Figure 6.

dCas9‐KRAB‐mediated silencing of ZEB1 reprograms the transcriptional and epigenetic state of mesenchymal breast cancer cells. (A) Significance (‐log₁₀(FDR)) for the top biological terms obtained from Gene Ontology analysis using 87 638 differentially methylated probes mapping to 17 052 genes in SUM159. Bars are colored by the proportion of differentially methylated probes relative to the total number of probes associated with this gene set (DE/N). Several are enriched in cell–cell interaction and cell adhesion suggesting a role in MET. (B) Changes in transcript abundance (log₂(fold change); logFC_RNA) and probe‐level changes in methylation (logFC_Meth) for 26 genes that show differential transcript abundance between the All gRNA treatment and No gRNA in SUM159 cells, which also have at least one differentially‐methylated probe. Genes are annotated with chromatin accessibility (ATAC‐seq) data and whether or not they are known EMT markers (at left), and are ordered based on RNA‐seq logFC. Probes scatter markers are colored by TSS (orange) or gene‐body (green) annotation. (C) Transcript abundance for selected genes within treated MDA‐MB‐231‐luc tumor samples (condition at bottom) as measured by qRT‐PCR, normalized relative to untransduced wild type tumor samples. (D) Native chromatin immunoprecipitation (ChIP) and assay of transposase accessible chromatin (ATAC) sequencing identifying epigenetic changes at the ZEB1 promoter of All gRNA and No gRNA SUM159 cells. (E) ChIP seq Counts per million (CPM) mapped reads significance in H3K4me3 and H3K9me3 at the ZEB1 promoter peak where ****p ≤ 0.0001. LogFC: log fold‐change, FC: fold‐change, WT: wild type untransduced sample, Epi: epithelial, Mes: mesenchymal, TSS: transcriptional start site, rep: replicate, H3K4me3: histone H3 trimethylation at fourth lysine residue, H3K9me3: histone H3 trimethylation at ninth lysine residue.