Enhancer transcripts mark active estrogen receptor binding sites

Enhancers are genomic regulatory elements that (1) carry sequence information for transcription factor binding, (2) may be located far from TSSs, (3) regulate gene expression regardless of location and orientation, and (4) play key roles in controlling tissue-specific gene expression (Bulger and Groudine 2011; Ong and Corces 2011). Current models posit that enhancers function by promoting communication with target gene promoters through chromatin loops or by tracking of enhancer-bound transcription factors through intervening chromatin to target gene promoters (Bulger and Groudine 2011; Ong and Corces 2011; Kolovos et al. 2012). Recent studies have focused intense interest on the properties of enhancers, beyond the binding of sequence-specific transcription factors, which might give clues to their mechanisms of action and aid in their identification. In this regard, histone modifications (e.g., H3 lysine 4 monomethyl, H3K4me1; H3 lysine 27 acetyl, H3K27ac), histone variants (e.g., H2A.Z), coactivators (e.g., EP300, CREBBP, Mediator), and an open chromatin architecture (e.g., DNase I hypersensitivity) have been identified as genomic features that mark or identify enhancers (Melgar et al. 2011; Natoli and Andrau 2012). Differential association of these features with enhancers in a given cell may define distinct classes of enhancers that specify distinct gene regulatory mechanisms and biological outcomes (Creyghton et al. 2010; Ghisletti et al. 2010; Rada-Iglesias et al. 2011; Wang et al. 2011; Zentner et al. 2011; Pham et al. 2012; Rada-Iglesias et al. 2012; Shen et al. 2012; Vahedi et al. 2012; Whyte et al. 2012; Xu et al. 2012; Ostuni et al. 2013). Enhancer profiles may even provide useful clinical signatures for cancer diagnosis and prognosis (Akhtar-Zaidi et al. 2012; Ross-Innes et al. 2012). More recently, several studies have shown that many enhancers overlap with sites of RNA Pol II loading, active RNA Pol II transcription, and the production of enhancer RNAs (“eRNAs”) (De Santa et al. 2010; Kim et al. 2010; Hah et al. 2011; Wang et al. 2011; Djebali et al. 2012). A common signature of enhancer transcription is the production of short (i.e., ∼1 to 2 kb) eRNAs that are transcribed bidirectionally (Kim et al. 2010). We and others have recently shown that the genomic binding sites for the estrogen receptor (ESR1) and other steroid hormone receptors overlap with sites of transcription (Hah et al. 2011; Wang et al. 2011). The role of transcription in enhancer function is unknown, but the act of transcription may help to create an open chromatin environment that promotes enhancer function (Natoli and Andrau 2012). Alternatively, the stable accumulation of eRNAs may play a functional, perhaps even structural, role and may facilitate gene looping (Orom et al. 2010; Orom and Shiekhattar 2011; Natoli and Andrau 2012; Lai et al. 2013; Melo et al. 2013). In the studies described herein, we used Global Run-On Sequencing (GRO-seq), a method that assays the location and orientation of all active RNA polymerases genome-wide (Core et al. 2008), to generate a global profile of active transcription at ESR1 binding sites (ERBSs) in MCF-7 human breast cancer cells in response to a short time course of E2 treatment. We integrated the data from our GRO-seq assays with data from a variety of other genomic assays (e.g., ChIP-seq, DNase-seq, ChIA-PET) using a novel computational pipeline to provide a comprehensive and global view of ESR1 enhancers and their regulation by E2 in MCF-7 cells. Together, our studies have shed new light on the activity of ESR1 at its enhancer sites and provide new insights about enhancer function in general, including the potential roles of enhancer transcription.