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

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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References

  1. Greer, P.L. & Greenberg, M.E. From synapse to nucleus: calcium-dependent gene transcription in the control of synapse development and function. Neuron 59, 846–860 (2008).

    Article  CAS  Google Scholar 

  2. Leslie, J.H. & Nedivi, E. Activity-regulated genes as mediators of neural circuit plasticity. Prog. Neurobiol. 94, 223–237 (2011).

    Article  CAS  Google Scholar 

  3. Hensch, T.K. Critical period plasticity in local cortical circuits. Nat. Rev. Neurosci. 6, 877–888 (2005).

    Article  CAS  Google Scholar 

  4. Alberini, C.M. Transcription factors in long-term memory and synaptic plasticity. Physiol. Rev. 89, 121–145 (2009).

    Article  CAS  Google Scholar 

  5. Day, J.J. & Sweatt, J.D. Epigenetic mechanisms in cognition. Neuron 70, 813–829 (2011).

    Article  CAS  Google Scholar 

  6. Lyons, M.R. & West, A.E. Mechanisms of specificity in neuronal activity-regulated gene transcription. Prog. Neurobiol. 94, 259–295 (2011).

    Article  CAS  Google Scholar 

  7. O'Brien, R.J. et al. Synaptic clustering of AMPA receptors by the extracellular immediate-early gene product Narp. Neuron 23, 309–323 (1999).

    Article  CAS  Google Scholar 

  8. Hong, E.J., McCord, A.E. & Greenberg, M.E. A biological function for the neuronal activity-dependent component of Bdnf transcription in the development of cortical inhibition. Neuron 60, 610–624 (2008).

    Article  CAS  Google Scholar 

  9. Lin, Y. et al. Activity-dependent regulation of inhibitory synapse development by Npas4. Nature 455, 1198–1204 (2008).

    Article  CAS  Google Scholar 

  10. Ebert, D.H. & Greenberg, M.E. Activity-dependent neuronal signalling and autism spectrum disorder. Nature 493, 327–337 (2013).

    Article  CAS  Google Scholar 

  11. Greenberg, M.E. & Ziff, E.B. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature 311, 433–438 (1984).

    Article  CAS  Google Scholar 

  12. Greenberg, M.E., Ziff, E.B. & Greene, L.A. Stimulation of neuronal acetylcholine receptors induces rapid gene transcription. Science 234, 80–83 (1986).

    Article  CAS  Google Scholar 

  13. Eferl, R. & Wagner, E.F. AP-1: a double-edged sword in tumorigenesis. Nat. Rev. Cancer 3, 859–868 (2003).

    Article  CAS  Google Scholar 

  14. Banerji, J., Rusconi, S. & Schaffner, W. Expression of a β-globin gene is enhanced by remote SV40 DNA sequences. Cell 27, 299–308 (1981).

    Article  CAS  Google Scholar 

  15. Levine, M. Transcriptional enhancers in animal development and evolution. Curr. Biol. 20, R754–R763 (2010).

    Article  CAS  Google Scholar 

  16. Heintzman, N.D. et al. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat. Genet. 39, 311–318 (2007).

    CAS  Google Scholar 

  17. Visel, A. et al. ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457, 854–858 (2009).

    Article  CAS  Google Scholar 

  18. Kim, T.K. et al. Widespread transcription at neuronal activity-regulated enhancers. Nature 465, 182–187 (2010).

    Article  CAS  Google Scholar 

  19. Maurano, M.T. et al. Systematic localization of common disease-associated variation in regulatory DNA. Science 337, 1190–1195 (2012).

    Article  CAS  Google Scholar 

  20. Visel, A. et al. A high-resolution enhancer atlas of the developing telencephalon. Cell 152, 895–908 (2013).

    Article  CAS  Google Scholar 

  21. Telese, F., Gamliel, A., Skowronska-Krawczyk, D., Garcia-Bassets, I. & Rosenfeld, M.G. “Seq-ing” insights into the epigenetics of neuronal gene regulation. Neuron 77, 606–623 (2013).

    Article  CAS  Google Scholar 

  22. Creyghton, M.P. et al. Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl. Acad. Sci. USA 107, 21931–21936 (2010).

