Figure 6. Epigenetic regulation of TBXT occurs independently of an ATF4-stress response and is key to the lethality induced by H3K27 demethylase inhibitors.

(A-B) ATF4, DDIT3, INHBE and TBXT regulation over a time course in response to KDOBA67 in UCH1, by qPCR of nascent RNA (A) and total RNA (B). Two independent experiments, three replicates per condition each experiment. Results for total RNA for UM-Chor and MUG-Chor are reported in Figure S7. (C) Volcano plot summarising differential gene expression by RNA-seq in UCH1 cells treated with KDOBA67 for 48 hours in the presence or absence of ISRIB. Blue, not significant; Orange, P_adj <0.05. Three biological replicates. (D) Proliferation of UCH1 cells in response to treatment with KDOBA67 for 48 hours in the presence or absence of ISRIB. Three independent experiments, three replicates per condition per experiment. (E) Venn diagram showing the number of genes regulated by KDOBA67 treatment in UCH1 cells (by RNA-seq, Figure 2) that overlap with genes previously identified as direct targets of TBXT in UCH1 cells using ChIP-seq in (20). (F) Overexpression of TBXT-HA in UCH1 cells is not affected by KDOBA67 treatment: similar levels of HA by western blot in cells treated with KDOBA67 or DMSO. (G) Overexpression of TBXT-HA or control EV in UCH1 cells is achieved in >95% of cells: expression of dTomato by fluorescence microscopy of EV and TBXT-HA cells treated with KDOBA67 or DMSO (representative images, scale bar 400μm). (H) Overexpression of TBXT-HA mitigates the reduction in cell viability in response to treatment with KDOBA67 for four days in UCH1 and UM-Chor. 4 replicates per condition in three (UCH1) and two (UM-Chor) independent experiments. *p ≤0.05, **p ≤0.01, ***p ≤0.001, ****p ≤0.0001.