Abstract P6-08-05: Genome-wide identification of transcripts regulated by estrogen in MCF-7 cells using BrU-seq

Abstract

Abstract Estrogen promotes estrogen receptor (ER)-positive breast cancer cell growth by modulating transcription through ligand activated ER. The spectrum of early ER target genes has been identified through gene expression microarray and more recently with RNA-seq analyses. These methods, although comprehensive, fall short in identifying many ER-regulated transcripts which are either not present in microarray; too abundant to determine estrogen regulated changes among steady-state pools of RNAs; or have very short half-lives and low abundance such as some non-coding RNAs, including RNAs generated by enhancers bound by ER (eRNAs). Global run-on sequencing (GRO-seq) has been used to identify ER target genes and eRNAs in breast cancer cells. It is technically cumbersome and is performed under non-physiological condition. We have applied BrU-seq technique to identify global transcripts regulated by estrogen in MCF-7 breast cancer cells. The newly synthesized RNAs in MCF-7 cells treated with or without estrogen for various time points, were labeled with 5'-bromo-uridine (BrU). The labeled RNAs were purified by immunoprecipitation using an anti-BrdU antibody and then subjected to RNA sequencing analysis, which provides greater sensitivity than regular RNA-seq analysis from steady-state total RNA samples. Robust estrogen-regulated transcripts can be detected within 30 min of treatment. Significantly more transcripts (both coding and non-coding RNAs) were identified as estrogen targets using BrU-seq analysis than RNA-seq analysis from total RNA pools. Many of these estrogen-regulated transcripts are located close to ER binding sites identified from ER ChIP-seq analysis, indicating a functional role for these ER binding sites in regulating RNA transcription. For estrogen targets identified through both analyses, the magnitude of expression changes (both up- and down-regulation) is usually greater from BrU-seq than from the regular RNA-seq analysis. The dynamics of RNA synthesis and degradation for some unstable RNAs (such as eRNAs) can be interrogated through our data set. Also, RNA pol II elongation rate can be deduced from some long ER target genes (>100 kb) such as GREB1. In addition to identifying estrogen targets, BrU-seq should be a very useful technique in assessing transcriptional changes triggered by various signal transduction pathways. Citation Format: Sun J, Capobianco E, Tsinoremas N, Lippman M. Genome-wide identification of transcripts regulated by estrogen in MCF-7 cells using BrU-seq [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-08-05.