Figure 5: KLF4 binds YAP1 promoter and recruits the BRG1-embedded BAF complex to initiate YAP1 expression.

(a) HCC primary spheres were collected for co-IP assays with KLF4 antibody. (b) ChIP assay was performed using Klf4 antibody. (c) The interaction of KLF4 with YAP1 promoter was verified by EMSA assay. (d) HCC primary sphere cells were crosslinked with formaldehyde for ChIP assay with KLF4 antibody, followed by glycerol gradient ultracentrifugation. Elution gradients were concentrated for western blotting (upper panels) and PCR (lower panels) analyses. (e,f) KLF4 KO cells were established using CRISPR/Cas9 technology and allowed for sphere formation, followed by ChIP assay using BRG1 (e) and H3K4me3 (f) antibodies. (g) BRG1 was overexpressed in YAP1 promoter mutant (YAP1p Mut) and wild-type (WT) cells, followed by sphere formation. Oncosphere cells were used for ChIP assays with H3K4me3 and H3K27ac antibodies, followed by examination for YAP1 promoter enrichment with real-time PCR. (h) LncBRM or BRG1 was overexpressed in YAP1 promoter mutant and WT cells and collected for RNA extraction. YAP1 mRNA expression was detected with northern blotting. ACTB served as a loading control. Data are shown as means±s.d. Two-tailed Student’s t-test was used for statistical analysis; *P<0.05, **P<0.01 and ***P<0.001. Data are representative of three independent experiments.