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Genome-wide identification and characterization of functional neuronal activity–dependent enhancers

Nature Neuroscience volume 17, pages 1330–1339 (2014)Cite this article

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Abstract

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.

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Figure 1: Genome-wide analysis of H3K27ac ChIP-seq peaks.
Figure 2: H3K27ac dynamics at activity-regulated enhancers.
Figure 3: Functional analysis of enhancers with distinct H3K27ac dynamics.
Figure 4: FOS binding is highly enriched at neuronal activity–regulated enhancers.
Figure 5: AP-1 transcription factors are required for the proper function of activity-regulated enhancers.
Figure 6: FOS activates an extensive gene program in neurons that regulates synaptic development and function.
Figure 7: Integrated genomic analysis identifies direct targets of FOS.
Figure 8: Expression of FOS direct target genes in mouse visual cortex.

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Acknowledgements

We thank all the members of M.E.G.'s lab for their scientific support and helpful discussions. This work was funded by the US National Institutes of Health (NIH project 5R37NS028829-25 to M.E.G.), the National Institute of General Medical Sciences award number T32GM007753 (A.N.M.) and National Cancer Institute Institutional Training grant T32CA009361 (T.V.). T.V. and H.S. are both Howard Hughes Medical Institute Fellows of the Damon Runyon Cancer Research Foundation. E.L. is supported by the National Science Foundation Graduate Research Fellowship under grant numbers DGE0946799 and DGE1144152. The content of this study is solely the responsibility of the authors and does not necessarily represent the official views of the funding sources mentioned.

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Author notes

  1. Athar N Malik and Thomas Vierbuchen: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA

    Athar N Malik, Thomas Vierbuchen, Alex A Rubin, Emi Ling, Cameron H Couch, Hume Stroud, Ivo Spiegel, Kyle Kai-How Farh, David A Harmin & Michael E Greenberg

  2. MD-PhD Program, Harvard Medical School, Boston, Massachusetts, USA

    Athar N Malik

  3. Division of Health Sciences and Technology, Harvard Medical School and Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Athar N Malik

  4. Department of Ophthalmology, Children's Hospital Boston, Harvard University, Boston, Massachusetts, USA

    Martin Hemberg

  5. Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts, USA

    Emi Ling

  6. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    Kyle Kai-How Farh

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Contributions

A.N.M. and M.E.G. conceived the study. A.N.M. performed experiments with assistance from T.V., A.A.R., E.L., I.S. and C.H.C. A.N.M. performed analysis of microarray experiments. A.N.M. performed analysis of genome-wide sequencing experiments with assistance from M.H., H.S., K.K.-H.F. and D.A.H. A.N.M. generated figures with assistance from T.V. T.V., A.N.M. and M.E.G. wrote the manuscript.

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Correspondence to Michael E Greenberg.

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Integrated supplementary information

Supplementary Figure 1 Reproducibility of H3K27ac ChIP-seq

(a) Reproducibility of H3K27ac ChIP-seq signal (no KCl treatment) at individual H3K27ac peaks genome-wide between two biological replicate samples, with the ChIP performed independently for each biological replicate (ρ = 0.91, Spearman’s rank correlation coefficient). H3K27ac peaks were identified by MACS (MACS default parameters; p = 1 X 10–5). (b) Reproducibility of H3K27ac ChIP-seq signal (2 h KCl treatment) at individual H3K27ac peaks genome-wide (ρ = 0.94, Spearman’s rank correlation coefficient). (c) H3K4me1 ChIP-seq signal at gene distal DHS sites enriched for H3K27ac before and after 2 h membrane depolarization by KCl (ρ = 0.96, Spearman’s rank correlation coefficient). (d) H3K27ac ChIP-seq signal at gene distal DHS sites enriched for H3K27ac before and after 2 h membrane depolarization by KCl (ρ = 0.77, Spearman’s rank correlation coefficient). (e) Classification of gene distal DHS sites with distinct H3K27ac dynamics in response to neuronal activity (see methods).

