Summary of study design, data integration, and main findings. (Top left) ESR1 binding sites were identified from ESR1 ChIP-seq data in cell lines and breast cancer samples. The activities of 14,299 ERBS were then assessed using STARR-seq. After excluding uncommon sites, 7576 ERBS were classified into three groups: (1) E2-induced ERBS, with increased activity upon E2; (2) constitutively active ERBS, with E2-independent activity; and (3) inactive ERBS, with no detectable enhancer activity. (Top right) Publicly available multi-omic data from MCF-7 cells and patient samples were integrated to define molecular features of each ERBS subgroup. (Bottom) Induced ERBS become accessible upon E2 stimulation and showed ESR1 motif enrichment, E2-driven H3K27ac signal gain, distinct coregulator recruitment, and strong enhancer and transcriptional activity. Constitutive and inactive ERBS reside in pre-accessible chromatin and rarely contain an ESR1 motif. Unlike inactive ERBS, constitutive ERBS are prebound by coregulators and associated with enhancers, promoters, and transcriptional activity. Clinical data integration reveal that ERBS subclasses were associated with distinct breast cancer subtypes and clinical outcomes, with induced ERBS more frequently associated with a favorable prognosis, whereas the other subclasses correlated with poor outcome.
