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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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Specific requirement of e2 and e4 enhancer in KCl or BDNF-mediated c-fos transcription. (a) Effect of c-fos promoter-targeted CRISPRi (dCas9-KRAB) (P = 0.0189, t(2) = 12.637, F = 4.830 [SDP = 0.2718], n = 3 biological replicates). (b) Effect of e1, e2, e4, and e5 enhancer-targeted CRISPRi on their respective eRNAs (c-fos e1 eRNA, P = 0.0151, t(2) = 8.037, F = 4.147 [SDP = 0.2906], n = 2; c-fos e2 eRNA, P = 0.0393, t(4) = 3.015, F = 3.461 [SDP = 0.2242], n = 3; c-fos e4 eRNA, P = 0.0206, t(2) = 6.864, F = 25.000 [SDP = 0.1257], n = 2; c-fos e5 eRNA, P = 0.0104, t(2) = 9.711, F = 32.398 [SDP = 0.1107], n = 2 biological replicates). (c) Effect of c-fos e1, e2, e4, and e5 enhancer-targeted CRISPRi. Following the suppression of e1, e2, e4, and e5 enhancer by CRISPRi, cortical neurons were stimulated by KCl or BDNF, and expression levels of c-fos pre-mRNA and mRNA were measured using RT-qPCR (KCl stimulation: e2 c-fos pre-mRNA, P = 0.0433, t(4) = 2.918, F = 1.388 [SDP = 0.4187], n = 3; c-fos mRNA, P = 0.0102, t(8) = 3.340, F = 1.114 [SDP = 0.459], n = 5; e5 c-fos pre-mRNA P = 0.0077, t(2) = 11.350, F = 3.550 [SDP = 0.3106], n = 2, c-fos mRNA, P = 0.0207, t(2) = 6.845, F = 1.122 [SDP = 0.4817], n = 2; BDNF stimulation: e4 c-fos pre-mRNA, P = 0.0485, t(2) = 4.373, F = 1.807 [SDP = 0.4868], n = 2, c-fos mRNA, P = 0.0322, t(2) = 5.440, F = 137.97 [SDP = 0.0541], n = 2; e5 c-fos pre-mRNA, P = 0.0489, t(2) = 4.353, F = 17.288 [SDP = 0.1503], n = 2, c-fos mRNA, P = 0.0223, t(2) = 6.582, F = 26.669 [SDP = 0.1218], n = 2 biological replicates). All unpaired t-test. 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.
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Figure 5: Specific requirement of e2 and e4 enhancer in KCl or BDNF-mediated c-fos transcription. (a) Effect of c-fos promoter-targeted CRISPRi (dCas9-KRAB) (P = 0.0189, t(2) = 12.637, F = 4.830 [SDP = 0.2718], n = 3 biological replicates). (b) Effect of e1, e2, e4, and e5 enhancer-targeted CRISPRi on their respective eRNAs (c-fos e1 eRNA, P = 0.0151, t(2) = 8.037, F = 4.147 [SDP = 0.2906], n = 2; c-fos e2 eRNA, P = 0.0393, t(4) = 3.015, F = 3.461 [SDP = 0.2242], n = 3; c-fos e4 eRNA, P = 0.0206, t(2) = 6.864, F = 25.000 [SDP = 0.1257], n = 2; c-fos e5 eRNA, P = 0.0104, t(2) = 9.711, F = 32.398 [SDP = 0.1107], n = 2 biological replicates). (c) Effect of c-fos e1, e2, e4, and e5 enhancer-targeted CRISPRi. Following the suppression of e1, e2, e4, and e5 enhancer by CRISPRi, cortical neurons were stimulated by KCl or BDNF, and expression levels of c-fos pre-mRNA and mRNA were measured using RT-qPCR (KCl stimulation: e2 c-fos pre-mRNA, P = 0.0433, t(4) = 2.918, F = 1.388 [SDP = 0.4187], n = 3; c-fos mRNA, P = 0.0102, t(8) = 3.340, F = 1.114 [SDP = 0.459], n = 5; e5 c-fos pre-mRNA P = 0.0077, t(2) = 11.350, F = 3.550 [SDP = 0.3106], n = 2, c-fos mRNA, P = 0.0207, t(2) = 6.845, F = 1.122 [SDP = 0.4817], n = 2; BDNF stimulation: e4 c-fos pre-mRNA, P = 0.0485, t(2) = 4.373, F = 1.807 [SDP = 0.4868], n = 2, c-fos mRNA, P = 0.0322, t(2) = 5.440, F = 137.97 [SDP = 0.0541], n = 2; e5 c-fos pre-mRNA, P = 0.0489, t(2) = 4.353, F = 17.288 [SDP = 0.1503], n = 2, c-fos mRNA, P = 0.0223, t(2) = 6.582, F = 26.669 [SDP = 0.1218], n = 2 biological replicates). All unpaired t-test. 