Supplementary Figure 2 Additional functional testing of neuronal activity–regulated enhancers

(a) Average neuronal activity-dependent induction of luciferase for each class of enhancers when measured in the Nptx2 reporter. Results shown are a summary of the data presented in Fig. 3a. *p = 0.0012 Student’s t-test, two-tailed, **p = 2.44X10–5 Student’s t-test, two-tailed. (b-g) The activity of four enhancers selected from each H3K27ac enhancer group initially tested in the Nptx2 reporter were tested in two additional luciferase reporter plasmids with distinct promoters (SV40 promoter: d-e, minP promoter: f-g). c, e and g show the average fold-induction of luciferase expression from the enhancers shown individually in b, d and f. Error bars for each graph represent standard error (n = 3 biological replicates, 3 technical replicates per experiment).

Supplementary Figure 3 Additional functional testing of enhancers with distinct transcription factor binding and chromatin features

(a) Functional testing of distinct classes of putative enhancers using the Nptx2 reporter. *p = 7.54X10–8 Student’s t-test, two-tailed; Comparison between the average fold induction of luciferase of (FOS + CBP + Increasing H3K27ac) enhancers and FOS only enhancers. **p = 0.0003 Student’s t-test, two-tailed; Comparison between (FOS + CBP + Increasing H3K27ac) enhancers and (FOS + Increasing H3K27ac) enhancers. ***p = 1.69X10–7 Student’s t-test, two-tailed; Comparison between (FOS + CBP + Increasing H3K27ac) enhancers and (FOS + CBP) enhancers.

Supplementary Figure 4 De novo motif finding analysis of H3K27ac enhancer classes

Position weight matrices for motifs identified by MEME de novo motif search from each class of distal H3K27ac sites genome-wide. Analyses were performed with a search window of 150 bp from the center of DHS that exhibit the indicated H3K27ac behaviors (as defined in Supplementary Fig. 1E). The upper E-value is the output of the MEME de novo motif finding algorithm. Each identified de novo motif was input into JASPAR to identify related transcription factor position weight matrices. The lower E-value indicates the certainty of the match between the identified de novo motifs and the JASPAR position weight matrices.

Supplementary Figure 5 Additional validation of AP-1 transcription factor ChIP-seq data

(a) Aggregate plot of FOS ChIP-seq binding before and after 2h membrane depolarization at all FOS peaks called using MACS. Signal is shown for both anti-FOS antibodies (sc-52 and sc-7202). (b) Aggregate plot of FOS binding before and after membrane depolarization at all FOS binding sites. The effect of FOS shRNA infection on ChIP-seq signal at FOS binding sites is indicated from two biological replicates (sc-52 antibody). (c) Aggregate plot of FOSB and JUNB binding at all FOS binding sites

Supplementary Figure 6 Overlap between FOS binding and distinct chromatin features

(a) Overlap between H3K27ac peaks and CBP/H3K4me1 enriched sites. Each distinct H3K27ac behavior in response to membrane depolarization is indicated. The category “other” denotes H3K27ac peaks that overlap with CBP/H3K4me1 sites but do not met criteria for one of the specific categories (i.e. increasing, decreasing, etc.) (b-c) Fraction of sites in (a) bound by FOS. (d) Overlap between CBP/H3K4me1 sites with no H3K27ac enrichment and FOS binding (see Fig. 2c) (e) Venn diagram indicating the overlap between all increasing H3K27ac sites, CBP/H3K4me1 sites and FOS binding.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Table 1 (PDF 1359 kb)

Supplementary Methods Checklist (PDF 374 kb)

Supplementary Table 2

Activity-regulated genes changed by FOS shRNA and list of FOS direct target genes identified by integrative genomic analysis (XLSX 105 kb)

Supplementary Table 3

ChIP-seq and RNA-Seq quantifications for each CBP/H3K4me1-enriched site from Kim et al.18 (XLSX 4330 kb)

Supplementary Table 4

Primer sequences (XLSX 60 kb)

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Malik, A., Vierbuchen, T., Hemberg, M. et al. Genome-wide identification and characterization of functional neuronal activity–dependent enhancers. Nat Neurosci 17, 1330–1339 (2014). https://doi.org/10.1038/nn.3808

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