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Stimulus-specific combinatorial functionality of neuronal c-fos enhancers.

Joo JY, Schaukowitch K, Farbiak L, Kilaru G, Kim TK - Nat. Neurosci. (2015)

Bottom Line: Here we demonstrate that multiple enhancers surrounding the c-fos gene are crucial for ensuring robust c-fos response to various stimuli.Accordingly, the functional requirement of key transcription factors varies depending on the type of stimulation.Combinatorial enhancer activation also occurs in the brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

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. Its induction mechanism and available reporter mouse lines are based exclusively on c-fos promoter 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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The c-fos enhancer activities measured by eRNA analysis and luciferase reporter assay. (a) Expression of c-fos mRNA and eRNA in cortical neurons induced by KCl 30 min, BDNF 1 h, and Forskolin 1 h. The induction levels of the five c-fos eRNAs and mRNA were measured using RT-qPCR and normalized to Gapdh mRNA (c-fos mRNA, KCl: P = 0.0418, t(2) = 4.736, n = 3, BDNF: P = 0.0333, t(2) = 5.339, n = 3, Forskolin: P = 0.0448, t(2) = 4.566, n = 3, unpaired t-test with Welch correction; c-fos eRNA, KCl, e1: P = 0.0418, t(4) = 6.654, F = 10.187 [SDP = 0.0894], n = 3, e2: P = 0.008, t(4) = 11.103, F = 3.149 [SDP = 0.3267], n = 3, e5: P = 0.0264, t(4) = 3.436, F = 26.341 [SDP = 0.0566], n = 3, unpaired t-test, BDNF, e1: P = 0.0329, t(4) = 3.200, F = 16.876 [SDP = 0.0599], n = 3, e4: P = 0.0271, t(4) = 3.408, F = 17.578 [SDP = 0.0538], n = 3, unpaired t-test, e5 P = 0.0214, t(2) = 6.723, n = 3, unpaired t-test with Welch correction, Forskolin, e1: P = 0.0498, t(4) = 2.718, F = 6.333 [SDP = 0.1364], n = 3, e5: P = 0.0485, t(4) = 2.805, F = 4.048 [SDP = 0.1981], n = 3 biological replicates, unpaired t-test). (b,c) Combinational c-fos enhancer activities measured by the luciferase reporter assay. Each of the c-fos enhancers was fused directly upstream of the c-fos promoter, which then together drives luciferase expression. Stimulus-induced luciferase activities were measured after 6 h of KCl, BDNF, or Forskolin treatment in cortical neurons. (b, KCl, e1: P = 0.0242. t(3) = 6.306, n = 4, e2: P = 0.0103. t(3) = 5.784, n = 4, e5: P = 0.0191. t(2) = 7.140, n = 3, BDNF, e1: P = 0.0350. t(2) = 5.207, n = 3, e4: P = 0.0056. t(2) = 13.349, n = 3, e5: P = 0.0391. t(2) = 4.907, n = 3, Forskolin, e1 P = 0.0087. t(2) = 10.633, n = 3, e5: P = 0.0438. t(2) = 4.618, n = 3 biological replicates, unpaired t-test with Welch correction; c, KCl, e1+e2+e4+e5: P = 0.0001, t(5) = 12.256, n = 6, e1+e2+e5: P = 0.0246, t(2) = 6.259, n = 2, e1+e4+e5: P = 0.0240, t(2) = 6.576, n = 2, e1+e5: P = 0.0128, t(2) = 8.764, n = 2 biological replicates, unpaired t-test with Welch correction, BDNF, e1+e2+e4+e5: P = 0.0007, t(7) = 5.818, n = 6, unpaired t-test with Welch correction, e1+e2+e5: P = 0.0034, t(3) = 8.537, F = 1.411 [SDP = 0.3569], n = 3, e1+e4+e5: P = 0.0004, t(3) = 18.125, F = 6.346 [SDP = 0.1280], n = 3, e1+e5: P = 0.00303, t(3) = 2.822, F = 63.970 [SDP = 0.0946], n = 3, biological replicates, unpaired t-test, Forskolin, e1+e2+e4+e5: P = 0.0001, t(5) = 12.601, n = 6, unpaired t-test with Welch correction, e1+e2+e5: P = 0.0189, t(2) = 7.164, F = 126.28 [SDP = 0.0565], n = 3 biological replicates, unpaired t-test). Error bars indicate SEM; p values are from a two-tailed t test. NS, not significant. SDP is the P value from comparing the standard deviations for both groups. SDP > 0.05 means the two SDs are not significantly different and can be used for an unpaired t-test.
