Fig. 5

circRHOT1 recruits TIP60 to initiate NR2F6 expression. a ChIP assays indicated that TIP60 is associated with the NR2F6 promoter on the same region as circRHOT1. b, c TIP60 knockout significantly reduced the enrichment of H2K27Ac (b) and RNA pol II (c) on the NR2F6 promoter. d, e TIP60 knockout significantly inhibited the mRNA (d) and protein levels (e) of NR2F6 in HCC cells. f, g RNA IP (f) and RNA FISH (g) results showed that circRHOT1 knockout suppresses the association of TIP60 with the NR2F6 promoter in HCC cells. Scale bar, 10 μm. h The indicated HCC cells were lysed and treated with 1% formaldehyde for crosslinking. Next, anti-TIP60 was incubated with the treated lysates for ChIP assays, followed by size fractionation via sucrose gradient ultracentrifugation. The eluate gradients were examined by western blotting and PCR assays. Pro: promoter. i circRHOT1 and TIP60 double knockout suppressed the mRNA level of NR2F6 more seriously. j The protein levels of TIP60 and NR2F6 were determined by western blotting in HCC tissues. k The expression of NR2F6 was inversely correlated with the expression of circRHOT1 and TIP60 in HCC tissues by qRT-PCR and according to the GSE45436 dataset. ***p < 0.001. All the data are representative of at least three independent experiments and presented as the means ± SD