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Stimulus-specific combinatorial functionality of neuronal c-fos enhancers

A Corrigendum to this article was published on 29 March 2016

This article has been updated


The c-fos gene (also known as Fos) is induced by a broad range of stimuli and is a reliable marker for neural activity. Here we demonstrate that multiple enhancers surrounding the c-fos gene are crucial for ensuring robust c-fos response to various stimuli. Membrane depolarization, brain-derived neurotrophic factor (BDNF) and forskolin activate distinct subsets of the enhancers to induce c-fos transcription in neurons, suggesting that stimulus-specific combinatorial activation of multiple enhancers underlies the broad inducibility of the c-fos gene. Accordingly, the functional requirement of key transcription factors varies depending on the type of stimulation. Combinatorial enhancer activation also occurs in the brain. Providing a comprehensive picture of the c-fos induction mechanism beyond the minimal promoter, our study should help in understanding the physiological nature of c-fos induction in relation to neural activity and plasticity.

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Figure 1: Time-course analysis of five c-fos eRNAs and mRNA.
Figure 2: The c-fos enhancer activities measured by eRNA analysis and luciferase reporter assay.
Figure 3: Stimulus-dependent interactions between the c-fos enhancers and the promoter.
Figure 4: Function of CREB, MEF2A, NPAS4 and SRF in KCl- or BDNF-mediated induction of c-fos mRNA and eRNAs.
Figure 5: Specific requirement of e2 and e4 enhancer in KCl- or BDNF-mediated c-fos transcription.
Figure 6: Characterization of c-fos enhancer activities in vivo.
Figure 7: Stimulus-specific combinatorial action of c-fos enhancers in vivo.

Change history

  • 22 January 2016

    In the version of this article initially published, it was stated that the c-fos gene-based TRAP mouse line described in ref. 42 utilized only the c-fos promoter region to drive CreERT2. In fact, that line was generated by CreERT2 knock-in to the endogenous c-fos locus. Accordingly, the sentence “Its induction mechanism and available reporter mouse lines are based exclusively on c-fos promoter activity” has been deleted from the Abstract, and the following sentences have been deleted from the third paragraph of the Discussion: “However, these mice utilize only the c-fos promoter region to induce the reporter fluorescent protein and do not include any of the c-fos enhancers we have characterized. On the basis of our findings, the promoter-only reporters might not faithfully recapitulate the expression characteristics of the endogenous c-fos gene in vivo triggered by sensory or pharmacological stimuli.” The changes have been made in the HTML and PDF versions of the article.


  1. 1

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

    CAS  Article  Google Scholar 

  2. 2

    Frey, U., Frey, S., Schollmeier, F. & Krug, M. Influence of actinomycin D, a RNA synthesis inhibitor, on long-term potentiation in rat hippocampal neurons in vivo and in vitro. J. Physiol. (Lond.) 490, 703–711 (1996).

    CAS  Article  Google Scholar 

  3. 3

    Sheng, M. & Greenberg, M.E. The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron 4, 477–485 (1990).

    CAS  Article  Google Scholar 

  4. 4

    Bartel, D.P., Sheng, M., Lau, L.F. & Greenberg, M.E. Growth factors and membrane depolarization activate distinct programs of early response gene expression: dissociation of fos and jun induction. Genes Dev. 3, 304–313 (1989).

    CAS  Article  Google Scholar 

  5. 5

    Heinz, S., Romanoski, C.E., Benner, C. & Glass, C.K. The selection and function of cell type-specific enhancers. Nat. Rev. Mol. Cell Biol. 16, 144–154 (2015).

    CAS  Article  Google Scholar 

  6. 6

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

    CAS  Article  Google Scholar 

  7. 7

    Kim, T.K. & Shiekhattar, R. Architectural and Functional Commonalities between Enhancers and Promoters. Cell 162, 948–959 (2015).

    CAS  Article  Google Scholar 

  8. 8

    Kaikkonen, M.U. et al. Remodeling of the enhancer landscape during macrophage activation is coupled to enhancer transcription. Mol. Cell 51, 310–325 (2013).

    CAS  Article  Google Scholar 

  9. 9

    Dixon, J.R. et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485, 376–380 (2012).

    CAS  Article  Google Scholar 

  10. 10

    Sexton, T. et al. Three-dimensional folding and functional organization principles of the Drosophila genome. Cell 148, 458–472 (2012).

    CAS  Article  Google Scholar 

  11. 11

    Jin, F. et al. A high-resolution map of the three-dimensional chromatin interactome in human cells. Nature 503, 290–294 (2013).

    CAS  Article  Google Scholar 

  12. 12

    Sanyal, A., Lajoie, B.R., Jain, G. & Dekker, J. The long-range interaction landscape of gene promoters. Nature 489, 109–113 (2012).

