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NF-κB mediates Gadd45β expression and DNA demethylation in the hippocampus during fear memory formation.

Jarome TJ, Butler AA, Nichols JN, Pacheco NL, Lubin FD - Front Mol Neurosci (2015)

Bottom Line: Here, we found that learning in a fear conditioning paradigm increased Gadd45β gene expression and brain-derivedneurotrophic factor (BDNF) DNA demethylation in area CA1 of the hippocampus, both of which were prevented with pharmacological inhibition of NF-κB activity.Further experiments found that conditional mutations in p65/RelA impaired fear memory formation but did not alter changes in Gadd45β expression.Together, these results support a novel transcriptional role for NF-κB in regulation of Gadd45β expression and DNA demethylation in hippocampal neurons during fear memory.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Alabama at Birmingham Birmingham, AL, USA.

ABSTRACT
Gadd45-mediated DNA demethylation mechanisms have been implicated in the process of memory formation. However, the transcriptional mechanisms involved in the regulation of Gadd45 gene expression during memory formation remain unexplored. NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) controls transcription of genes in neurons and is a critical regulator of synaptic plasticity and memory formation. In silico analysis revealed several NF-κB (p65/RelA and cRel) consensus sequences within the Gadd45β gene promoter. Whether NF-κB activity regulates Gadd45 expression and associated DNA demethylation in neurons during memory formation is unknown. Here, we found that learning in a fear conditioning paradigm increased Gadd45β gene expression and brain-derivedneurotrophic factor (BDNF) DNA demethylation in area CA1 of the hippocampus, both of which were prevented with pharmacological inhibition of NF-κB activity. Further experiments found that conditional mutations in p65/RelA impaired fear memory formation but did not alter changes in Gadd45β expression. The learning-induced increases in Gadd45β mRNA levels, Gadd45β binding at the BDNF gene and BDNF DNA demethylation were blocked in area CA1 of the c-rel knockout mice. Additionally, local siRNA-mediated knockdown of c-rel in area CA1 prevented fear conditioning-induced increases in Gadd45β expression and BDNF DNA demethylation, suggesting that c-Rel containing NF-κB transcription factor complex is responsible for Gadd45β regulation during memory formation. Together, these results support a novel transcriptional role for NF-κB in regulation of Gadd45β expression and DNA demethylation in hippocampal neurons during fear memory.

No MeSH data available.


Related in: MedlinePlus

Knockout of c-rel prevents fear conditioning-induced changes in Gadd45β expression and BDNF DNA methylation in the hippocampus. (A) Wild-type (WT) or c-rel knockout (c-rel−/−) mice were fear conditioned and area CA1 collected 1 h later. (B) Basal expression of Gadd45β was not altered in c-rel−/− mice (n = 4–5 per group). (C)Gadd45β expression was increased in WT, but not c-rel−/−, mice following fear conditioning (n = 8 per group). (D) Chromatin immunoprecipitation revealed an increase in Gadd45β binding at BDNF Promoter 4 following fear conditioning, which was lost in c-rel−/− mice (n = 4 per group). (E) Bisulfite sequencing analysis of CpG sites in the mouse BDNF Promoter 4 region revealed a fear conditioning-induced decrease in BDNF Promoter 4 DNA methylation in area CA1 that was prevented in c-rel−/− mice (n = 3–4 per group). *p < 0.05 from WT. #p < 0.05 from WT-FC.
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Figure 4: Knockout of c-rel prevents fear conditioning-induced changes in Gadd45β expression and BDNF DNA methylation in the hippocampus. (A) Wild-type (WT) or c-rel knockout (c-rel−/−) mice were fear conditioned and area CA1 collected 1 h later. (B) Basal expression of Gadd45β was not altered in c-rel−/− mice (n = 4–5 per group). (C)Gadd45β expression was increased in WT, but not c-rel−/−, mice following fear conditioning (n = 8 per group). (D) Chromatin immunoprecipitation revealed an increase in Gadd45β binding at BDNF Promoter 4 following fear conditioning, which was lost in c-rel−/− mice (n = 4 per group). (E) Bisulfite sequencing analysis of CpG sites in the mouse BDNF Promoter 4 region revealed a fear conditioning-induced decrease in BDNF Promoter 4 DNA methylation in area CA1 that was prevented in c-rel−/− mice (n = 3–4 per group). *p < 0.05 from WT. #p < 0.05 from WT-FC.

