Abstract

We used genome-wide sequencing methods to study stimulus-dependent enhancer function in mouse cortical neurons. We identified 12,000 neuronal activity-regulated enhancers that are bound by the general transcriptional co-activator CBP in an activity-dependent manner. A function of CBP at enhancers may be to recruit RNA polymerase II (RNAPII), as we also observed activity-regulated RNAPII binding to thousands of enhancers. Notably, RNAPII at enhancers transcribes bi-directionally a novel class of enhancer RNAs (eRNAs) within enhancer domains defined by the presence of histone H3 monomethylated at lysine 4. The level of eRNA expression at neuronal enhancers positively correlates with the level of messenger RNA synthesis at nearby genes, suggesting that eRNA synthesis occurs specifically at enhancers that are actively engaged in promoting mRNA synthesis. These findings reveal that a widespread mechanism of enhancer activation involves RNAPII binding and eRNA synthesis.

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Gene Expression Omnibus

Data deposits

Sequencing data have been submitted to the GEO repository under accession numbers GSE21161 (for all ChIP-Seq and RNA-Seq data) and HM047267 (for circularized Arc enhancer RNA). The bigWig files for genome browser visualization are posted online (see Supplementary Table 6).

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Acknowledgements

We thank members of the Greenberg laboratory for discussions and for critical reading of the manuscript. We thank S. Vasquez for preparing dissociated mouse cortical neurons. We thank L. Hu for generating antibodies. We thank the Molecular Genetics Core Facility at Children’s Hospital Boston, including H. Schneider and S. Burgess, for operation of their SOLiD 3.0 sequencer (I.D.D.R.C). We thank the support and R&D teams at Life Technologies including S. Ranade, R. David, J. Ni, C. Barbacioru, M. Barker, G. Costa and K. McKernan. M.E.G. acknowledges the support of the Nancy Lurie Marks Family Foundation. We thank M. Dehoff for technical support in the Arc knockout experiments. This work was supported by the National Institutes of Health grants NS028829 (M.E.G.), R21EY019710 (G.K.), DP2OD006461 (G.K.) and MH-053608 (P.F.W.). This work was also supported by The Lefler postdoctoral fellowship (T-K.K.) and The Jane Coffin Childs Memorial Funds (T-K.K.), The Helen Hay Whitney postdoctoral fellowship (J.M.G.), The Children’s Hospital Ophthalmology Foundation (G.K.), The Whitehall Foundation (G.K.), and The Klingenstein Fund (G.K.)

Author information

Author notes

    • Tae-Kyung Kim
    • , Martin Hemberg
    •  & Jesse M. Gray

    These authors contributed equally to this work.

    • Tae-Kyung Kim
    •  & Eirene Markenscoff-Papadimitriou

    Present addresses: University of Texas Southwestern Medical Center, Department of Neuroscience, 5323 Harry Hines Blvd, Dallas, Texas 75390-9111, USA (T.-K.K.); Graduate Program in Neuroscience, University of California San Francisco, 1550 4th Street, San Francisco, California 94158, USA (E.M.-P.).

Affiliations

  1. Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, Massachusetts 02115, USA

    • Tae-Kyung Kim
    • , Jesse M. Gray
    • , Allen M. Costa
    • , Daniel M. Bear
    • , David A. Harmin
    • , Mike Laptewicz
    • , Eirene Markenscoff-Papadimitriou
    •  & Michael E. Greenberg
  2. Department of Ophthalmology, Children’s Hospital Boston, Center for Brain Science and Swartz Center for Theoretical Neuroscience, Harvard University, 300 Longwood Avenue, Boston, Massachusetts 02115, USA

    • Martin Hemberg
    •  & Gabriel Kreiman
  3. The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA

    • Jing Wu
    •  & Paul F. Worley
  4. Children’s Hospital Informatics Program at the Harvard-MIT Division of Health Sciences and Technology, 300 Longwood Avenue, Boston, Massachusetts 02115, USA

    • David A. Harmin
  5. Molecular Genetics Core facility, Children’s Hospital Boston, 300 Longwood Avenue, Boston, Massachusetts 02115, USA

    • Kellie Barbara-Haley
  6. Epicentre Biotechnologies, 726 Post Road, Madison, Wisconsin 53713, USA

    • Scott Kuersten
  7. Institute for Molecular and Cellular Cognition (IMCC), Center for Molecular Neurobiology (ZMNH), University Medical Center Hamburg-Eppendorf (UKE), Falkenried 94, 20251 Hamburg, Germany

    • Dietmar Kuhl
  8. Department of Neurochemistry, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan

    • Haruhiko Bito

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Contributions

Author Contributions T-K.K., J.M.G. and M.E.G. conceived and designed experiments. T-K.K., J.M.G., M.H., G.K. and M.E.G. wrote the manuscript. T-K.K. optimized the protocol for ChIP-Seq library preparation to be suitable for the SOLiD sequencer and made all ChIP-Seq libraries used in this study. S.K. invented the library construction methodology used for all RNA sequencing reported here. J.M.G., A.M.C. and E.M.-P. made all RNA-Seq libraries. M.H., J.M.G. and D.A.H. performed bioinformatic analyses. K.B.-H. carried out the SOLiD bead preparation and sequencing. T-K.K., J.W., P.F.W. and A.M.C. performed the Arc knockout experiment. D.M.B. performed the luciferase experiments. M.L. performed the RNA circularization experiment. H.B. provided the pArc7000 plasmid. D.K. provided the Arc knockout mouse. All authors reviewed the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Michael E. Greenberg.

Supplementary information

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  1. 1.

    Supplementary Figures

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  1. 1.

    This file contains Supplementary Figures 1-11 with legends.

    This zipped file comprises Supplementary Tables as follows: Supplementary Table S1 shows the number of CBP sites that were removed at each step of filtering in order to produce a list of high-confidence enhancers. Supplementary Table S2 shows ChIP-Seq, RNA-Seq, and other data associated with each CBP peak, with ~41,000 CBP peaks as rows and nearly 200 columns of information about each peak. Supplementary Tables S3a and S3b show lists of TF peaks found in the 2hour KCl stimulated and unstimulated conditions, with positive values for any given factor indicating the presence of a peak. Supplementary Table S4 shows the number of extragenic enhancers that have TFs, RNAPII, or eRNAs detected, as well as the number of enhancers with any two of these features detected. Supplementary Table S5 shows the number of ChIP-Seq/RNA-Seq reads for each experiment and the antibody used for each ChIP-Seq experiment. Supplementary Table S6 contains text that can be pasted into the UCSC Genome Browser to display the raw ChIP-Seq/RNA-Seq sequencing data using the mm9 mouse genome. Supplementary Table S7 shows DAVID analysis of the genes whose promoters bind CREB. Supplementary Table S8 shows a list of genes with expression changes that were significant following 6 hours KCl stimulation, based on RNA-Seq biological replicate 1. Supplementary Tables S9a and 9b show DAVID analysis of strongly up-and down-regulated genes. Supplementary Table S10 shows primers used for RT-qPCR validation.

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https://doi.org/10.1038/nature09033

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