    CAS  Article  Google Scholar 

  13. 13

    Schwarzer, W. & Spitz, F. The architecture of gene expression: integrating dispersed cis-regulatory modules into coherent regulatory domains. Curr. Opin. Genet. Dev. 27, 74–82 (2014).

    CAS  Article  Google Scholar 

  14. 14

    Flavell, S.W. & Greenberg, M.E. Signaling mechanisms linking neuronal activity to gene expression and plasticity of the nervous system. Annu. Rev. Neurosci. 31, 563–590 (2008).

    CAS  Article  Google Scholar 

  15. 15

    Wu, H. et al. Tissue-specific RNA expression marks distant-acting developmental enhancers. PLoS Genet. 10, e1004610 (2014).

    Article  Google Scholar 

  16. 16

    Andersson, R. et al. An atlas of active enhancers across human cell types and tissues. Nature 507, 455–461 (2014).

    CAS  Article  Google Scholar 

  17. 17

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

    CAS  Article  Google Scholar 

  18. 18

    Core, L.J. et al. Analysis of nascent RNA identifies a unified architecture of initiation regions at mammalian promoters and enhancers. Nat. Genet. 46, 1311–1320 (2014).

    CAS  Article  Google Scholar 

  19. 19

    Pekowska, A. et al. H3K4 tri-methylation provides an epigenetic signature of active enhancers. EMBO J. 30, 4198–4210 (2011).

    CAS  Article  Google Scholar 

  20. 20

    Koch, F. et al. Transcription initiation platforms and GTF recruitment at tissue-specific enhancers and promoters. Nat. Struct. Mol. Biol. 18, 956–963 (2011).

    CAS  Article  Google Scholar 

  21. 21

    Schaukowitch, K. et al. Enhancer RNA facilitates NELF release from immediate early genes. Mol. Cell 56, 29–42 (2014).

    CAS  Article  Google Scholar 

  22. 22

    Arner, E. et al. Gene regulation. Transcribed enhancers lead waves of coordinated transcription in transitioning mammalian cells. Science 347, 1010–1014 (2015).

    CAS  Article  Google Scholar 

  23. 23

    Malik, A.N. et al. Genome-wide identification and characterization of functional neuronal activity-dependent enhancers. Nat. Neurosci. 17, 1330–1339 (2014).

    CAS  Article  Google Scholar 

  24. 24

    Heintzman, N.D. et al. Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature 459, 108–112 (2009).

    CAS  Article  Google Scholar 

  25. 25

    Dekker, J. The three 'C' s of chromosome conformation capture: controls, controls, controls. Nat. Methods 3, 17–21 (2006).

    CAS  Article  Google Scholar 

  26. 26

    Thomas-Chollier, M. et al. RSAT 2011: regulatory sequence analysis tools. Nucleic Acids Res. 39 (suppl. 2), W86–W91 (2011).

    CAS  Article  Google Scholar 

  27. 27

    Misra, R.P. et al. L-type voltage-sensitive calcium channel activation stimulates gene expression by a serum response factor-dependent pathway. J. Biol. Chem. 269, 25483–25493 (1994).

    CAS  PubMed  Google Scholar 

  28. 28

    Ramamoorthi, K. et al. Npas4 regulates a transcriptional program in CA3 required for contextual memory formation. Science 334, 1669–1675 (2011).

    CAS  Article  Google Scholar 

  29. 29

    Sheng, M., Dougan, S.T., McFadden, G. & Greenberg, M.E. Calcium and growth factor pathways of c-Fos transcriptional activation require distinct upstream regulatory sequences. Mol. Cell. Biol. 8, 2787–2796 (1988).

    CAS  Article  Google Scholar 

  30. 30

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

    CAS  Article  Google Scholar 

  31. 31

    Gilbert, L.A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

    CAS  Article  Google Scholar 

  32. 32

    Gilbert, L.A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).

    CAS  Article  Google Scholar 

  33. 33

    Lagha, M., Bothma, J.P. & Levine, M. Mechanisms of transcriptional precision in animal development. Trends Genet. 28, 409–416 (2012).

    CAS  Article  Google Scholar 

  34. 34

    Morgan, J.I., Cohen, D.R., Hempstead, J.L. & Curran, T. Mapping patterns of c-fos expression in the central nervous system after seizure. Science 237, 192–197 (1987).

    CAS  Article  Google Scholar 

  35. 35

    Monteggia, L.M. et al. Essential role of brain-derived neurotrophic factor in adult hippocampal function. Proc. Natl. Acad. Sci. USA 101, 10827–10832 (2004).

    CAS  Article  Google Scholar 

  36. 36

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

    CAS  Article  Google Scholar 

  37. 37

    Kawashima, T., Okuno, H. & Bito, H. A new era for functional labeling of neurons: activity-dependent promoters have come of age. Front. Neural Circuits 8, 37 (2014).

