Identifying REST-bound silencers

We first defined REST+ cCREs by selecting all cCREs that contained a REST motif site and overlapped the summits of at least five ChIP-seq peaks. We then analyzed REST+ cCREs within each underlying cCRE class (e.g., promoter, distal enhancer, proximal enhancer, CA-TF) using class-matched functional, chromatin, and expression evidence. REST-bound promoter cCREs were excluded because they showed elevated transcriptional activity and nearby gene expression inconsistent with a repressive role. Among the remaining classes, distal enhancers and a small subset of proximal enhancers uniquely showed evidence of enhancer activity in neuronal contexts together with silencer activity in REST-expressing cells. All other REST+ cCRE classes exhibited exclusively silencer-like behavior. Based on these consistent, class-specific patterns, we consolidated REST+ cCREs into two primary functional categories: REST+ enhancer/silencer cCREs and REST+ silencer cCREs.

Although both classes are associated with neuronal gene regulation, they differ in key sequence and evolutionary features. REST+ enhancer/silencer cCREs show modest enrichment for enhancer-associated transcription factor motifs and are more evolutionarily conserved in flanking regions than REST+ silencer cCREs. In contrast, REST+ silencer cCREs are more enriched for long interspersed nuclear elements (particularly L1), suggesting more recent evolutionary origins. These differences imply that REST+ enhancer/silencer cCREs may represent older regulatory elements that retain both activating and repressive potential, whereas REST+ silencer cCREs may encode repression more exclusively. Further work will be needed to define the mechanistic basis of these distinctions.

The transgenic mouse enhancer assay measures enhancer activity at a specific developmental time point (E11.5) and in a limited set of tissues. Some REST+ enhancer/silencer cCREs may function as enhancers at other developmental stages, in different tissues, or under specific cellular conditions not captured by this assay. Importantly, the overall enhancer validation rate for REST+ enhancer/silencer cCREs is comparable to that of class-matched enhancers lacking REST binding, supporting the conclusion that these elements have bona fide enhancer potential despite context-dependent activity.

Yes. While REST is best known for repressing neuronal genes in non-neuronal cells, REST+ enhancer/silencer cCREs also show enrichment for active chromatin signatures in select non-neuronal contexts. These include endocrine tissues such as the pancreas, where REST plays a known role in regulating cell differentiation, as well as specific cancer cell lines such as HCT116. Although the functional significance of REST-associated enhancers in these non-neuronal contexts is not yet fully understood, their reproducible chromatin signatures suggest broader roles for REST-mediated regulation that warrant further investigation.