Building on our analysis of REST-associated silencers, we next asked whether silencer activity could be identified genome-wide using functional data alone. Using CAPRA to analyze genome-wide STARR-seq data, we identified thousands of cCREs with reproducible negative activity scores, consistent with transcriptional repression. These STARR-defined silencers extend beyond canonical NRSEs and are enriched within newly added cCRE classes, underscoring the importance of an expanded classification framework for capturing diverse repressive regulatory elements.
Frequently Asked Questions
WG-STARR-seq assays were originally designed to detect enhancer activity using a minimal promoter, and therefore may miss silencers that require a strong or specific promoter context to exert repression. Dedicated silencer assays have recently been developed to address this limitation. However, the advantage of using a single, widely applied assay is that it enables systematic, genome-wide comparison of activating and repressive activities within a unified experimental framework. This consistency allows us to directly contrast enhancers, silencers, and dual-function elements using the same underlying data.
Although STARR-seq assays regulatory activity outside of native chromatin context, multiple lines of evidence support the biological relevance of STARR silencer cCREs. Genes located near these elements show reduced expression in K562 cells, consistent with a repressive role. In addition, integration with CRISPRi-FlowFISH data identified cCREs that act as silencers when perturbed in their endogenous genomic context. One such example overlaps both a STARR silencer and a REST-associated enhancer/silencer and represses gene expression through long-range chromatin interactions. Together, these results indicate that at least a subset of STARR silencers function as bona fide silencers in vivo.
Genes proximal to STARR silencer cCREs are enriched for tissue- and lineage-specific developmental programs, including nervous system and renal development. This pattern suggests that silencers play an important role in repressing context-specific gene expression programs in inappropriate cellular environments, complementing the role of enhancers in activating lineage-specific transcription.