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Figure 2: The c-fos enhancer activities measured by eRNA analysis and luciferase reporter assay. (a) Expression of c-fos mRNA and eRNA in cortical neurons induced by KCl 30 min, BDNF 1 h, and Forskolin 1 h. The induction levels of the five c-fos eRNAs and mRNA were measured using RT-qPCR and normalized to Gapdh mRNA (c-fos mRNA, KCl: P = 0.0418, t(2) = 4.736, n = 3, BDNF: P = 0.0333, t(2) = 5.339, n = 3, Forskolin: P = 0.0448, t(2) = 4.566, n = 3, unpaired t-test with Welch correction; c-fos eRNA, KCl, e1: P = 0.0418, t(4) = 6.654, F = 10.187 [SDP = 0.0894], n = 3, e2: P = 0.008, t(4) = 11.103, F = 3.149 [SDP = 0.3267], n = 3, e5: P = 0.0264, t(4) = 3.436, F = 26.341 [SDP = 0.0566], n = 3, unpaired t-test, BDNF, e1: P = 0.0329, t(4) = 3.200, F = 16.876 [SDP = 0.0599], n = 3, e4: P = 0.0271, t(4) = 3.408, F = 17.578 [SDP = 0.0538], n = 3, unpaired t-test, e5 P = 0.0214, t(2) = 6.723, n = 3, unpaired t-test with Welch correction, Forskolin, e1: P = 0.0498, t(4) = 2.718, F = 6.333 [SDP = 0.1364], n = 3, e5: P = 0.0485, t(4) = 2.805, F = 4.048 [SDP = 0.1981], n = 3 biological replicates, unpaired t-test). (b,c) Combinational c-fos enhancer activities measured by the luciferase reporter assay. Each of the c-fos enhancers was fused directly upstream of the c-fos promoter, which then together drives luciferase expression. Stimulus-induced luciferase activities were measured after 6 h of KCl, BDNF, or Forskolin treatment in cortical neurons. (b, KCl, e1: P = 0.0242. t(3) = 6.306, n = 4, e2: P = 0.0103. t(3) = 5.784, n = 4, e5: P = 0.0191. t(2) = 7.140, n = 3, BDNF, e1: P = 0.0350. t(2) = 5.207, n = 3, e4: P = 0.0056. t(2) = 13.349, n = 3, e5: P = 0.0391. t(2) = 4.907, n = 3, Forskolin, e1 P = 0.0087. t(2) = 10.633, n = 3, e5: P = 0.0438. t(2) = 4.618, n = 3 biological replicates, unpaired t-test with Welch correction; c, KCl, e1+e2+e4+e5: P = 0.0001, t(5) = 12.256, n = 6, e1+e2+e5: P = 0.0246, t(2) = 6.259, n = 2, e1+e4+e5: P = 0.0240, t(2) = 6.576, n = 2, e1+e5: P = 0.0128, t(2) = 8.764, n = 2 biological replicates, unpaired t-test with Welch correction, BDNF, e1+e2+e4+e5: P = 0.0007, t(7) = 5.818, n = 6, unpaired t-test with Welch correction, e1+e2+e5: P = 0.0034, t(3) = 8.537, F = 1.411 [SDP = 0.3569], n = 3, e1+e4+e5: P = 0.0004, t(3) = 18.125, F = 6.346 [SDP = 0.1280], n = 3, e1+e5: P = 0.00303, t(3) = 2.822, F = 63.970 [SDP = 0.0946], n = 3, biological replicates, unpaired t-test, Forskolin, e1+e2+e4+e5: P = 0.0001, t(5) = 12.601, n = 6, unpaired t-test with Welch correction, e1+e2+e5: P = 0.0189, t(2) = 7.164, F = 126.28 [SDP = 0.0565], n = 3 biological replicates, unpaired t-test). Error bars indicate SEM; p values are from a two-tailed t test. NS, not significant. SDP is the P value from comparing the standard deviations for both groups. SDP > 0.05 means the two SDs are not significantly different and can be used for an unpaired t-test.

