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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

Abstract

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.

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Acknowledgements

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). https://doi.org/10.1038/nn.4170

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