Limits...
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.

Show MeSH

Related in: MedlinePlus

Stimulus-specific combinatorial action of c-fos enhancers in vivo. (a) Schematic diagram of BDNF stereotaxic injection procedure. (b) c-fos mRNA and eRNA induction in the hippocampus following BDNF injection. c-fos mRNA and eRNA were measured using RT-qPCR 1 h after PBS or BDNF injection (c-fos mRNA, Hippocampus: P = 0.0236, t(3) = 4.267, n = 4 mice, unpaired t-test with Welch correction; c-fos e4, P = 0.0001, t(6) = 10.373, F = 5.082 [SDP = 0.1074], n = 4 mice, unpaired t-test, c-fos e5, P = 0.0402, t(3) = 3.475, n = 4 mice, unpaired t-test with Welch correction). (c) c-fos mRNA and eRNA induction in the hippocampus following KA-evoked seizure from wild-type and BDNF-KO mice. c-fos mRNA and eRNA were measured using RT-qPCR 1 h after saline or KA administration (c-fos mRNA, Hippocampus: P = 0.0216, t(4) = 3.660, F = 3.756 [SDP = 0.2103], n = 3 mice; c-fos e4, P = 0.0476, t(4) = 2.825, F = 10.113 [SDP = 0.0900], n = 3 mice, all unpaired t-test). The levels of mRNA and eRNA were normalized to the level of Gapdh mRNA. Error bars indicate SEM; p values are from a two-tailed t test. 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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4696896&req=5

Figure 7: Stimulus-specific combinatorial action of c-fos enhancers in vivo. (a) Schematic diagram of BDNF stereotaxic injection procedure. (b) c-fos mRNA and eRNA induction in the hippocampus following BDNF injection. c-fos mRNA and eRNA were measured using RT-qPCR 1 h after PBS or BDNF injection (c-fos mRNA, Hippocampus: P = 0.0236, t(3) = 4.267, n = 4 mice, unpaired t-test with Welch correction; c-fos e4, P = 0.0001, t(6) = 10.373, F = 5.082 [SDP = 0.1074], n = 4 mice, unpaired t-test, c-fos e5, P = 0.0402, t(3) = 3.475, n = 4 mice, unpaired t-test with Welch correction). (c) c-fos mRNA and eRNA induction in the hippocampus following KA-evoked seizure from wild-type and BDNF-KO mice. c-fos mRNA and eRNA were measured using RT-qPCR 1 h after saline or KA administration (c-fos mRNA, Hippocampus: P = 0.0216, t(4) = 3.660, F = 3.756 [SDP = 0.2103], n = 3 mice; c-fos e4, P = 0.0476, t(4) = 2.825, F = 10.113 [SDP = 0.0900], n = 3 mice, all unpaired t-test). The levels of mRNA and eRNA were normalized to the level of Gapdh mRNA. Error bars indicate SEM; p values are from a two-tailed t test. 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 wondered if our finding from the in vitro study that e2 and e4 are uniquely responsive to KCl and BDNF, respectively, could be observed in the intact brain in some way. However, unlike our in vitro culture system in which we could make a particular signaling dominate in the c-fos response, any sensory or chemical stimulation to the intact brain would involve multiple signaling pathways that would intricately intersect with each other. Despite this confounding issue, we attempted to investigate BDNF-induced activation of c-fos enhancers in the hippocampus. We directly injected recombinant BDNF proteins into the hippocampus and analyzed both c-fos mRNA and eRNAs 1 h later (Fig. 7a). A direct BDNF injection induced c-fos mRNA ~10 fold in the hippocampus compared to control PBS injection (Fig. 7b). In parallel, the e4 and e5 activities were mainly increased by BDNF. Notably, this pattern of enhancer activation is similar to the one from in vitro neuronal culture stimulated by BDNF except for the e1 enhancer despite the considerable differences present between the two experimental systems such as cell types and developmental stage. We next investigated whether any of BDNF-activated enhancers would be functionally important for BDNF-induced c-fos expression. We utilized Bdnf conditional KO mice in which the Bdnf gene has been selectively deleted in neurons during late embryogenesis 35. We then induced seizure by KA in both Bdnf KO and littermate control mice, and compared the expression levels of the c-fos mRNA and eRNAs between the two groups (Fig. 7c). While KA-induced seizure increased c-fos expression in the hippocampus, the induction level of the c-fos gene was dramatically diminished in the conditional Bdnf KO mice. This result implies that the downstream signaling pathways triggered by KA and BDNF significantly overlap or intersect with each other. Interestingly, KA-induced activation of e4 enhancer was severely impaired by Bdnf deletion whereas e5 enhancer activity was unaltered. These results strongly suggest that e4 enhancer is selectively responsive to BDNF-induced signaling pathways both in vitro and in vivo.