Mentions: We reasoned that the previously observed broad inducibility of the c-fos gene might be enabled by synergistic and/or combinatorial actions of the surrounding enhancers. As the first step to test this idea, we treated neuronal cultures with three different stimuli – 55 mM KCl, Brain derived nerve growth factor (BDNF), and Forskolin which stimulates adenylate cyclase – to induce c-fos transcription, and then measured eRNA induction levels, which is an indicator of enhancer activity (Fig. 2a) 6, 12, 15, 16. All three stimuli effectively increased c-fos mRNA levels (Fig. 2aupper panel). Consistent with the RNA-seq profile, KCl-mediated membrane depolarization induced eRNA expression mainly from e1, e2, and e5. On the other hand, BDNF and Forskolin activated different subsets of the c-fos enhancers (e1, e4 and e5 by BDNF, and e1 and e5 by Forskolin) (Fig. 2alower panel). In all cases, eRNA expression from the e3 enhancer was not reliably detected, which is consistent with the previous observation that the H3K27ac level is the lowest at e3 among the c-fos enhancers (Fig. 1andSupplementary Fig. 1) 17, 23. This result suggests that different stimuli promote c-fos transcription through the activation of distinct subsets of the surrounding enhancers.


Stimulus-specific combinatorial functionality of neuronal c-fos enhancers.

Joo JY, Schaukowitch K, Farbiak L, Kilaru G, Kim TK - Nat. Neurosci. (2015)

