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Fig. 1 | Epigenetics & Chromatin

Fig. 1

From: Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner

Fig. 1

Hit-and-run epigenetic editing by Ezh2-dCas9, but not KRAB-dCas9, with targeted DNA methylation establishes and maintains a local heterochromatic environment. a Schematic representation of epi-dCas9. dCas9 fusions to effector domains (ED) contained N-terminal and C-terminal nuclear localization domains (NLSs), as well as an N-terminal 3XFLAG epitope tag with a 15-aa linker [(GGS)5] separating dCas9 and the EDs. Effector domains KRAB, Ezh2 and DNMT3A with domains are shown next to epigenetic marks they induce. A detailed view around the human HER2 promoter region shows an annotated CpG island (green bar), gRNAs targeting HER2 promoter region (blue boxes) and the regions interrogated by bisulfite cloning (gray bar) and ChIP-qPCR (black bar). The transcription start site (TSS) is indicated. UCSC browser tracks of DNAse1 hypersensitivity and H3K27ac ChIP-seq enrichment at the HER2 locus (human genome assembly hg19) are also shown. b Diagram of experimental design to assay for long-term repression using flow cytometry to select for low HER2 expression (HER2−) 4 days after transient transfection assays. Relative epi-dCas9 expression was determined by RT-qPCR in HCT116 cells after co-transfection of plasmids expressing dCas9,KRAB-dCas9 and Ezh2-dCas9 with three gRNAs targeted to the HER2 promoter (Dunnett’s test P < 0.001; n = 3 independent experiments per epi-dCas9 fusion; mean ± SEM). Representative flow cytometry data for HER2 expression in HCT116 cells treated with plasmids expressing Ezh2 + D3A + D3L (Ezh2-dCas9, D3A-dCas9 and overexpressed D3L) or KRAB + D3A + D3L (KRAB-dCas9, D3A-dCas9 and overexpressed D3L) in the presence of three gRNAs targeting the HER2 promoter. HER2− cells collected are indicated. Untreated control to identify HER2+ population is shown on the left. c Long-term repression of HER2 mRNA levels in HER2− cells was monitored for 50 days using RT-qPCR after co-transfection of plasmids expressing the indicated epi-dCas9 fusions and three gRNAs targeted to the HER2 promoter (n = 3 independent experiments each; mean ± SEM). d Average methylation levels were determined by bisulfite cloning at the HER2 promoter 10 days after transfection with the indicated Ezh2-dCas9 and KRAB-dCas9 cocktails or dCas9 (no effector domain). Each line represents a single clone, filled and empty circles represent a single methylated or unmethylated CpG, respectively. Average % methylation is indicated for each treatment. e Methylation level of CpG probes along 7 kb of the HER2 locus 17 days after HCT116 cells were co-transfected with indicated epi-dCas9 and three gRNAs targeting the HER2 promoter. CpG Island is shown in green. f Transient (4 days) and persistent (24 days) histone methylation was determined by ChIP-qPCR at the HER2 promoter in HCT116 cells co-transfected with either dCas9 with no ED or the indicated epi-dCas9 treatment cocktail and three gRNAs targeted to the HER2 promoter (Student’s t test, *P < 0.05, ***P < 0.001; n = 2–4; mean ± SEM). Due to higher cell number required for ChIP-qPCR, cells were cross-linked immediately after puromycin selection (4 days); sort for HER2− cells by FACS was omitted. H3K27me3 and H3K9me3 ChIP enrichment was assayed for Ezh2 + D3A + D3L and KRAB + D3A + D3L, respectively and compared to dCas9 with no ED. Decrease of H3K27ac enrichment was measured by ChIP-qPCR at the HER2 promoter in HCT116 at 4 and 24 days after co-transfection with three gRNAs targeted to the HER2 promoter and dCas9 with no ED or the epi-dCas9 treatment cocktail Ezh2 + D3A + D3L (Student’s t test, **P < 0.01, *P < 0.05; n = 2–3; mean ± SEM)

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