Enhancer Journal Club: Enhancer transcripts mark active estrogen receptor binding sites
Something rather contradictory for the Enhancer Journal Club series, the Hah et al. paper, published in May from the Kraus lab at UT Southwestern. This paper is really interesting because it has very different conclusions to the previous three papers I've blogged about, and I'm fascinated to see if and how the field can resolve these discrepancies.
As always, I've posted a non-technical summary at ScienceGist: http://sciencegist.com/gists/172
The abstract for this paper is as follows:
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.
Like the Li et al. paper (Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation), this paper identifies putative enhancers in MCF-7 cells by looking for sites of overlap between estrogen receptor binding (identified by ChIP) and primary transcription (identified by GRO-seq). Similarly, they find that most of the features of active enhancers associated with these sites (H3K4me1, H3K27ac, bidirectional transcription and DNA loop formation) are up-regulated by treatment with estradiol, which occurs in parallel to the up-regulation of genes in the vicinity of these putative enhancers.
Where the paper gets interesting is the attempt to disentangle some of the mechanistic details at work here. The obvious question is whether the transcription of the enhancer RNA "causes" the increased deposition of the active enhancer marks, or indeed the transcriptional up-regulation of the neighbouring gene. Whilst the Li and Lam papers tackle this for specific enhancers by targeting the associated eRNA with siRNA/antisense oligonucleotides, in this paper the authors attempt a global intervention by pre-incubating the cells in the general transcriptional inhibitor flavopiridol before treating with E2. They find that even when the cells are unable to respond to E2 by up-regulating the transcription of the eRNA, the E2 treatment is still able to induce higher occupancy of RNA Pol II, P300 and H3K27ac, as well as an increase in enhancer/promoter looping. Although the flavopiridol has a potentially global effect, they only identify its effects by ChIP-qPCR or 3C assays over a small number of genes. Additionally, the intervention is not quite as elegant as using siRNAs targeting the eRNA specifically, because the flavopiridol also inhibits transcription of the target gene, so they are unable to link eRNA production to the real functional output of gene regulation.
Of course in both of these contrasting papers, the authors demonstrate effects on a limited number of individual genes, so it is always possible that the two sets of genes use different mechanisms. I do wonder if there is a model which would explain both of these sets of results, but at the moment they do seem to be mutually exclusive - one claiming to show that removing the eRNA abolishes enhancer/promoter looping, and the other that looping occurs even when transcription of the eRNA is inhibited. Overall, very perplexing!
Let me know your thoughts in the comments...
Original paper: http://genome.cshlp.org/content/23/8/1210.long