Limits...
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

Inhibition of NF-κB activity abolishes learning-induced changes in Gadd45β expression and BDNF DNA demethylation in the hippocampus. (A)In silico analysis revealed several NF-κB consensus sequences in the Gadd45β promoter. (B) Pharmacological inhibition of NF-κB activity with DDTC abolished fear conditioning-induced increases in Gadd45β expression in area CA1 (n = 3–6 per group). (C) Bisulfite sequencing analysis of CpG sites in the rat BDNF Promoter 4 region revealed that inhibition of NF-κB with DDTC completely prevented the fear conditioning-induced decreases in BDNF Promoter 4 DNA methylation in area CA1 (n = 4–6 per group). *p < 0.05 from Naïve. #p < 0.05 from FC.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4584956&req=5

Figure 2: Inhibition of NF-κB activity abolishes learning-induced changes in Gadd45β expression and BDNF DNA demethylation in the hippocampus. (A)In silico analysis revealed several NF-κB consensus sequences in the Gadd45β promoter. (B) Pharmacological inhibition of NF-κB activity with DDTC abolished fear conditioning-induced increases in Gadd45β expression in area CA1 (n = 3–6 per group). (C) Bisulfite sequencing analysis of CpG sites in the rat BDNF Promoter 4 region revealed that inhibition of NF-κB with DDTC completely prevented the fear conditioning-induced decreases in BDNF Promoter 4 DNA methylation in area CA1 (n = 4–6 per group). *p < 0.05 from Naïve. #p < 0.05 from FC.

Mentions: Considering that NF-κB is a transcription factor that plays a critical role in memory formation (Yeh et al., 2002; Lubin and Sweatt, 2007; Federman et al., 2013), we next determined whether or not Gadd45β expression was being regulated by NF-κB transcriptional activity during the memory consolidation period. In silico analysis revealed several NF-κB consensus sequences within the Gadd45β gene (Figure 2A). We found that pharmacological inhibition of NF-κB signaling activity with diethyldithiocarbamate (DDTC) abolished the fear conditioning-induced increases in Gadd45β expression in area CA1 (F(3,11) = 34.47, p < 0.001; Figure 2B), suggesting that NF-κB signaling was critical for the increased transcription of Gadd45β following learning. Since Gadd45β regulates DNA demethylation during memory formation, we next tested if pharmacological blockade of NF-κB activity with DDTC prevented DNA demethylation of the BDNF gene, a well-established regulator of memory formation that undergoes dynamic activity-dependent and promoter-specific changes in DNA methylation levels (Lee et al., 2004; Bekinschtein et al., 2007; Lubin et al., 2008; Peters et al., 2010). Remarkably, we found that while fear conditioning resulted in decreased BDNF Promoter 4 DNA methylation, inhibiting NF-κB activity completely attenuated this effect (F(2,12) = 4.589, p < 0.05; Figure 2C). Together, these results suggest that NF-κB controls Gadd45β expression and BDNF DNA demethylation in the hippocampus 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)

Inhibition of NF-κB activity abolishes learning-induced changes in Gadd45β expression and BDNF DNA demethylation in the hippocampus. (A)In silico analysis revealed several NF-κB consensus sequences in the Gadd45β promoter. (B) Pharmacological inhibition of NF-κB activity with DDTC abolished fear conditioning-induced increases in Gadd45β expression in area CA1 (n = 3–6 per group). (C) Bisulfite sequencing analysis of CpG sites in the rat BDNF Promoter 4 region revealed that inhibition of NF-κB with DDTC completely prevented the fear conditioning-induced decreases in BDNF Promoter 4 DNA methylation in area CA1 (n = 4–6 per group). *p < 0.05 from Naïve. #p < 0.05 from FC.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4584956&req=5

Figure 2: Inhibition of NF-κB activity abolishes learning-induced changes in Gadd45β expression and BDNF DNA demethylation in the hippocampus. (A)In silico analysis revealed several NF-κB consensus sequences in the Gadd45β promoter. (B) Pharmacological inhibition of NF-κB activity with DDTC abolished fear conditioning-induced increases in Gadd45β expression in area CA1 (n = 3–6 per group). (C) Bisulfite sequencing analysis of CpG sites in the rat BDNF Promoter 4 region revealed that inhibition of NF-κB with DDTC completely prevented the fear conditioning-induced decreases in BDNF Promoter 4 DNA methylation in area CA1 (n = 4–6 per group). *p < 0.05 from Naïve. #p < 0.05 from FC.
Mentions: Considering that NF-κB is a transcription factor that plays a critical role in memory formation (Yeh et al., 2002; Lubin and Sweatt, 2007; Federman et al., 2013), we next determined whether or not Gadd45β expression was being regulated by NF-κB transcriptional activity during the memory consolidation period. In silico analysis revealed several NF-κB consensus sequences within the Gadd45β gene (Figure 2A). We found that pharmacological inhibition of NF-κB signaling activity with diethyldithiocarbamate (DDTC) abolished the fear conditioning-induced increases in Gadd45β expression in area CA1 (F(3,11) = 34.47, p < 0.001; Figure 2B), suggesting that NF-κB signaling was critical for the increased transcription of Gadd45β following learning. Since Gadd45β regulates DNA demethylation during memory formation, we next tested if pharmacological blockade of NF-κB activity with DDTC prevented DNA demethylation of the BDNF gene, a well-established regulator of memory formation that undergoes dynamic activity-dependent and promoter-specific changes in DNA methylation levels (Lee et al., 2004; Bekinschtein et al., 2007; Lubin et al., 2008; Peters et al., 2010). Remarkably, we found that while fear conditioning resulted in decreased BDNF Promoter 4 DNA methylation, inhibiting NF-κB activity completely attenuated this effect (F(2,12) = 4.589, p < 0.05; Figure 2C). Together, these results suggest that NF-κB controls Gadd45β expression and BDNF DNA demethylation in the hippocampus 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