Fig. 4.

Functional importance of TOX2-SE in NKTL cells. A Enhancer activity was identified in a reporter assay for TOX2-eNC (a low H3K27Ac region outside of TOX2-SE on Chr20), TOX2-e1, TOX2-e2 and TOX2-e3 regions, respectively. The position of each region on chr20 was indicated (not in size scale). Enhancer activity is expressed as relative fold change of TOX2-SE regions (-e1, -e2, -e3) vs control region (-eNC). Three biologically independent assays were performed. Error bars represent SD. **p < 0.001. B A schematic diagram of the pairs of sgRNAs designed to target 3 valley bases (P1, P2 and P3) on H3K27Ac track of the SE region of TOX2. Two pairs of sgRNAs were used to direct the dCas9-KRAB transcription repression system to target 2 sites of each valley base (T1-2 for P1; T3-4 for P2; T5-6 for P3). C Decreased mRNA expression of TOX2 target gene after activation of pairs of sgRNAs (T1, T3-6) guided dCas9–KRAB repression system targeting the TOX2-SE region (n = 3 biologically independent samples of NKYS cells). dCas9: stable NKYS-dCas9 cells without pairs of sgRNAs. Dox: doxycycline. Student’s t-test was applied for all statistical comparisons of TOX2 expression in cells + Dox versus -Dox (**p < 0.01). D TOX2, PRL-3, and apoptosis-related proteins were analyzed by Western blot in NKYS-dCas9 cells after transfected with pairs of sgRNA in condition of + Dox or -Dox. Detection of β-actin protein was used as an internal loading control. Three independent experiments were conducted and representative blot images were shown. E Cell proliferation assays with different pairs of sgRNA transfected NKYS-dCas9 cells with or without Dox induction. The number of cells over 9 days was recorded under each condition as indicated. Data of three biological replicates (mean ± SD) were used to construct these growth curves. **p < 0.001 for the different of -Dox versus + Dox group