Comprehensive functional annotation of ESR1-driven enhancers in breast cancer reveals hierarchical activity independent of genomic and epigenomic contexts

STARR-seq defines functional ERBS subgroups. (A) Schematic of ESR1 STARR-seq. A total of 14,299 ESR1-bound sites identified from human breast tumors and cell lines were cloned into hSTARR-ORI plasmid, transfected into MCF-7, treated with DMSO or E2 for 6 h, and analyzed by RNA-seq to measure enhancer-driven self-transcription. (B) Post-filtering heat maps showing STARR-seq signal (purple, log₂ fold change over input plasmid library) and ESR1 ChIP-seq in full-medium (red, GSE32222) (Ross-Innes et al. 2012) and after 10 min E2 stimulation (blue, GSE54855) (Guertin et al. 2014). ChIP-seq signals are represented as reads per genomic content (RPGC). Regions are grouped by STARR-seq-defined functional classes. (C) Volcano plot of STARR-seq signal log₂(fold change, E2 vs. DMSO). Dashed lines represent significance thresholds (|log₂(fold change)| > 1 and P_adj < 0.05). (D) Bar plot of ERBS distribution and counts (in white). Green, blue, and yellow bars represent induced, constitutive, and inactive ERBS, respectively. (E) Snapshot of the XBP1 locus, showing STARR-seq data (RNA over DNA fold change), and ChIP-seq for ESR1 in full-medium (GSE32222) (Ross-Innes et al. 2012), ESR1 with and without 10 min E2 stimulation (GSE54855) (Guertin et al. 2014), FOXA1 with and without 45 min E2 stimulation (GSE23852), and RNAPII with and without 10 min E2 stimulation (GSE62789) (Honkela et al. 2015). Green, blue and yellow boxes represent induced, constitutive, and inactive ERBS, respectively. Numbers represent signal intensity. (F) ESR1 ChIP-seq signal across ERBS subtypes during E2 time course (GSE54855) (Guertin et al. 2014). Significance of Tukey's honest significant difference test following a one-way ANOVA is indicated. (G) Percentage of ERBS with consensus ESR1 motifs. (H) RNAPII ChIP-seq signal across ERBS subtypes during E2 time course (GSE62789) (Honkela et al. 2015). Significance of Tukey's honest significant difference test following a one-way ANOVA is indicated. (I) Gene expression fold changes for genes linked to each ERBS subgroup after 24 h E2 treatment in MCF-7 (GSE99680) (Chi et al. 2019). Significance of Wilcoxon test is indicated. (J) Hypergeometric test showing enriched hallmark pathways per ERBS subgroup. Bar colors reflect adjusted P-values, with gray indicating nonsignificance (>0.05).