Fig. 4

FUS promotes back-splicing program of circEZH2 and KLF5 activates FUS transcription. a RT-qPCR was used to determine the expression of circEZH2 after transfected with siRNA targeting EIF4A3, FUS, PTBP1, U2AF65 and control. b Schematic diagram showed that FUS binding downstream intron4 region of pre-EZH2. c TCGA analysis of FUS expression in different stages and subtypes of BRCA.. d Clinical BC samples from SYSUCC were used by RT-qPCR analysis revealing the spearman correlation evaluation between circEZH2 and FUS transcripts. e RT-qPCR was performed to reveal the assoicated expression of circEZH2 when FUS was overexpressed or knocked down in BC cells. f Western blots were used to determine the resultants of RNA pull-down assay using biotin-preEZH2-probe. g Diagram revealing five truncated biotinylated pre-EZH2 probes were constructed. h RNA pull down assays were performed by five truncated probes and the resultants analyzed by western-blot were displayed. i Lysates of BT-549 were subjected to RIP by anti-FUS antibody and anti-IgG antibody and the enrichments of IP and IgG groups were displayed. j Flow illustration showed that firstly pc-HA-EZH2, a novel back-splicing formation validation vector by adding a HA label to the 5′-second EZH2 exon to differentiate the internal circEZH2 was constructed. Secondly, FUS binding region was mutated of pc-HA-EZH2 as mutant type (MT). After that MT and WT vector were transfected into HEK293T with/without knockdown of FUS. Eventually, the back-splicing efficiency of circEZH2 was deternmined by divergent primers across HA tag region and RT-qPCR while linear HA-EZH2 was used as reference. k The back-splicing efficiency of circEZH2 was identified by RT-qPCR in WT and MT groups. l In SYSUCC clinical human BC tissues, expression of KLF5 was positively correlated with FUS as determined by Spearman’s correlation analysis. m RT-qPCR was used to find the associated expression of KLF5, circEZH2 and FUS in change the expression of KLF5. n Eight ChIP-seq datasets showed that there were binding peaks within 2k bp upstream promotor of FUS by KLF5. o Top, JASPAR predicted potential three binding regions between KLF5 and the promotor of FUS. Bottom, each of three binding sites were mutated in FUS promotor dual-luciferase plasmid while wild type without mutation was positive control and all mutated group was negative control. The results of each group were quantitated by dual-luciferase assays. The data was showed as the mean ± SD and all experiments were repeated at least three times, ns no significance, **P < 0.01, ***P < 0.001