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: The eRNA induction profile, enhancer reporter assay, and TF knockdown consistently suggest that e2 and e4 enhancers are selectively activated by KCl and BDNF, respectively. In order to further verify this intriguing observation, we utilized the CRISPRi system in which a catalytically inactivated mutant form of Cas9 (dCaps9) is fused to the KRAB (Krüppel-associated box) domain of Kox1 31. The KRAB domain protein can recruit KAP1 protein, which act as a scaffold for various heterochromatin-inducing factors such as heterochromatin protein 1 (HP1) and the histone methyltransferase SETDB1. The dCas9-KRAB fusion protein guided by sgRNA has been shown to efficiently silence expression of target genes 31, 32. We used CRISPRi to selectively silence the activity of individual c-fos enhancers and examined how that affected c-fos induction in response to KCl or BDNF. We first directed dCas9-KRAB to the c-fos transcription start site (TSS) and observed a decrease in the level of KCl-induced c-fos transcription, but not other IEGs, demonstrating the specificity of the system (Fig. 5a). The dCas9-KRAB caused a significant decrease in KCl- or BDNF-induced eRNA transcription (Fig. 5b). CRISPRi-mediated inhibition appears to specific to targeted enhancer as blocking e2 enhancer only reduced the activity of e2 enhancer but not other c-fos enhancers (Supplementary Fig 10). Based on the CRISPRi analysis with e5 enhancer, it appears that CRISPRi could also impair the physical interaction between the targeted enhancer and the promoter (Supplementary Fig 11). We then examined if the CRISPRi-mediated inhibition of individual enhancers affects the c-fos expression induced by either KCl or BDNF (Fig. 5c). Levels of pre-mRNA were measured in parallel with mRNAs in this analysis in order to evaluate the impact of CRISPRi more specifically on de novo transcription activity at the c-fos gene. CRISPRi at e2 and e5 caused a significant decrease in the induction levels of both RNA populations of the c-fos gene whereas suppression of e4 and e5 enhancer activities impaired BDNF-induced c-fos expression. The induction of other IEGs (Arc, Gadd45b, and Egr-1) was not affected by any CRISPRi (Supplementary Fig. 12). Together with eRNA induction profiles and luciferase reporter data, CRISPRi study demonstrates that e2 and e4 enhancers functionally contribute to the c-fos gene induction by specifically responding to KCl and BDNF stimulation, respectively. On the other hand, blocking e1 activity in the native context by CRISPRi did not have any impact on c-fos induction by either stimulus despite its inducible interaction with the c-fos promoter and eRNA transcription. We also showed by luciferase assay that e1 alone could enhance reporter expression in response to all three stimuli. Based on these results, we propose that e1 might act as a “shadow enhancer” for c-fos expression in native context, whose function is to ensure precise and robust transcription of target genes during extreme or adverse conditions that may occur throughout development and/or by dynamic environmental changes, where otherwise its function is dispensable by functionally redundant primary enhancers 33.


