Figure 3 | Identification of distinct functional categories of REST-bound cCREs. 

a, Computational pipeline for identifying REST-bound cCREs (REST+ cCREs). We overlapped cCREs with REST ChIP-seq peaks and selected all cCREs that overlap at least five peak summits and an annotated REST motif site. b, Schematic for characterizing REST+ cCREs into two categories: REST+ enhancer/silencer cCREs (N = 2,534) and REST+ silencer cCREs (N = 2,253). c, Representative result from the mouse transgenic enhancer assay showing activity of REST+ enhancer cCRE EH38E1910506 in mouse brain regions (hindbrain activity in 4/6 embryos; midbrain activity in 6/6 embryos). e, Bar graphs denoting the percentage of cCREs tested in transgenic mouse enhancer assays with positive activity. cCREs are stratified into four groups based on REST+ category (enhancer/silencer in yellow: N = 22, silencer in purple: N = 15) and REST binding (+ and dark bars indicate REST+, − and light bars indicate REST−). Statistical significance is calculated by a two-sided Fisher’s exact test between REST+ categories (N = 22 and N = 15 for enhancer/silencer and silencer, respectively) and class matched controls (N = 5,428 and N = 448). REST+ silencer cCREs are depleted for activity compared to REST+ enhancer/silencers (p = 0.001) and matched control cCREs (p = 0.02). g, Bar graphs showing the percentage of REST+ enhancer/silencer cCREs with transgenic mouse enhancer assay activity in specific tissues vs. other tested enhancers (as defined in f). Statistical significance is calculated by a two-sided Fisher’s exact test. REST+ enhancer/silencer cCREs are enriched for activity in hindbrain (p = 0.02) and midbrain (p = 0.05). h, Density plot of the distributions of CAPRA STARR scores for all cCREs (black), REST+ enhancer/silencer cCREs (yellow), and REST+ silencer cCREs (purple). Both groups of REST+ cCREs have median STARR scores less than zero, suggesting silencer activity for both groups. Statistical significance is calculated using a two-sided Wilcoxon test comparing each REST+ category to the background set of all cCREs.