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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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Time course analysis of five c-fos eRNAs and mRNA. (a) A UCSC genome browser view of the c-fos genomic locus with RNA-seq and ChIP-Seq data (adapted from Kim et al.10 and Malik et al. 48). + and − indicate the presence or absence of KCl. Blue and gray vertical bars indicate the locations of the c-fos enhancers and the promoter. (b) Expression levels of c-fos eRNA and mRNA in cortical neurons after KCl stimulation for various time points. Cortical neurons were depolarized with 55 mM KCl at DIV 6 and mRNA and eRNA levels were measured using RT-qPCR and normalized to Gapdh mRNA (n = 3 biological replicates). Error bars indicate SEM.
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Figure 1: Time course analysis of five c-fos eRNAs and mRNA. (a) A UCSC genome browser view of the c-fos genomic locus with RNA-seq and ChIP-Seq data (adapted from Kim et al.10 and Malik et al. 48). + and − indicate the presence or absence of KCl. Blue and gray vertical bars indicate the locations of the c-fos enhancers and the promoter. (b) Expression levels of c-fos eRNA and mRNA in cortical neurons after KCl stimulation for various time points. Cortical neurons were depolarized with 55 mM KCl at DIV 6 and mRNA and eRNA levels were measured using RT-qPCR and normalized to Gapdh mRNA (n = 3 biological replicates). Error bars indicate SEM.

Mentions: Our previous genome-wide study revealed that the c-fos gene is surrounded by five activity-regulated enhancers (Fig. 1a) 6. These enhancers exhibit a high level of the well-defined enhancer mark, H3K4me1 whereas the c-fos promoter region is enriched by H3K4me3. Upon membrane depolarization of mouse cortical neurons, all of the c-fos enhancers (e1 through e5) are inducibly bound by the general transcription coactivator, CBP, and several activity-regulated TFs in various combinations (Supplementary Fig. 1). RNAPII is also inducibly recruited to each of the c-fos enhancers and synthesizes eRNAs, which have been shown to positively correlate with the activity of the enhancers from which they are expressed 6, 12, 15, 16. These enhancers that we identified in primary culture of mouse cortical neurons appear to also be active in the intact brain throughout development, as all of them are tightly aligned with the peaks of an active enhancer marker, H3K27ac (Supplementary Fig. 1) 17. While all five enhancers show comparable levels of the H3K4me1 mark, the induction levels of RNAPII and eRNAs at individual enhancers are different, in that e1, e2, and e5 show much stronger transcription activity than e3 and e4 when neurons are depolarized by KCl. Notably we observed that the active promoter mark, H3K4me3 was also enriched at e2 and e5 enhancers, which transcribe eRNAs at much higher levels than the other enhancers. Several recent studies also reported the same observation that enhancers with very strong transcription activity are enriched by both H3K4me3 and H3K4me1 marks 18–20. It has been suggested that the H3K4me3/me1 ratio positively correlates with transcription level, independent of transcript stability. We have confirmed the induction of these eRNAs by RT-qPCR (Fig. 1b) and also observed that, just like the kinetic relationship we have previously shown between eRNA and mRNA from the Arc and Gadd45b genes 21, expression of all c-fos eRNAs peaked earlier than that of the c-fos mRNA. These results are consistent with a recent study of genome-scale CAGE (cap analysis of gene expression) analysis showing that enhancer transcription occurs most rapidly among the transcriptional changes during cellular differentiation or activation 22.


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

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

Time course analysis of five c-fos eRNAs and mRNA. (a) A UCSC genome browser view of the c-fos genomic locus with RNA-seq and ChIP-Seq data (adapted from Kim et al.10 and Malik et al. 48). + and − indicate the presence or absence of KCl. Blue and gray vertical bars indicate the locations of the c-fos enhancers and the promoter. (b) Expression levels of c-fos eRNA and mRNA in cortical neurons after KCl stimulation for various time points. Cortical neurons were depolarized with 55 mM KCl at DIV 6 and mRNA and eRNA levels were measured using RT-qPCR and normalized to Gapdh mRNA (n = 3 biological replicates). Error bars indicate SEM.
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Related In: Results  -  Collection

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Figure 1: Time course analysis of five c-fos eRNAs and mRNA. (a) A UCSC genome browser view of the c-fos genomic locus with RNA-seq and ChIP-Seq data (adapted from Kim et al.10 and Malik et al. 48). + and − indicate the presence or absence of KCl. Blue and gray vertical bars indicate the locations of the c-fos enhancers and the promoter. (b) Expression levels of c-fos eRNA and mRNA in cortical neurons after KCl stimulation for various time points. Cortical neurons were depolarized with 55 mM KCl at DIV 6 and mRNA and eRNA levels were measured using RT-qPCR and normalized to Gapdh mRNA (n = 3 biological replicates). Error bars indicate SEM.
Mentions: Our previous genome-wide study revealed that the c-fos gene is surrounded by five activity-regulated enhancers (Fig. 1a) 6. These enhancers exhibit a high level of the well-defined enhancer mark, H3K4me1 whereas the c-fos promoter region is enriched by H3K4me3. Upon membrane depolarization of mouse cortical neurons, all of the c-fos enhancers (e1 through e5) are inducibly bound by the general transcription coactivator, CBP, and several activity-regulated TFs in various combinations (Supplementary Fig. 1). RNAPII is also inducibly recruited to each of the c-fos enhancers and synthesizes eRNAs, which have been shown to positively correlate with the activity of the enhancers from which they are expressed 6, 12, 15, 16. These enhancers that we identified in primary culture of mouse cortical neurons appear to also be active in the intact brain throughout development, as all of them are tightly aligned with the peaks of an active enhancer marker, H3K27ac (Supplementary Fig. 1) 17. While all five enhancers show comparable levels of the H3K4me1 mark, the induction levels of RNAPII and eRNAs at individual enhancers are different, in that e1, e2, and e5 show much stronger transcription activity than e3 and e4 when neurons are depolarized by KCl. Notably we observed that the active promoter mark, H3K4me3 was also enriched at e2 and e5 enhancers, which transcribe eRNAs at much higher levels than the other enhancers. Several recent studies also reported the same observation that enhancers with very strong transcription activity are enriched by both H3K4me3 and H3K4me1 marks 18–20. It has been suggested that the H3K4me3/me1 ratio positively correlates with transcription level, independent of transcript stability. We have confirmed the induction of these eRNAs by RT-qPCR (Fig. 1b) and also observed that, just like the kinetic relationship we have previously shown between eRNA and mRNA from the Arc and Gadd45b genes 21, expression of all c-fos eRNAs peaked earlier than that of the c-fos mRNA. These results are consistent with a recent study of genome-scale CAGE (cap analysis of gene expression) analysis showing that enhancer transcription occurs most rapidly among the transcriptional changes during cellular differentiation or activation 22.

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