Extended Data Fig. 9. Distinct transcriptional responses follow treatment with epigenetic inhibitors.

a) Overrepresented GO terms of biological processes for differentially up- or down-regulated gene sets following single and dual inhibitor treatments. Genes up-regulated upon DNMT1i treatment are enriched in germline-associated processes while genes up-regulated upon loss of H3K27me3 are associated with morphogenesis. Treatment with both inhibitors leads to a discrete response affecting genes involved in cell cycle regulation and chromosome segregation. b) Clustering of knockout, wild type and inhibitor samples based on their RNA-seq profiles (see Methods). c) Distribution of log2-transformed TPMs for specific gene sets (differentially expressed genes in our seven day inhibitor treatments or genes associated with hypermethylated CGIs, number of genes indicated in figure panel). Eed KO mimics the transcriptional response to the EZH2i treatment, as do our PRC1 knockouts. Loss of either or both repressive pathways does not lead to expression of genes associated with hypermethylated CGIs, although a subtle upward trend can be observed after double treatment. Lines denote the median, edges denote the IQR and whiskers denote either 1.5 × IQR and minima/maxima are represented by dots. d) Heatmaps of MINUTE-ChIP signal and DNA methylation in WT TSCs at significantly up- or down-regulated genes after 7 days of inhibitor treatment. Genes up-regulated after treatment with DNMT1i are mostly methylated in TSCs and become expressed after inhibitor-triggered loss of methylation. In contrast, neither EZH2i nor dual inhibitor treatment seem to affect the expression of genes with hypermethylated promoter CGIs. EZH2i sensitive genes show no substantial enrichment for H3K27me3, H2AK119ub1 or DNA methylation and therefore may be more indicative of indirect responses.