target sites 23 days after treatment with indicated epi-dCas9. As a control, HER2 expression was knocked down by HER2 siRNA and compared to siRNA control treatment. Expression levels were calculated relative to GAPDH (Tuckey’s test, ***P < 0.001; n = 3; mean ± SEM)