    Article  Google Scholar 

  38. 38

    Fleischmann, A. et al. Impaired long-term memory and NR2A-type NMDA receptor-dependent synaptic plasticity in mice lacking c-Fos in the CNS. J. Neurosci. 23, 9116–9122 (2003).

    CAS  Article  Google Scholar 

  39. 39

    Lamprecht, R. & Dudai, Y. Transient expression of c-Fos in rat amygdala during training is required for encoding conditioned taste aversion memory. Learn. Mem. 3, 31–41 (1996).

    CAS  Article  Google Scholar 

  40. 40

    Mileusnic, R., Anokhin, K. & Rose, S.P. Antisense oligodeoxynucleotides to c-fos are amnestic for passive avoidance in the chick. Neuroreport 7, 1269–1272 (1996).

    CAS  Article  Google Scholar 

  41. 41

    Barth, A.L., Gerkin, R.C. & Dean, K.L. Alteration of neuronal firing properties after in vivo experience in a fosGFP transgenic mouse. J. Neurosci. 24, 6466–6475 (2004).

    CAS  Article  Google Scholar 

  42. 42

    Guenthner, C.J., Miyamichi, K., Yang, H.H., Heller, H.C. & Luo, L. Permanent genetic access to transiently active neurons via TRAP: targeted recombination in active populations. Neuron 78, 773–784 (2013).

    CAS  Article  Google Scholar 

  43. 43

    Andrey, G. et al. A switch between topological domains underlies HoxD genes collinearity in mouse limbs. Science 340, 1234167 (2013).

    Article  Google Scholar 

  44. 44

    Chipumuro, E. et al. CDK7 inhibition suppresses super-enhancer-linked oncogenic transcription in MYCN-driven cancer. Cell 159, 1126–1139 (2014).

    CAS  Article  Google Scholar 

  45. 45

    Whyte, W.A. et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319 (2013).

    CAS  Article  Google Scholar 

  46. 46

    Hnisz, D. et al. Convergence of developmental and oncogenic signaling pathways at transcriptional super-enhancers. Mol. Cell 58, 362–370 (2015).

    CAS  Article  Google Scholar 

  47. 47

    Pinheiro, E.M. et al. Lpd depletion reveals that SRF specifies radial versus tangential migration of pyramidal neurons. Nat. Cell Biol. 13, 989–995 (2011).

    CAS  Article  Google Scholar 

  48. 48

    Rexach, J.E. et al. Dynamic O-GlcNAc modification regulates CREB-mediated gene expression and memory formation. Nat. Chem. Biol. 8, 253–261 (2012).

    CAS  Article  Google Scholar 

  49. 49

    Flavell, S.W. et al. Activity-dependent regulation of MEF2 transcription factors suppresses excitatory synapse number. Science 311, 1008–1012 (2006).

    CAS  Article  Google Scholar 

  50. 50

    Pereira, A.H. et al. MEF2C silencing attenuates load-induced left ventricular hypertrophy by modulating mTOR/S6K pathway in mice. PLoS ONE 4, e8472 (2009).

    Article  Google Scholar 

  51. 51

    Heigwer, F., Kerr, G. & Boutros, M. E-CRISP: fast CRISPR target site identification. Nat. Methods 11, 122–123 (2014).

    CAS  Article  Google Scholar 

  52. 52

    Sanjana, N.E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).

    CAS  Article  Google Scholar 

  53. 53

    Flavell, S.W. et al. Genome-wide analysis of MEF2 transcriptional program reveals synaptic target genes and neuronal activity-dependent polyadenylation site selection. Neuron 60, 1022–1038 (2008).

    CAS  Article  Google Scholar 

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We thank L. Monteggia and members of her laboratory for providing conditional Bdnf KO mice. We thank M. Greenberg for providing MEF2A and MEF2D antibodies used for ChIP experiments. We also thank W. Xu for advice on stereotaxic injection, and C. Green and J. Stubblefield for providing the dark controlled-environment chamber. This work was supported by the US National Science Foundation (NSF) BRAIN EAGER Award (IOS1451034) and the US National Institute of Neurological Disorders and Stroke (NINDS) under award number R01NS085418 (T.-K.K.).

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T.-K.K. and J.-Y.J. designed the project; J.-Y.J. performed CRISPRi, 3C assays, luciferase reporter assay, ChIP, immunocytochemistry, in vivo experiments, in vitro analysis of transcription factor knockdown and eRNA and mRNA expression analysis. K.S. performed western blot and expression analysis for c-fos eRNA in NIH 3T3 cells. L.F. performed primary cortical neuron culture, helped to make lentivirus constructs and helped in CRISPRi and shRNA cloning. G.K. performed bioinformatics analysis. J.-Y.J., K.S. and T.-K.K. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Tae-Kyung Kim.

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Joo, JY., Schaukowitch, K., Farbiak, L. et al. Stimulus-specific combinatorial functionality of neuronal c-fos enhancers. Nat Neurosci 19, 75–83 (2016).

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