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. 2020 Aug 18;48(17):9550–9570. doi: 10.1093/nar/gkaa671

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© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Activity dependence of enhancers and eRNAs. (A) Illustration of activity-dependent TAPE identification. (B) Volcano plots showing differentially expressed TAPEs in primary neuronal cultures generated from rat cortex, hippocampus, and striatum. (C) mRNA levels from TAPE-linked genes are predicted by the direction of eRNA changes at upregulated and downregulated TAPEs (Wilcoxon signed rank test with theoretical median = 0 for upregulated TAPEs, n = 77, P < 0.0001, and downregulated TAPEs n = 15, P < 0.0001). Chromatin accessibility decreases at gene promoters that correspond to upregulated TAPEs (Wilcoxon signed rank test with theoretical median = 0 for upregulated TAPEs, n = 78, P < 0.0001, and downregulated TAPEs, n = 15, P < 0.0730). ATAC-seq signal also decreases at both up- and downregulated TAPEs (Wilcoxon signed rank test with theoretical median = 0 for upregulated TAPEs, n = 78, P < 0.0001, and downregulated TAPEs, n = 15, P < 0.0103). (D–F) Genomic locus of Fos, Fosb, and Nr4a1 genes and their surrounding enhancer regions. (G–I) Time course experiments following neuronal depolarization with 25 mM KCl revealed that eRNAs are induced prior (Fos eRNA1, eRNA3, and Nr4a1 eRNA) or at the same time (Fosb eRNA) as mRNAs are induced (two-way ANOVA for Fos eRNA1, F(6,153) = 41.81, P < 0.0001, Fos eRNA3 F(6,154) = 37.87, P < 0.0001, Fos mRNA, F(6,154) = 456, P < 0.0001, Nr4a1 eRNA, F(6,154) = 31.4, P < 0.0001, Nr4a1 mRNA, F(6,154) = 311.3, P < 0.0001, Fosb eRNA, F(6,154) = 8.341, P < 0.0001, Fosb mRNA, F(6,154) = 98.34, P < 0.0001, Sidak's post hoc test for multiple comparisons). Inverted triangles represent P < 0.05 as compared to vehicle treated controls. (J) Summary of KCl time course experiments plotted as percentage of maximal response. (K) RT-qPCR analysis of eRNA and mRNA expression in response to 1 hr treatment with KCl, AMPA, and NMDA reveals activity-dependent induction of Fos eRNA1 and eRNA3, while FSK and TTX treatment had no effects on Fos eRNA expression (Kurskal–Wallis test for eRNA1 KCl F (3,32) = 25.04, P < 0.0001, AMPA F(3,32) = 20.81, P = 0.0001, NMDA F(3,32) = 17.79, P = 0.0005, FSK F(3,32) = 1.967, P = 0.5793, eRNA3 KCl F(3,32) = 26.52, P < 0.0001, AMPA F(3,32) = 26.11, P < 0.0001, NMDA F(3,32) = 15.66, P = 0.0013, FSK F(3,32) = 5.961, P = 0.1135, and mRNA KCl F(3,32) = 28.26, P < 0.0001, AMPA F(3,32) = 29.79, P < 0.0001, NMDA F(3,32) = 29.79, P < 0.0005, FSK F(3,32) = 30.28, P < 0.0001, with Dunn's post hoc test for multiple comparisons, and unpaired t-test for eRNA1 TTX t(14) = 0.1740, P = 0.8644, eRNA3 TTX t(14) = 1.461, P = 0.166, and mRNA TTX t(14) = 2.346, P = 0.0342). Data expressed as mean ± s.e.m. Multiple comparisons, *P < 0.05, **P < 0.01, *** P <0.001, ****P < 0.0001.