We have integrated and analyzed a large number of data sets from a variety of 
genomic assays using a novel computational pipeline to provide a global view of 
estrogen receptor 1 (ESR1; a.k.a. ERα) enhancers in MCF-7 human breast cancer 
cells. Using this approach, we have defined a class of primary transcripts 
(eRNAs) that are transcribed uni- or bidirectionally from estrogen receptor 
binding sites (ERBSs) with an average transcription unit length of ∼3-5 kb. The 
majority are up-regulated by short treatments with estradiol (i.e., 10, 25, or 
40 min) with kinetics that precede or match the induction of the target genes. 
The production of eRNAs at ERBSs is strongly correlated with the enrichment of a 
number of genomic features that are associated with enhancers (e.g., H3K4me1, 
H3K27ac, EP300/CREBBP, RNA polymerase II, open chromatin architecture), as well 
as enhancer looping to target gene promoters. In the absence of eRNA production, 
strong enrichment of these features is not observed, even though ESR1 binding is 
evident. We find that flavopiridol, a CDK9 inhibitor that blocks transcription 
elongation, inhibits eRNA production but does not affect other molecular 
indicators of enhancer activity, suggesting that eRNA production occurs after 
the assembly of active enhancers. Finally, we show that an enhancer 
transcription "signature" based on GRO-seq data can be used for de novo enhancer 
prediction across cell types. Together, our studies shed new light on the 
activity of ESR1 at its enhancer sites and provide new insights about enhancer 
function.