Mentions: In our in silicio analysis we found multiple c-Rel consensus sites in the Gadd45β promoter, suggesting that c-Rel containing NF-κB complexes may be responsible for the learning-dependent increases in Gadd45β expression in area CA1. To test this, we examined Gadd45β expression in c-rel knockout (c-rel−/−) mice (Figure 4A), which we have previously shown to have impaired hippocampus-dependent but not hippocampus-independent fear memory (Levenson et al., 2004; O’Riordan et al., 2006; Ahn et al., 2008). First, we examined if a loss of c-rel during development resulted in long-term changes in Gadd45β expression. However, we did not observe altered basal levels of Gadd45β expression in area CA1 (t(7) = 0.022, p = 0.982; Figure 4B) of c-rel−/− mice, suggesting normal Gadd45β expression in the hippocampus. Next, we tested if c-rel−/− mice have altered Gadd45β expression in the hippocampus in response to learning. We found an increase in Gadd45β expression in fear conditioned WT mice relative to naïve controls (t(14) = 2.256, p < 0.05). Surprisingly, this increase in Gadd45β mRNA levels were not present in c-rel−/− mice (t(14) = 0.177, p = 0.862), suggesting that c-Rel containing NF-κB complexes were critical for the NF-κB-dependent regulation of Gadd45β expression following learning (Figure 4C). Since Gadd45β regulates DNA demethylation and we found that pharmacological inhibition of NF-κB signaling activity with DDTC prevented learning-induced DNA demethylation of BDNF Promoter 4, we next tested if BDNF Promoter 4 DNA demethylation was altered in c-rel−/− mice. Using chromatin immunoprecipitation, we found an increase in Gadd45β protein levels at BDNF Promoter 4 in area CA1 following fear conditioning that was abolished in c-rel−/− mice (F(2,9) = 4.543, p < 0.05; Figure 4D) associated with BDNF Promoter 4 DNA demethylation (F(2,7) = 4.504, p = 0.055), suggesting that there is a loss of learning-dependent Gadd45β accumulation at the BDNF gene in the hippocampus of c-rel knockout mice. Remarkably, we found that while fear conditioning resulted in decreased BDNF Promoter 4 DNA methylation relative to controls (t(4) = 3.039, p < 0.05), this did not occur in c-rel−/− mice (t(5) = 0.454, p = 0.668; Figure 4E). This suggests that c-Rel is likely responsible for the NF-κB-dependent regulation of Gadd45β expression and BDNF Promoter 4 DNA demethylation during memory formation.


NF-κB mediates Gadd45β expression and DNA demethylation in the hippocampus during fear memory formation.

Jarome TJ, Butler AA, Nichols JN, Pacheco NL, Lubin FD - Front Mol Neurosci (2015)