The c-fos enhancer activities measured by eRNA analysis and luciferase reporter assay. (a) Expression of c-fos mRNA and eRNA in cortical neurons induced by KCl 30 min, BDNF 1 h, and Forskolin 1 h. The induction levels of the five c-fos eRNAs and mRNA were measured using RT-qPCR and normalized to Gapdh mRNA (c-fos mRNA, KCl: P = 0.0418, t(2) = 4.736, n = 3, BDNF: P = 0.0333, t(2) = 5.339, n = 3, Forskolin: P = 0.0448, t(2) = 4.566, n = 3, unpaired t-test with Welch correction; c-fos eRNA, KCl, e1: P = 0.0418, t(4) = 6.654, F = 10.187 [SDP = 0.0894], n = 3, e2: P = 0.008, t(4) = 11.103, F = 3.149 [SDP = 0.3267], n = 3, e5: P = 0.0264, t(4) = 3.436, F = 26.341 [SDP = 0.0566], n = 3, unpaired t-test, BDNF, e1: P = 0.0329, t(4) = 3.200, F = 16.876 [SDP = 0.0599], n = 3, e4: P = 0.0271, t(4) = 3.408, F = 17.578 [SDP = 0.0538], n = 3, unpaired t-test, e5 P = 0.0214, t(2) = 6.723, n = 3, unpaired t-test with Welch correction, Forskolin, e1: P = 0.0498, t(4) = 2.718, F = 6.333 [SDP = 0.1364], n = 3, e5: P = 0.0485, t(4) = 2.805, F = 4.048 [SDP = 0.1981], n = 3 biological replicates, unpaired t-test). (b,c) Combinational c-fos enhancer activities measured by the luciferase reporter assay. Each of the c-fos enhancers was fused directly upstream of the c-fos promoter, which then together drives luciferase expression. Stimulus-induced luciferase activities were measured after 6 h of KCl, BDNF, or Forskolin treatment in cortical neurons. (b, KCl, e1: P = 0.0242. t(3) = 6.306, n = 4, e2: P = 0.0103. t(3) = 5.784, n = 4, e5: P = 0.0191. t(2) = 7.140, n = 3, BDNF, e1: P = 0.0350. t(2) = 5.207, n = 3, e4: P = 0.0056. t(2) = 13.349, n = 3, e5: P = 0.0391. t(2) = 4.907, n = 3, Forskolin, e1 P = 0.0087. t(2) = 10.633, n = 3, e5: P = 0.0438. t(2) = 4.618, n = 3 biological replicates, unpaired t-test with Welch correction; c, KCl, e1+e2+e4+e5: P = 0.0001, t(5) = 12.256, n = 6, e1+e2+e5: P = 0.0246, t(2) = 6.259, n = 2, e1+e4+e5: P = 0.0240, t(2) = 6.576, n = 2, e1+e5: P = 0.0128, t(2) = 8.764, n = 2 biological replicates, unpaired t-test with Welch correction, BDNF, e1+e2+e4+e5: P = 0.0007, t(7) = 5.818, n = 6, unpaired t-test with Welch correction, e1+e2+e5: P = 0.0034, t(3) = 8.537, F = 1.411 [SDP = 0.3569], n = 3, e1+e4+e5: P = 0.0004, t(3) = 18.125, F = 6.346 [SDP = 0.1280], n = 3, e1+e5: P = 0.00303, t(3) = 2.822, F = 63.970 [SDP = 0.0946], n = 3, biological replicates, unpaired t-test, Forskolin, e1+e2+e4+e5: P = 0.0001, t(5) = 12.601, n = 6, unpaired t-test with Welch correction, e1+e2+e5: P = 0.0189, t(2) = 7.164, F = 126.28 [SDP = 0.0565], n = 3 biological replicates, unpaired t-test). Error bars indicate SEM; p values are from a two-tailed t test. NS, not significant. SDP is the P value from comparing the standard deviations for both groups. SDP > 0.05 means the two SDs are not significantly different and can be used for an unpaired t-test.
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Figure 2: The c-fos enhancer activities measured by eRNA analysis and luciferase reporter assay. (a) Expression of c-fos mRNA and eRNA in cortical neurons induced by KCl 30 min, BDNF 1 h, and Forskolin 1 h. The induction levels of the five c-fos eRNAs and mRNA were measured using RT-qPCR and normalized to Gapdh mRNA (c-fos mRNA, KCl: P = 0.0418, t(2) = 4.736, n = 3, BDNF: P = 0.0333, t(2) = 5.339, n = 3, Forskolin: P = 0.0448, t(2) = 4.566, n = 3, unpaired t-test with Welch correction; c-fos eRNA, KCl, e1: P = 0.0418, t(4) = 6.654, F = 10.187 [SDP = 0.0894], n = 3, e2: P = 0.008, t(4) = 11.103, F = 3.149 [SDP = 0.3267], n = 3, e5: P = 0.0264, t(4) = 3.436, F = 26.341 [SDP = 0.0566], n = 3, unpaired t-test, BDNF, e1: P = 0.0329, t(4) = 3.200, F = 16.876 [SDP = 0.0599], n = 3, e4: P = 0.0271, t(4) = 3.408, F = 17.578 [SDP = 0.0538], n = 3, unpaired t-test, e5 P = 0.0214, t(2) = 6.723, n = 3, unpaired t-test with