Super-silencer perturbation by EZH2 and REST inhibition leads to large loss of chromatin interactions and reduction in cancer growth [Cut&Run]

Human silencers have been shown to exist and regulate developmental gene expression. However, the functional importance of human silencers needs to be elucidated, such as whether they can form “super-silencers” and whether they are linked to cancer progression. Here, through interrogating two putative silencer components of FGF18 gene, we found that two nearby silencers can cooperate via compensatory chromatin interactions to form a “super-silencer”. Furthermore, double knockout of two silencers exhibited synergistic upregulation of FGF18 expression and changes of cell identity. To perturb the “super-silencers”, we applied combinational treatment of an EZH2 inhibitor GSK343, and a REST inhibitor, X5050 (“GR”). We found that GR led to severe loss of TADs and loops, while the use of one inhibitor by itself only showed mild changes. Such changes in TADs and loops were associated with reduced CTCF and TOP2A mRNA levels. Moreover, GSK343 and X5050 synergistically upregulated super-silencer-controlled genes related to cell cycle, apoptosis and DNA damage, leading to anticancer effects both in vitro and in vivo. Overall, our data demonstrated the first example of a “super-silencer” and showed that combinational usage of GSK343 and X5050 to disrupt “super-silencers” could potentially lead to cancer ablation. Silencer knockout (KO) Hi-C data has two biological replicates for each condition including EV, S1KO, S2KO, and DKO. Drug treatment Hi-C data has two biological replicates for each drug-treated condition, including DMSO, GSK343, X5050, GR 8h, GR 24h, and GR 72h. Drug treatment Hi-C data has two biological replicates for each DMSO- and etoposide-treated K562 cells (72 hr). siRNA Hi-C data has two biological replicates for each siScramble- and siCTCF-treated K562 cells (48 h). Silencer KO 4C has two biological replicates for each condition (EV, S1KO, and DKO). Silencer KO RNA-seq has two biological replicates for different KO cells (EV, S1KO S2KO, and DKO). H3K27me3 and H3K27ac ChIP-seq for silencer KO cells (EV, S1KO, and DKO) have two biological replicates. H3K27me3 HiChIP in wild-type K562 cells has one biological replicate. Cut and Run CTCF has two biological replicates for CTCF antibodies in EV, S1KO, S2KO, and DKO cells. Cut and Run H3K27me3 has two biological replicates for H3K27me3 antibodies (24 h) in THP1 and K562 cells. ATAC-seq has two biological replicates for each EV, S1KO, S2KO, and DKO cells. ATAC-seq has two biological replicates for each DMSO- and GR-treated K562 cells (72 h). Drug-treated K562 cells RNA-seq has two biological replicates including DMSO, GSK343, X5050, GR 8 h, GR 24 h, and GR 72 h.