H3K27me3 chromatin heterogeneity reveals variable cell responses to estrogen and endocrine treatment

Gene expression heterogeneity generates subpopulations of tumor cells that can evade therapeutic pressure. This heterogeneity has been observed in both primary Estrogen Receptor alpha positive (ERα+) breast tumors and cell lines. Therefore, understanding the mechanisms regulating expression heterogeneity is critical towards developing effective therapies. A key contributor to gene expression variability is the stochastic nature of transcription. Transcription occurs in a probabilistic, burst-like manner, in which gene activation occurs intermittently, producing RNA in pulses and interspersed with transcriptional off-periods. The estrogen-responsive gene TFF1 is expressed in the majority of ERα⁺ breast tumors and exemplifies such heterogeneity, with transcriptional inactivity ranging from minutes to several days. Here, we identify the molecular mechanism underlying the wide range in TFF1 expression by analyzing cells sorted based on their TFF1 activity levels. We observed that TFF1 inactive (TFF1 low ) cells exhibit a repressive chromatin state marked by H3K27me3 at the TFF1 promoter and enhancer. Despite global similarity in ERα binding, occupancy at the TFF1 regulatory elements was selectively reduced in TFF1 low cells, resulting in fewer active alleles and diminished transcriptional bursting frequency. Conversely, TFF1 high cells exhibited more active TFF1 alleles and hyperbursting. These cells also retained sensitivity to endocrine therapy, while TFF1 low cells displayed reduced drug responsiveness. Genome-wide, differentially enriched H3K27me3 regions correlated with variable expression of estrogen-responsive genes, highlighting a broader regulatory mechanism that links chromatin state to expression variability. Together, our findings establish how repressive chromatin dynamics contribute to gene expression heterogeneity and endocrine resistance in ERα⁺ breast cancer. Graphical abstract