Welch correction, Forskolin, e1: P = 0.0498, t(4) = 2.718, F = 6.333 [SDP = 0.1364], n = 3, e5: P = 0.0485, t(4) = 2.805, F = 4.048 [SDP = 0.1981], n = 3 biological replicates, unpaired t-test). (b,c) Combinational c-fos enhancer activities measured by the luciferase reporter assay. Each of the c-fos enhancers was fused directly upstream of the c-fos promoter, which then together drives luciferase expression. Stimulus-induced luciferase activities were measured after 6 h of KCl, BDNF, or Forskolin treatment in cortical neurons. (b, KCl, e1: P = 0.0242. t(3) = 6.306, n = 4, e2: P = 0.0103. t(3) = 5.784, n = 4, e5: P = 0.0191. t(2) = 7.140, n = 3, BDNF, e1: P = 0.0350. t(2) = 5.207, n = 3, e4: P = 0.0056. t(2) = 13.349, n = 3, e5: P = 0.0391. t(2) = 4.907, n = 3, Forskolin, e1 P = 0.0087. t(2) = 10.633, n = 3, e5: P = 0.0438. t(2) = 4.618, n = 3 biological replicates, unpaired t-test with Welch correction; c, KCl, e1+e2+e4+e5: P = 0.0001, t(5) = 12.256, n = 6, e1+e2+e5: P = 0.0246, t(2) = 6.259, n = 2, e1+e4+e5: P = 0.0240, t(2) = 6.576, n = 2, e1+e5: P = 0.0128, t(2) = 8.764, n = 2 biological replicates, unpaired t-test with Welch correction, BDNF, e1+e2+e4+e5: P = 0.0007, t(7) = 5.818, n = 6, unpaired t-test with Welch correction, e1+e2+e5: P = 0.0034, t(3) = 8.537, F = 1.411 [SDP = 0.3569], n = 3, e1+e4+e5: P = 0.0004, t(3) = 18.125, F = 6.346 [SDP = 0.1280], n = 3, e1+e5: P = 0.00303, t(3) = 2.822, F = 63.970 [SDP = 0.0946], n = 3, biological replicates, unpaired t-test, Forskolin, e1+e2+e4+e5: P = 0.0001, t(5) = 12.601, n = 6, unpaired t-test with Welch correction, e1+e2+e5: P = 0.0189, t(2) = 7.164, F = 126.28 [SDP = 0.0565], n = 3 biological replicates, unpaired t-test). Error bars indicate SEM; p values are from a two-tailed t test. NS, not significant. SDP is the P value from comparing the standard deviations for both groups. SDP > 0.05 means the two SDs are not significantly different and can be used for an unpaired t-test.
Mentions: We reasoned that the previously observed broad inducibility of the c-fos gene might be enabled by synergistic and/or combinatorial actions of the surrounding enhancers. As the first step to test this idea, we treated neuronal cultures with three different stimuli – 55 mM KCl, Brain derived nerve growth factor (BDNF), and Forskolin which stimulates adenylate cyclase – to induce c-fos transcription, and then measured eRNA induction levels, which is an indicator of enhancer activity (Fig. 2a) 6, 12, 15, 16. All three stimuli effectively increased c-fos mRNA levels (Fig. 2aupper panel). Consistent with the RNA-seq profile, KCl-mediated membrane depolarization induced eRNA expression mainly from e1, e2, and e5. On the other hand, BDNF and Forskolin activated different subsets of the c-fos enhancers (e1, e4 and e5 by BDNF, and e1 and e5 by Forskolin) (Fig. 2alower panel). In all cases, eRNA expression from the e3 enhancer was not reliably detected, which is consistent with the previous observation that the H3K27ac level is the lowest at e3 among the c-fos enhancers (Fig. 1andSupplementary Fig. 1) 17, 23. This result suggests that different stimuli promote c-fos transcription through the activation of distinct subsets of the surrounding enhancers.

Bottom Line: Here we demonstrate that multiple enhancers surrounding the c-fos gene are crucial for ensuring robust c-fos response to various stimuli.Accordingly, the functional requirement of key transcription factors varies depending on the type of stimulation.Combinatorial enhancer activation also occurs in the brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

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. Its induction mechanism and available reporter mouse lines are based exclusively on c-fos promoter 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.

Show MeSH
Related in: MedlinePlus