Abstract P1-05-02: Estrogen-regulated enhancer RNAs control enhancer assembly and function in breast cancer cells

Abstract Approximately 70% of breast cancers express estrogen receptor alpha (ERα), a hormone-regulated transcription factor, making it an excellent target for endocrine therapy. When endogenous and exogenous ligands engage ERα, the receptor binds to regulatory regions in the genome known as enhancers, where it (1) associates with coregulatory proteins, (2) increases histone modifications associated with active enhancers, (3) promotes chromatin looping, (4) stimulates enhancer transcription and the production of enhancer RNAs (eRNAs), and (5) ultimately regulates target gene transcription. We have previously used global run-on sequencing (GRO-seq), a high-throughput method that identifies the location and orientation of all transcriptionally active RNA polymerases across the genome, to identify active enhancers based on enhancer transcription and enrichment of the aforementioned genomic features. In ERα-positive breast cancer cells, estrogen signaling promotes the synthesis of thousands of eRNAs that originate from ERα enhancers. We are interested in understanding the functions of these estrogen-regulated eRNAs and how they contribute to the etiology of breast cancer. Recent studies on the roles of eRNAs have suggested that they may (1) recruit regulatory proteins to enhancers; (2) promote RNA polymerase II transition; (3) regulate histone modification; and (4) increase chromosome looping. One challenge facing the field is the lack of accurate eRNA annotations and cloned full-length eRNAs; eRNAs have typically been studied as signals from genomic assays on browser tracks, not as physical entities (e.g., cDNAs) - in other words, “ghosts.” We have employed precision nuclear run-on of capped RNA (PRO-cap) to determine the transcription start sites of all expressed eRNAs. In addition, we have also used ultra-deep RNA-sequencing of polyA-depleted and polyA-enriched RNA fractions to detect eRNAs and examine for the potential splicing of eRNAs. Combining these genomic data, our annotations provide critical information for the large-scale cloning of eRNAs and testing them either by (1) tethering them to their cognate ERα enhancers by using CRISPR/dCas9 technology (i.e., cis) or (2) ectopically expressing them (i.e., trans) in MCF-7 breast cancer cells. When we tether eRNAs to their cognate ERα enhancers by using dCas9 and CRISPR guide RNAs, various aspects of ERα enhancer assembly and target gene expression are enhanced. Currently we are devising strategies to screen for functional eRNAs on a genome-wide basis to determine how they contribute to ERα enhancer assembly and function on a global scale, and ultimately identify the eRNAs that drive breast cancer initiation and growth. This work is supported by grants from the NIH/NIDDK (DK058110) and CPRIT (RP160319) to W.L.K. Citation Format: Tim Y Hou, William L Kraus. Estrogen-regulated enhancer RNAs control enhancer assembly and function in breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-05-02.