An epigenetic CRISPR screen identifies ARID1A as an upstream regulator of enhancer RNA expression
Estrogen Receptor-alpha (ER) exerts its transcriptional activity by binding primarily to cis-regulatory DNA elements called enhancers, thereby promoting breast cancer progression. These regions are transcribed to produce noncoding RNAs called enhancer RNAs (eRNAs), whose presence strongly indicates enhancer and transcription factor activity. We show that RNA-in situ hybridization (RNA-ISH) using RNAscope probes specific to the eRNAs transcribed from ER target gene enhancers can efficiently detect the regulation of its expression with treatments using ER antagonists and locked nucleic acids. Interestingly, our RNA-FISH analyses show that the expression of eRNA associated with GREB1 (eGREB1) correlates with tamoxifen resistance in explants derived from breast cancer patient xenografts. Additionally, using a high-throughput multiplex RNA-FISH assay coupled with CRISPR-Cas9 based knockout of 237 epigenetic proteins (EpiCRISPeR), we identified the upstream regulators of estrogen-induced RNAs (GREB1 eRNA and its corresponding mRNA) and a proliferation marker MKI67 simultaneously in a single-cell-based manner. The EpiCRISPeR assay identified FOXA1 and FACT complex subunits, SSRP1 and SUPT16H, as topmost genes, which are important for eRNA expression and a major chromatin remodelling complex subunit, ARID1A, suppresses eRNA expression. ARID1A indirectly regulates transcriptional elongation of its target genes by controlling the recruitment of other chromatin remodelling complexes and BRD4. Overall, using a highly sensitive and robust RNA-FISH approach, our study identified upstream epigenetic regulators of eRNAs, enabling better understanding of eRNA regulation.