The 11th International Symposium on 3D Genomics

Human silencers are crucial in regulating developmental gene expression, yet the function and mechanism of the recently identified "super-silencers" are not well understood, especially regarding the working modes of their component silencers. Focusing on two such components, silencer1 (S1) and silencer2 (S2), within a "super-silencer" at the FGF18 gene locus, this study explores their potential working model. Using CRISPR-Cas9 technology, we created individual knockouts for S1 (S1KO) and S2 (S2KO), as well as a combinatorial double KO (DKO). Our results showed that both S1KO and S2KO led to an upregulation in FGF18 expression, and DKO showed an upregulation that surpassed the sum of S1KO and S2KO effects. This suggests that S1 and S2 components may work synergistically as a "super-silencer". Furthermore, this synergy was not limited to gene expression regulation. DKO also exhibited synergistic effects on phenotypic changes and growth inhibition. Interestingly, these silencer components primarily influenced genes and loops within its vicinity, which is potentially linked to the recruitment of additional CTCF binding sites and altered chromatin accessibility. Overall, our findings provide the first demonstrated example of a "super-silencer", revealing that silencer components can act synergistically as a "super-silencer" to repress key genes, such as FGF18, crucial for maintaining cancer cell identity. This highlights the significant potential of "super-silencers" in oncological research and opens new avenues for exploring gene regulation mechanisms in cancer.