Knockout of c-rel prevents fear conditioning-induced changes in Gadd45β expression and BDNF DNA methylation in the hippocampus. (A) Wild-type (WT) or c-rel knockout (c-rel−/−) mice were fear conditioned and area CA1 collected 1 h later. (B) Basal expression of Gadd45β was not altered in c-rel−/− mice (n = 4–5 per group). (C)Gadd45β expression was increased in WT, but not c-rel−/−, mice following fear conditioning (n = 8 per group). (D) Chromatin immunoprecipitation revealed an increase in Gadd45β binding at BDNF Promoter 4 following fear conditioning, which was lost in c-rel−/− mice (n = 4 per group). (E) Bisulfite sequencing analysis of CpG sites in the mouse BDNF Promoter 4 region revealed a fear conditioning-induced decrease in BDNF Promoter 4 DNA methylation in area CA1 that was prevented in c-rel−/− mice (n = 3–4 per group). *p < 0.05 from WT. #p < 0.05 from WT-FC.
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Figure 4: Knockout of c-rel prevents fear conditioning-induced changes in Gadd45β expression and BDNF DNA methylation in the hippocampus. (A) Wild-type (WT) or c-rel knockout (c-rel−/−) mice were fear conditioned and area CA1 collected 1 h later. (B) Basal expression of Gadd45β was not altered in c-rel−/− mice (n = 4–5 per group). (C)Gadd45β expression was increased in WT, but not c-rel−/−, mice following fear conditioning (n = 8 per group). (D) Chromatin immunoprecipitation revealed an increase in Gadd45β binding at BDNF Promoter 4 following fear conditioning, which was lost in c-rel−/− mice (n = 4 per group). (E) Bisulfite sequencing analysis of CpG sites in the mouse BDNF Promoter 4 region revealed a fear conditioning-induced decrease in BDNF Promoter 4 DNA methylation in area CA1 that was prevented in c-rel−/− mice (n = 3–4 per group). *p < 0.05 from WT. #p < 0.05 from WT-FC.
Mentions: In our in silicio analysis we found multiple c-Rel consensus sites in the Gadd45β promoter, suggesting that c-Rel containing NF-κB complexes may be responsible for the learning-dependent increases in Gadd45β expression in area CA1. To test this, we examined Gadd45β expression in c-rel knockout (c-rel−/−) mice (Figure 4A), which we have previously shown to have impaired hippocampus-dependent but not hippocampus-independent fear memory (Levenson et al., 2004; O’Riordan et al., 2006; Ahn et al., 2008). First, we examined if a loss of c-rel during development resulted in long-term changes in Gadd45β expression. However, we did not observe altered basal levels of Gadd45β expression in area CA1 (t(7) = 0.022, p = 0.982; Figure 4B) of c-rel−/− mice, suggesting normal Gadd45β expression in the hippocampus. Next, we tested if c-rel−/− mice have altered Gadd45β expression in the hippocampus in response to learning. We found an increase in Gadd45β expression in fear conditioned WT mice relative to naïve controls (t(14) = 2.256, p < 0.05). Surprisingly, this increase in Gadd45β mRNA levels were not present in c-rel−/− mice (t(14) = 0.177, p = 0.862), suggesting that c-Rel containing NF-κB complexes were critical for the NF-κB-dependent regulation of Gadd45β expression following learning (Figure 4C). Since Gadd45β regulates DNA demethylation and we found that pharmacological inhibition of NF-κB signaling activity with DDTC prevented learning-induced DNA demethylation of BDNF Promoter 4, we next tested if BDNF Promoter 4 DNA demethylation was altered in c-rel−/− mice. Using chromatin immunoprecipitation, we found an increase in Gadd45β protein levels at BDNF Promoter 4 in area CA1 following fear conditioning that was abolished in c-rel−/− mice (F(2,9) = 4.543, p < 0.05; Figure 4D) associated with BDNF Promoter 4 DNA demethylation (F(2,7) = 4.504, p = 0.055), suggesting that there is a loss of learning-dependent Gadd45β accumulation at the BDNF gene in the hippocampus of c-rel knockout mice. Remarkably, we found that while fear conditioning resulted in decreased BDNF Promoter 4 DNA methylation relative to controls (t(4) = 3.039, p < 0.05), this did not occur in c-rel−/− mice (t(5) = 0.454, p = 0.668; Figure 4E). This suggests that c-Rel is likely responsible for the NF-κB-dependent regulation of Gadd45β expression and BDNF Promoter 4 DNA demethylation during memory formation.

Bottom Line: Here, we found that learning in a fear conditioning paradigm increased Gadd45β gene expression and brain-derivedneurotrophic factor (BDNF) DNA demethylation in area CA1 of the hippocampus, both of which were prevented with pharmacological inhibition of NF-κB activity.Further experiments found that conditional mutations in p65/RelA impaired fear memory formation but did not alter changes in Gadd45β expression.Together, these results support a novel transcriptional role for NF-κB in regulation of Gadd45β expression and DNA demethylation in hippocampal neurons during fear memory.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Alabama at Birmingham Birmingham, AL, USA.

ABSTRACT
Gadd45-mediated DNA demethylation mechanisms have been implicated in the process of memory formation. However, the transcriptional mechanisms involved in the regulation of Gadd45 gene expression during memory formation remain unexplored. NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) controls transcription of genes in neurons and is a critical regulator of synaptic plasticity and memory formation. In silico analysis revealed several NF-κB (p65/RelA and cRel) consensus sequences within the Gadd45β gene promoter. Whether NF-κB activity regulates Gadd45 expression and associated DNA demethylation in neurons during memory formation is unknown. Here, we found that learning in a fear conditioning paradigm increased Gadd45β gene expression and brain-derivedneurotrophic factor (BDNF) DNA demethylation in area CA1 of the hippocampus, both of which were prevented with pharmacological inhibition of NF-κB activity. Further experiments found that conditional mutations in p65/RelA impaired fear memory formation but did not alter changes in Gadd45β expression. The learning-induced increases in Gadd45β mRNA levels, Gadd45β binding at the BDNF gene and BDNF DNA demethylation were blocked in area CA1 of the c-rel knockout mice. Additionally, local siRNA-mediated knockdown of c-rel in area CA1 prevented fear conditioning-induced increases in Gadd45β expression and BDNF DNA demethylation, suggesting that c-Rel containing NF-κB transcription factor complex is responsible for Gadd45β regulation during memory formation. Together, these results support a novel transcriptional role for NF-κB in regulation of Gadd45β expression and DNA demethylation in hippocampal neurons during fear memory.

No MeSH data available.


Related in: MedlinePlus