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

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

Stimulus-specific combinatorial action of c-fos enhancers in vivo. (a) Schematic diagram of BDNF stereotaxic injection procedure. (b) c-fos mRNA and eRNA induction in the hippocampus following BDNF injection. c-fos mRNA and eRNA were measured using RT-qPCR 1 h after PBS or BDNF injection (c-fos mRNA, Hippocampus: P = 0.0236, t(3) = 4.267, n = 4 mice, unpaired t-test with Welch correction; c-fos e4, P = 0.0001, t(6) = 10.373, F = 5.082 [SDP = 0.1074], n = 4 mice, unpaired t-test, c-fos e5, P = 0.0402, t(3) = 3.475, n = 4 mice, unpaired t-test with Welch correction). (c) c-fos mRNA and eRNA induction in the hippocampus following KA-evoked seizure from wild-type and BDNF-KO mice. c-fos mRNA and eRNA were measured using RT-qPCR 1 h after saline or KA administration (c-fos mRNA, Hippocampus: P = 0.0216, t(4) = 3.660, F = 3.756 [SDP = 0.2103], n = 3 mice; c-fos e4, P = 0.0476, t(4) = 2.825, F = 10.113 [SDP = 0.0900], n = 3 mice, all unpaired t-test). The levels of mRNA and eRNA were normalized to the level of Gapdh mRNA. Error bars indicate SEM; p values are from a two-tailed t test. 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696896&req=5

Figure 7: Stimulus-specific combinatorial action of c-fos enhancers in vivo. (a) Schematic diagram of BDNF stereotaxic injection procedure. (b) c-fos mRNA and eRNA induction in the hippocampus following BDNF injection. c-fos mRNA and eRNA were measured using RT-qPCR 1 h after PBS or BDNF injection (c-fos mRNA, Hippocampus: P = 0.0236, t(3) = 4.267, n = 4 mice, unpaired t-test with Welch correction; c-fos e4, P = 0.0001, t(6) = 10.373, F = 5.082 [SDP = 0.1074], n = 4 mice, unpaired t-test, c-fos e5, P = 0.0402, t(3) = 3.475, n = 4 mice, unpaired t-test with Welch correction). (c) c-fos mRNA and eRNA induction in the hippocampus following KA-evoked seizure from wild-type and BDNF-KO mice. c-fos mRNA and eRNA were measured using RT-qPCR 1 h after saline or KA administration (c-fos mRNA, Hippocampus: P = 0.0216, t(4) = 3.660, F = 3.756 [SDP = 0.2103], n = 3 mice; c-fos e4, P = 0.0476, t(4) = 2.825, F = 10.113 [SDP = 0.0900], n = 3 mice, all unpaired t-test). The levels of mRNA and eRNA were normalized to the level of Gapdh mRNA. Error bars indicate SEM; p values are from a two-tailed t test. 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 wondered if our finding from the in vitro study that e2 and e4 are uniquely responsive to KCl and BDNF, respectively, could be observed in the intact brain in some way. However, unlike our in vitro culture system in which we could make a particular signaling dominate in the c-fos response, any sensory or chemical stimulation to the intact brain would involve multiple signaling pathways that would intricately intersect with each other. Despite this confounding issue, we attempted to investigate BDNF-induced activation of c-fos enhancers in the hippocampus. We directly injected recombinant BDNF proteins into the hippocampus and analyzed both c-fos mRNA and eRNAs 1 h later (Fig. 7a). A direct BDNF injection induced c-fos mRNA ~10 fold in the hippocampus compared to control PBS injection (Fig. 7b). In parallel, the e4 and e5 activities were mainly increased by BDNF. Notably, this pattern of enhancer activation is similar to the one from in vitro neuronal culture stimulated by BDNF except for the e1 enhancer despite the considerable differences present between the two experimental systems such as cell types and developmental stage. We next investigated whether any of BDNF-activated enhancers would be functionally important for BDNF-induced c-fos expression. We utilized Bdnf conditional KO mice in which the Bdnf gene has been selectively deleted in neurons during late embryogenesis 35. We then induced seizure by KA in both Bdnf KO and littermate control mice, and compared the expression levels of the c-fos mRNA and eRNAs between the two groups (Fig. 7c). While KA-induced seizure increased c-fos expression in the hippocampus, the induction level of the c-fos gene was dramatically diminished in the conditional Bdnf KO mice. This result implies that the downstream signaling pathways triggered by KA and BDNF significantly overlap or intersect with each other. Interestingly, KA-induced activation of e4 enhancer was severely impaired by Bdnf deletion whereas e5 enhancer activity was unaltered. These results strongly suggest that e4 enhancer is selectively responsive to BDNF-induced signaling pathways both in vitro and in vivo.

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