At promoters, CBP recruits components of the basal transcription machinery, including RNAPII, thereby facilitating the assembly of functional transcription complexes that initiate mRNA synthesis17
. Since CBP binds to enhancers in an activity-dependent manner, we asked if CBP also recruits RNAPII to these enhancers. To address this issue, we used ChIP-Seq to identify RNAPII binding sites across the genome using two different RNAPII antibodies. Consistent with previous studies18,19
, a large number of RNAPII sites were found to be located near annotated TSSs (, and Supplementary Fig. 4a
). Surprisingly RNAPII also bound to ~3,000 activity-regulated enhancers (25%) (, , and Supplementary Figs. 1f, 3, 4a
), and the level of RNAPII binding was increased about 2-fold upon membrane depolarization (Supplementary Fig. 2
). While RNAPII has previously been reported to be present at several enhancers, including the β-globin and MHC class II gene enhancers20,21
, it has not been thought to play a widespread role in enhancer function. Given that CBP was previously known to recruit RNAPII to promoters and that increases in CBP and RNAPII binding coincide at thousands of enhancers in membrane depolarized neurons, it is likely that CBP plays a role in the activity-regulated increase in RNAPII binding at enhancers. However, the observation that RNAPII is present at only a subset of CBP-bound enhancers suggests that additional activation steps beyond CBP binding may be required for RNAPII recruitment to enhancers.
Enhancers bind RNA Polymerase II (RNAPII) and produce eRNAs
The presence of RNAPII at enhancers raises the possibility that RNA transcription may occur at enhancers. Alternatively, the detection of RNAPII at enhancers might be an indirect consequence of the interaction of enhancers with active promoters, such that promoter-bound RNAPII gets cross-linked to enhancer DNA during the preparation of cells for ChIP-Seq experiments. To distinguish between these two possibilities, we used high-throughput RNA sequencing (RNA-Seq) to determine whether enhancer-bound RNAPII drives RNA synthesis at enhancers. Since it was not clear whether enhancer-derived transcripts would be polyadenylated, we sequenced total RNA, obtained from unstimulated or membrane-depolarized neurons after ribosomal RNA was depleted. To distinguish possible enhancer-derived transcripts from mRNA transcripts, we sought evidence of RNA transcription specifically at those ~5,000 activity-regulated enhancers located outside of annotated genes (extragenic enhancers). Surprisingly, we detected short (< 2kb) RNAs at ~2,000 extragenic enhancers (, , ). We observed dynamic changes in the levels of these enhancer RNAs (eRNAs) upon membrane depolarization, with a mean increase of ~2-fold (). Synthesis of eRNAs appears to initiate near enhancer centers where CBP and RNAPII are bound and to proceed bi-directionally, extending to the ends of the H3K4me1-modified enhancer domain (, , ). Interestingly, we also detected eRNAs at ~1,000 of ~7,000 intragenic enhancers (Methods
). Although high levels of mRNA transcription across intragenic enhancers prevented accurate quantification of eRNAs in the sense orientation, antisense eRNAs at intragenic enhancers were detectable and were similar in level to eRNAs at extragenic enhancers (; Methods
). These observations suggest that enhancers are not only sites where transcription factors bind and recruit RNAPII that might subsequently be delivered to promoters, but that enhancers are also sites where RNA synthesis occurs.
eRNAs are transcribed bidirectionally, and their activity-dependent induction correlates with induction of nearby genes
The strand-specific synthesis of eRNAs () and the dynamic changes in the level of eRNAs in response to neuronal activity suggest that the detection of eRNAs is not due to the sequencing of residual genomic DNA that is present in our purified RNA samples. Nevertheless, to confirm the existence of activity-regulated eRNAs at enhancers, we employed an alternative method (DNase I treatment followed by RT-qPCR) to detect these RNA transcripts (Supplementary Fig. 6
). By RT-qPCR, we detected eRNAs at each of 18 enhancer loci tested. This result provides independent confirmation that the thousands of distinct eRNAs detected by RNA-Seq are bona fide
RNA transcripts that are induced in an activity-dependent manner from neuronal enhancers.
We did not detect eRNAs in RNA-Seq from polyA+ RNA fractions, suggesting that a large number of eRNAs may not be polyadenylated. While it is possible that some polyadenylated eRNAs are present but not detectable at our current sequencing depth, two independent lines of evidence suggest that a large number of eRNAs may not be polyadenylated. First, using RT-qPCR, we observed that eRNAs were detected at higher levels in randomly primed reactions compared to oligo-dT-primed RT reactions (data not shown). Second, conventional sequencing of a circularized eRNA from the arc enhancer confirmed that this transcript is not polyadenylated (). These experiments suggest that polyadenylation may not be a common feature of eRNA synthesis.
eRNA synthesis but not RNAPII binding at the arc enhancer requires the presence of the arc promoter
The detection of RNAPII binding and RNA synthesis at many enhancers could in principle result from mis-categorization of un-annotated promoters as enhancers. However, several lines of evidence suggest that both the extragenic and intragenic enhancers we have identified are indeed enhancers and are not un-annotated promoters. First, histone modification profiles at enhancers and annotated promoters are clearly distinguishable ( top and Supplementary Figs. 1c, 8a
). Activity-regulated enhancers have high H3K4me1 and low H3K4me3 levels, while promoters have lower H3K4me1 and high H3K4me3 levels. Second, the observation that eRNAs do not extend beyond the ~4kb enhancer domain suggests that the eRNAs are much shorter (<2kb for each strand) than transcripts initiated at most gene promoters (, ). Third, unlike promoters, enhancers do not produce detectable levels of polyadenylated RNA (). Fourth, a promoter prediction algorithm (ProSOM)22
revealed that fewer than 100 of ~12,000 enhancer regions are predicted to be promoters compared to 8,494 out of 27,857 annotated TSSs. Fifth, while sense transcription is more prevalent than antisense transcription at most promoters, transcription at enhancers appears to be less biased toward one particular strand (). Finally, a few enhancers, including the well-characterized β-globin enhancer, have previously been shown to recruit RNAPII and drive transcription23,24
. These findings argue against the possibility that RNAPII-bound enhancers that produce eRNAs are actually un-annotated promoters.