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

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

Specific requirement of e2 and e4 enhancer in KCl or BDNF-mediated c-fos transcription. (a) Effect of c-fos promoter-targeted CRISPRi (dCas9-KRAB) (P = 0.0189, t(2) = 12.637, F = 4.830 [SDP = 0.2718], n = 3 biological replicates). (b) Effect of e1, e2, e4, and e5 enhancer-targeted CRISPRi on their respective eRNAs (c-fos e1 eRNA, P = 0.0151, t(2) = 8.037, F = 4.147 [SDP = 0.2906], n = 2; c-fos e2 eRNA, P = 0.0393, t(4) = 3.015, F = 3.461 [SDP = 0.2242], n = 3; c-fos e4 eRNA, P = 0.0206, t(2) = 6.864, F = 25.000 [SDP = 0.1257], n = 2; c-fos e5 eRNA, P = 0.0104, t(2) = 9.711, F = 32.398 [SDP = 0.1107], n = 2 biological replicates). (c) Effect of c-fos e1, e2, e4, and e5 enhancer-targeted CRISPRi. Following the suppression of e1, e2, e4, and e5 enhancer by CRISPRi, cortical neurons were stimulated by KCl or BDNF, and expression levels of c-fos pre-mRNA and mRNA were measured using RT-qPCR (KCl stimulation: e2 c-fos pre-mRNA, P = 0.0433, t(4) = 2.918, F = 1.388 [SDP = 0.4187], n = 3; c-fos mRNA, P = 0.0102, t(8) = 3.340, F = 1.114 [SDP = 0.459], n = 5; e5 c-fos pre-mRNA P = 0.0077, t(2) = 11.350, F = 3.550 [SDP = 0.3106], n = 2, c-fos mRNA, P = 0.0207, t(2) = 6.845, F = 1.122 [SDP = 0.4817], n = 2; BDNF stimulation: e4 c-fos pre-mRNA, P = 0.0485, t(2) = 4.373, F = 1.807 [SDP = 0.4868], n = 2, c-fos mRNA, P = 0.0322, t(2) = 5.440, F = 137.97 [SDP = 0.0541], n = 2; e5 c-fos pre-mRNA, P = 0.0489, t(2) = 4.353, F = 17.288 [SDP = 0.1503], n = 2, c-fos mRNA, P = 0.0223, t(2) = 6.582, F = 26.669 [SDP = 0.1218], n = 2 biological replicates). All unpaired t-test. 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.
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Figure 5: Specific requirement of e2 and e4 enhancer in KCl or BDNF-mediated c-fos transcription. (a) Effect of c-fos promoter-targeted CRISPRi (dCas9-KRAB) (P = 0.0189, t(2) = 12.637, F = 4.830 [SDP = 0.2718], n = 3 biological replicates). (b) Effect of e1, e2, e4, and e5 enhancer-targeted CRISPRi on their respective eRNAs (c-fos e1 eRNA, P = 0.0151, t(2) = 8.037, F = 4.147 [SDP = 0.2906], n = 2; c-fos e2 eRNA, P = 0.0393, t(4) = 3.015, F = 3.461 [SDP = 0.2242], n = 3; c-fos e4 eRNA, P = 0.0206, t(2) = 6.864, F = 25.000 [SDP = 0.1257], n = 2; c-fos e5 eRNA, P = 0.0104, t(2) = 9.711, F = 32.398 [SDP = 0.1107], n = 2 biological replicates). (c) Effect of c-fos e1, e2, e4, and e5 enhancer-targeted CRISPRi. Following the suppression of e1, e2, e4, and e5 enhancer by CRISPRi, cortical neurons were stimulated by KCl or BDNF, and expression levels of c-fos pre-mRNA and mRNA were measured using RT-qPCR (KCl stimulation: e2 c-fos pre-mRNA, P = 0.0433, t(4) = 2.918, F = 1.388 [SDP = 0.4187], n = 3; c-fos mRNA, P = 0.0102, t(8) = 3.340, F = 1.114 [SDP = 0.459], n = 5; e5 c-fos pre-mRNA P = 0.0077, t(2) = 11.350, F = 3.550 [SDP = 0.3106], n = 2, c-fos mRNA, P = 0.0207, t(2) = 6.845, F = 1.122 [SDP = 0.4817], n = 2; BDNF stimulation: e4 c-fos pre-mRNA, P = 0.0485, t(2) = 4.373, F = 1.807 [SDP = 0.4868], n = 2, c-fos mRNA, P = 0.0322, t(2) = 5.440, F = 137.97 [SDP = 0.0541], n = 2; e5 c-fos pre-mRNA, P = 0.0489, t(2) = 4.353, F = 17.288 [SDP = 0.1503], n = 2, c-fos mRNA, P = 0.0223, t(2) = 6.582, F = 26.669 [SDP = 0.1218], n = 2 biological replicates). All unpaired t-test. 