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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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Characterization of c-fos enhancer activities in vivo. (a) c-fos mRNA expression in the cortex, hippocampus, and cerebellum following KA-evoked seizure. c-fos mRNA was measured using RT-qPCR 1 h after saline or KA administration (note that the y axes are scaled differently). c-fos eRNA induction in the cortex, hippocampus, and cerebellum following KA-evoked seizure (c-fos mRNA, Cortex: P = 0.0458, t(4) = 4.511, n = 5 mice, Hippocampus: P = 0.0067, t(5) = 4.455, n = 5 mice, Cerebellum: P = 0.0133, t(4) = 47.767, n = 5 mice; Cortex, c-fos e4, P = 0.0150, t(4) = 4.084, n = 5 mice, c-fos e5, P = 0.034, t(4) = 3.154, n = 5 mice; Hippocampus, c-fos e1, P = 0.0386, t(4) = 3.036, n = 5 mice, c-fos e2, P = 0.0419, t(5) = 2.717, n = 5 mice, c-fos e4, P = 0.0211, t(4) = 3.686, n = 5 mice, c-fos e5, P = 0.0028, t(5) = 5.454, n = 5 mice). (b) Expression of c-fos mRNA in the visual cortex of mice treated with light stimulation. c-fos eRNA induction in the visual cortex of mice following light stimulation. The levels of mRNA and eRNA were normalized to the level of Gapdh mRNA (c-fos mRNA, Visual cortex: P = 0.0069, t(2) = 11.966, n = 3 mice; c-fos e5, P = 0.0485, t(4) = 2.806, n = 3 mice). All unpaired t-test with Welch correction. Error bars indicate SEM; p values are from a two-tailed t test.
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Figure 6: Characterization of c-fos enhancer activities in vivo. (a) c-fos mRNA expression in the cortex, hippocampus, and cerebellum following KA-evoked seizure. c-fos mRNA was measured using RT-qPCR 1 h after saline or KA administration (note that the y axes are scaled differently). c-fos eRNA induction in the cortex, hippocampus, and cerebellum following KA-evoked seizure (c-fos mRNA, Cortex: P = 0.0458, t(4) = 4.511, n = 5 mice, Hippocampus: P = 0.0067, t(5) = 4.455, n = 5 mice, Cerebellum: P = 0.0133, t(4) = 47.767, n = 5 mice; Cortex, c-fos e4, P = 0.0150, t(4) = 4.084, n = 5 mice, c-fos e5, P = 0.034, t(4) = 3.154, n = 5 mice; Hippocampus, c-fos e1, P = 0.0386, t(4) = 3.036, n = 5 mice, c-fos e2, P = 0.0419, t(5) = 2.717, n = 5 mice, c-fos e4, P = 0.0211, t(4) = 3.686, n = 5 mice, c-fos e5, P = 0.0028, t(5) = 5.454, n = 5 mice). (b) Expression of c-fos mRNA in the visual cortex of mice treated with light stimulation. c-fos eRNA induction in the visual cortex of mice following light stimulation. The levels of mRNA and eRNA were normalized to the level of Gapdh mRNA (c-fos mRNA, Visual cortex: P = 0.0069, t(2) = 11.966, n = 3 mice; c-fos e5, P = 0.0485, t(4) = 2.806, n = 3 mice). All unpaired t-test with Welch correction. Error bars indicate SEM; p values are from a two-tailed t test.

Mentions: We next asked if combinatorial activation of c-fos enhancers also occurs in the brain. First we examined whether different brain regions activate distinctive sets of the c-fos enhancers to mediate c-fos induction upon chemically induced seizure. Young adult mice were injected intraperitoneally with Kainic acid (KA), which activates glutamate receptors to induce seizure. One hour after KA injection, a strong c-fos mRNA induction was detected in the cerebral cortex and hippocampus, and also weakly in the cerebellum (Fig. 6a) 34. We then measured individual eRNA levels in those brain regions. A single injection of KA led to induction of c-fos eRNAs from e4 and e5 in the cerebral cortex. On the other hand, eRNAs from all c-fos enhancers except e3 were induced in the hippocampus. We could not detect any induction of eRNA in the cerebellum, possibly because the KA-induced c-fos mRNA induction level was lower than other brain regions. We also investigated whether sensory stimulation can trigger eRNA transcription from the c-fos enhancers in the intact brain. We took dark-reared mice and stimulated their visual cortex by light for 1 h. The RT-qPCR analysis of the RNAs isolated from the visual cortex following visual stimulation detected eRNAs only from the e5 enhancer region (Fig. 6b). These results support our findings from in vitro neuronal culture that different combinations of enhancers are utilized to ensure c-fos induction in response to various stimuli, and also illustrate region-specific combinatorial activation of the c-fos enhancers.


