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
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).
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.)
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.