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: The eRNA induction profile, enhancer reporter assay, and TF knockdown consistently suggest that e2 and e4 enhancers are selectively activated by KCl and BDNF, respectively. In order to further verify this intriguing observation, we utilized the CRISPRi system in which a catalytically inactivated mutant form of Cas9 (dCaps9) is fused to the KRAB (Krüppel-associated box) domain of Kox1 31. The KRAB domain protein can recruit KAP1 protein, which act as a scaffold for various heterochromatin-inducing factors such as heterochromatin protein 1 (HP1) and the histone methyltransferase SETDB1. The dCas9-KRAB fusion protein guided by sgRNA has been shown to efficiently silence expression of target genes 31, 32. We used CRISPRi to selectively silence the activity of individual c-fos enhancers and examined how that affected c-fos induction in response to KCl or BDNF. We first directed dCas9-KRAB to the c-fos transcription start site (TSS) and observed a decrease in the level of KCl-induced c-fos transcription, but not other IEGs, demonstrating the specificity of the system (Fig. 5a). The dCas9-KRAB caused a significant decrease in KCl- or BDNF-induced eRNA transcription (Fig. 5b). CRISPRi-mediated inhibition appears to specific to targeted enhancer as blocking e2 enhancer only reduced the activity of e2 enhancer but not other c-fos enhancers (Supplementary Fig 10). Based on the CRISPRi analysis with e5 enhancer, it appears that CRISPRi could also impair the physical interaction between the targeted enhancer and the promoter (Supplementary Fig 11). We then examined if the CRISPRi-mediated inhibition of individual enhancers affects the c-fos expression induced by either KCl or BDNF (Fig. 5c). Levels of pre-mRNA were measured in parallel with mRNAs in this analysis in order to evaluate the impact of CRISPRi more specifically on de novo transcription activity at the c-fos gene. CRISPRi at e2 and e5 caused a significant decrease in the induction levels of both RNA populations of the c-fos gene whereas suppression of e4 and e5 enhancer activities impaired BDNF-induced c-fos expression. The induction of other IEGs (Arc, Gadd45b, and Egr-1) was not affected by any CRISPRi (Supplementary Fig. 12). Together with eRNA induction profiles and luciferase reporter data, CRISPRi study demonstrates that e2 and e4 enhancers functionally contribute to the c-fos gene induction by specifically responding to KCl and BDNF stimulation, respectively. On the other hand, blocking e1 activity in the native context by CRISPRi did not have any impact on c-fos induction by either stimulus despite its inducible interaction with the c-fos promoter and eRNA transcription. We also showed by luciferase assay that e1 alone could enhance reporter expression in response to all three stimuli. Based on these results, we propose that e1 might act as a “shadow enhancer” for c-fos expression in native context, whose function is to ensure precise and robust transcription of target genes during extreme or adverse conditions that may occur throughout development and/or by dynamic environmental changes, where otherwise its function is dispensable by functionally redundant primary enhancers 33.

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