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

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

Characterization of c-fos enhancer activities in vivo. (a) c-fos mRNA expression in the cortex, hippocampus, and cerebellum following KA-evoked seizure. c-fos mRNA was measured using RT-qPCR 1 h after saline or KA administration (note that the y axes are scaled differently). c-fos eRNA induction in the cortex, hippocampus, and cerebellum following KA-evoked seizure (c-fos mRNA, Cortex: P = 0.0458, t(4) = 4.511, n = 5 mice, Hippocampus: P = 0.0067, t(5) = 4.455, n = 5 mice, Cerebellum: P = 0.0133, t(4) = 47.767, n = 5 mice; Cortex, c-fos e4, P = 0.0150, t(4) = 4.084, n = 5 mice, c-fos e5, P = 0.034, t(4) = 3.154, n = 5 mice; Hippocampus, c-fos e1, P = 0.0386, t(4) = 3.036, n = 5 mice, c-fos e2, P = 0.0419, t(5) = 2.717, n = 5 mice, c-fos e4, P = 0.0211, t(4) = 3.686, n = 5 mice, c-fos e5, P = 0.0028, t(5) = 5.454, n = 5 mice). (b) Expression of c-fos mRNA in the visual cortex of mice treated with light stimulation. c-fos eRNA induction in the visual cortex of mice following light stimulation. The levels of mRNA and eRNA were normalized to the level of Gapdh mRNA (c-fos mRNA, Visual cortex: P = 0.0069, t(2) = 11.966, n = 3 mice; c-fos e5, P = 0.0485, t(4) = 2.806, n = 3 mice). All unpaired t-test with Welch correction. Error bars indicate SEM; p values are from a two-tailed t test.
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Figure 6: Characterization of c-fos enhancer activities in vivo. (a) c-fos mRNA expression in the cortex, hippocampus, and cerebellum following KA-evoked seizure. c-fos mRNA was measured using RT-qPCR 1 h after saline or KA administration (note that the y axes are scaled differently). c-fos eRNA induction in the cortex, hippocampus, and cerebellum following KA-evoked seizure (c-fos mRNA, Cortex: P = 0.0458, t(4) = 4.511, n = 5 mice, Hippocampus: P = 0.0067, t(5) = 4.455, n = 5 mice, Cerebellum: P = 0.0133, t(4) = 47.767, n = 5 mice; Cortex, c-fos e4, P = 0.0150, t(4) = 4.084, n = 5 mice, c-fos e5, P = 0.034, t(4) = 3.154, n = 5 mice; Hippocampus, c-fos e1, P = 0.0386, t(4) = 3.036, n = 5 mice, c-fos e2, P = 0.0419, t(5) = 2.717, n = 5 mice, c-fos e4, P = 0.0211, t(4) = 3.686, n = 5 mice, c-fos e5, P = 0.0028, t(5) = 5.454, n = 5 mice). (b) Expression of c-fos mRNA in the visual cortex of mice treated with light stimulation. c-fos eRNA induction in the visual cortex of mice following light stimulation. The levels of mRNA and eRNA were normalized to the level of Gapdh mRNA (c-fos mRNA, Visual cortex: P = 0.0069, t(2) = 11.966, n = 3 mice; c-fos e5, P = 0.0485, t(4) = 2.806, n = 3 mice). All unpaired t-test with Welch correction. Error bars indicate SEM; p values are from a two-tailed t test.
Mentions: We next asked if combinatorial activation of c-fos enhancers also occurs in the brain. First we examined whether different brain regions activate distinctive sets of the c-fos enhancers to mediate c-fos induction upon chemically induced seizure. Young adult mice were injected intraperitoneally with Kainic acid (KA), which activates glutamate receptors to induce seizure. One hour after KA injection, a strong c-fos mRNA induction was detected in the cerebral cortex and hippocampus, and also weakly in the cerebellum (Fig. 6a) 34. We then measured individual eRNA levels in those brain regions. A single injection of KA led to induction of c-fos eRNAs from e4 and e5 in the cerebral cortex. On the other hand, eRNAs from all c-fos enhancers except e3 were induced in the hippocampus. We could not detect any induction of eRNA in the cerebellum, possibly because the KA-induced c-fos mRNA induction level was lower than other brain regions. We also investigated whether sensory stimulation can trigger eRNA transcription from the c-fos enhancers in the intact brain. We took dark-reared mice and stimulated their visual cortex by light for 1 h. The RT-qPCR analysis of the RNAs isolated from the visual cortex following visual stimulation detected eRNAs only from the e5 enhancer region (Fig. 6b). These results support our findings from in vitro neuronal culture that different combinations of enhancers are utilized to ensure c-fos induction in response to various stimuli, and also illustrate region-specific combinatorial activation of the c-fos 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