Enhancer Journal Club: Genome-wide identification and characterization of functional neuronal activity–dependent enhancers

I noticed a nice paper this week in Nature Neuroscience from Michael Greenberg's lab at Harvard Medical School. The paper discusses the identification of enhancers in neurons which are activated (or deactivated) when those neurons are stimulated by membrane de-polarization. The original paper can be found at: http://www.nature.com/neuro/journal/vaop/ncurrent/full/nn.3808.html

The abstract is as follows:

Experience-dependent gene transcription is required for nervous system development and function. However, the DNA regulatory elements that control this program of gene expression are not well defined. Here we characterize the enhancers that function across the genome to mediate activity-dependent transcription in mouse cortical neurons. We find that the subset of enhancers enriched for monomethylation of histone H3 Lys4 (H3K4me1) and binding of the transcriptional coactivator CREBBP (also called CBP) that shows increased acetylation of histone H3 Lys27 (H3K27ac) after membrane depolarization of cortical neurons functions to regulate activity-dependent transcription. A subset of these enhancers appears to require binding of FOS, which was previously thought to bind primarily to promoters. These findings suggest that FOS functions at enhancers to control activity-dependent gene programs that are critical for nervous system function and provide a resource of functional cis-regulatory elements that may give insight into the genetic variants that contribute to brain development and disease.

It has been known for a while that neurons will respond to stimulation by activating the transcription of certain genes - this is known as "activity–dependent transcription". Whilst the signalling pathway leading from changes in membrane potential to the transcription of early-response genes has been the focus of a few studies, the way in which these few early-response genes activate a broader transcriptional program is not well understood. The authors postulated that the early-response might lead to activation of enhancers, which lie upstream of the broader transcriptional changes in stimulated cells.

They first set out to identify enhancers which are activated or deactivated after membrane depolarization by KCl treatment of cortical neurons. They perform ChIP-seq of H3K27Ac/me3, H3K4me1/3, CBP and RNA Pol II before and after KCl addition, plus RNA-seq before and at 1/6 hours post depolarization. They identify putative enhancers as CBP/H3K4me1-enriched sites >1 kb from an annotated TSS, and found that 1468 of them (12%) showed at least a twofold increase in H3K27Ac after depolarization, which they call "neuronal activity–regulated enhancers". In contrast, they only found 738 sites that showed at least a twofold decrease. They nicely validated the neuronal activity–regulated enhancers by showing that 14/14 tested regions showed greater activity in a luciferase assay after depolarization. In contrast, no putative enhancers with constant or decreased H3K27Ac after KCl treatment showed higher activity in the luciferase assay following the same treatment.

So these putative enhancer regions can activate luciferase in response to membrane depolarization, but the question remains whether they activate endogenous genes in vivo. To answer this they asked whether the nearest genes to neuronal activity–regulated enhancers also showed increases in transcription on neuronal activation. Whilst they do show a significant increase in the expression level of these genes, the increase is not that large. This could be because many of the enhancer regions don't regulate the nearest gene (either regulating a more distal gene or having no targets) or because the increase in expression in each target gene is small. Presenting the data as a bean or box plot of fold changes, rather than a bar chart of mean expression level could have gone some way to answering this question.

The next obvious question is which factors might be responsible for activating the neuronal activity–regulated enhancers. They perform a motif enrichment analysis and find that AP-1 motif is the most highly enriched, which normally binds FOS- and JUN-family proteins. This is nice as several of these transcription factors are known early-response genes, but on the other hand the AP-1 motif is normally thought of as a promoter motif and not as a component of distal enhancers. ChIP seq for FOS protein confirmed that 96% of the identified 12,594 were at distal sites, not at promoters. Whilst this strongly implicates FOS in the activation of these enhancers, only 42% of neuronal activity–regulated enhancers were directly bound by FOS. That would seem to indicate that FOS is neither necessary nor sufficient for enhancer activation in a large number of cases. The paper does a good job of showing that FOS is required for those enhancers where it binds, however. In a panel of eight such enhancers tested in luciferase assays, all eight showed reduced activity if the AP-1 binding site was mutated by a single base pair, or when cells were treated with an shRNA against FOS.

They extend this approach genome wide by looking for genes which are upregulated in response to membrane depolarization, but which show at least 33% lower induction in the presence of FOS shRNA. Interestingly, only 53 of the 187 genes identified by this approach had a FOS-bound enhancer within 100kb. This could indicate that many of the genes sensitive to FOS shRNA are indirect targets. Alternatively, since only one shRNA is used, these genes could be direct off-target effects or could lie downstream of off-target genes. One interesting final possibility is that many of the activity regulated enhancers act over distances of >100kb. Never the less, they test 14 of their 53 FOS direct targets in the visual cortex of dark-housed mice exposed to light. 10/14 tested genes showed induction under these conditions, validating that these genes can respond to neuronal stimulus in the intact brain.

Overall, I think it's very interesting that H3K27Ac can change quite dramatically after only 2 hours of depolarization with KCl, and that many of the new peaks which appear do seem to mark functional enhancers. One aspect that could be interesting to explore is the peaks which are lost after depolarization. The authors do perform motif analysis for these peaks, which identified candidate TFs like Atoh1 and SRF, but a deeper investigation of how enhancer repression might be involved in the response to neuronal stimulation could be quite revealing.