    CAS  Google Scholar 

  23. Rada-Iglesias, A. et al. A unique chromatin signature uncovers early developmental enhancers in humans. Nature 470, 279–283 (2011).

    Article  CAS  Google Scholar 

  24. Calo, E. & Wysocka, J. Modification of enhancer chromatin: what, how, and why? Mol. Cell 49, 825–837 (2013).

    Article  CAS  Google Scholar 

  25. Hsu, Y.C. & Perin, M.S. Human neuronal pentraxin II (NPTX2): conservation, genomic structure, and chromosomal localization. Genomics 28, 220–227 (1995).

    Article  CAS  Google Scholar 

  26. Shen, Y. et al. A map of the cis-regulatory sequences in the mouse genome. Nature 488, 116–120 (2012).

    Article  CAS  Google Scholar 

  27. Mouse ENCODE Consortium. et al. An encyclopedia of mouse DNA elements (Mouse ENCODE). Genome Biol. 13, 418 (2012).

  28. Crawford, G.E. et al. Genome-wide mapping of DNase hypersensitive sites using massively parallel signature sequencing (MPSS). Genome Res. 16, 123–131 (2006).

    Article  CAS  Google Scholar 

  29. Nord, A.S. et al. Rapid and pervasive changes in genome-wide enhancer usage during mammalian development. Cell 155, 1521–1531 (2013).

    Article  CAS  Google Scholar 

  30. Bailey, T.L. et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 37, W202–W208 (2009).

    Article  CAS  Google Scholar 

  31. Gerstein, M.B. et al. Architecture of the human regulatory network derived from ENCODE data. Nature 489, 91–100 (2012).

    Article  CAS  Google Scholar 

  32. Fleischmann, A. et al. Fra-1 replaces c-Fos–dependent functions in mice. Genes Dev. 14, 2695–2700 (2000).

    Article  CAS  Google Scholar 

  33. Kitano, J. et al. Tamalin, a PDZ domain–containing protein, links a protein complex formation of group 1 metabotropic glutamate receptors and the guanine nucleotide exchange factor cytohesins. J. Neurosci. 22, 1280–1289 (2002).

    Article  CAS  Google Scholar 

  34. Gu, Y. et al. Obligatory role for the immediate early gene NARP in critical period plasticity. Neuron 79, 335–346 (2013).

    Article  CAS  Google Scholar 

  35. Chang, M.C. et al. Narp regulates homeostatic scaling of excitatory synapses on parvalbumin-expressing interneurons. Nat. Neurosci. 13, 1090–1097 (2010).

    Article  CAS  Google Scholar 

  36. Beck, K.D., Powell-Braxton, L., Widmer, H.R., Valverde, J. & Hefti, F. Igf1 gene disruption results in reduced brain size, CNS hypomyelination, and loss of hippocampal granule and striatal parvalbumin-containing neurons. Neuron 14, 717–730 (1995).

    Article  CAS  Google Scholar 

  37. Sugo, N. et al. Nucleocytoplasmic translocation of HDAC9 regulates gene expression and dendritic growth in developing cortical neurons. Eur. J. Neurosci. 31, 1521–1532 (2010).

    PubMed  Google Scholar 

  38. Ronan, J.L., Wu, W. & Crabtree, G.R. From neural development to cognition: unexpected roles for chromatin. Nat. Rev. Genet. 14, 347–359 (2013).

    Article  CAS  Google Scholar 

  39. Zhou, Z. et al. Brain-specific phosphorylation of MeCP2 regulates activity-dependent Bdnf transcription, dendritic growth, and spine maturation. Neuron 52, 255–269 (2006).

    Article  CAS  Google Scholar 

  40. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    Article  CAS  Google Scholar 

  41. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    Article  Google Scholar 

  42. Rupp, R.A., Snider, L. & Weintraub, H. Xenopus embryos regulate the nuclear localization of XMyoD. Genes Dev. 8, 1311–1323 (1994).

    Article  CAS  Google Scholar 

  43. Heckman, K.L. & Pease, L.R. Gene splicing and mutagenesis by PCR-driven overlap extension. Nat. Protoc. 2, 924–932 (2007).

    Article  CAS  Google Scholar 

  44. Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

    Article  CAS  Google Scholar 

Download references

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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Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Michael E Greenberg.

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The authors declare no competing financial interests.

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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