Epigenetic regulation of BDNF gene transcription in the consolidation of fear memory

Abstract

Long-term memory formation requires selective changes in gene expression. Here, we determined the contribution of chromatin remodeling to learning-induced changes in brain-derived neurotrophic factor (bdnf) gene expression in the adult hippocampus. Contextual fear learning induced differential regulation of exon-specific bdnf mRNAs (I, IV, VI, IX) that was associated with changes in bdnf DNA methylation and altered local chromatin structure. Infusions of zebularine (a DNA methyltransferase inhibitor) significantly altered bdnf DNA methylation and triggered changes in exon-specific bdnf mRNA levels, indicating that altered DNA methylation is sufficient to drive differential bdnf transcript regulation in the hippocampus. In addition, NMDA receptor blockade prevented memory-associated alterations in bdnf DNA methylation, resulting in a block of altered bdnf gene expression in hippocampus and a deficit in memory formation. These results suggest epigenetic modification of the bdnf gene as a mechanism for isoform-specific gene readout during memory consolidation.

Figure 1.

Increased bdnf gene expression after context exposure and fear conditioning. A, Schematic representation of the contextual fear-conditioning test protocol. Animals were exposed to the training chamber and either received a series of footshocks after being exposed to the context (Context + Shock), being exposed to the context alone (Context + No Shock), or receiving only the footshock without being exposed to the context (No Context + Shock). B, Animals were reexposed to the training chamber 24 h later and tested for freezing behavior. n = 8–9/group; *p < 0.001 compared with shock-alone controls. C, bdnf mRNA in area CA1 of hippocampus is increased within 0.5 h of context exposure (t₍₈₎ = 2.48, p = 0.0381, n = 5) and context + shock (t₍₈₎ = 2.41, p = 0.0425, n = 5) compared with naive controls. At 2 h, bdnf mRNA expression peaks in area CA1 of hippocampus in fear-conditioned animals (t₍₉₎ = 3.15, p = 0.0117, n = 5–6). At 24 h, bdnf mRNA levels returned to baseline in area CA1 of hippocampus from both context (t₍₆₎ = 0.42, p = 0.6887, n = 4) and context + shock (t₍₇₎ = 1.97, p = 0.0894, n = 4–5) animals relative to naive controls. D, After context exposure alone, exon I and VI bdnf mRNA increased in area CA1 (exon I, t₍₃₎ = 3.42, p = 0.0418, n = 4; exon VI, t₍₅₎ = 2.66, p = 0.0449, n = 6) with a corresponding increase in total bdnf mRNA levels (exon IX, t₍₄₎ = 3.38, p = 0.0279, n = 5) relative to naive control. After fear conditioning, exon IV bdnf mRNA increased (t₍₃₎ = 5.88, p = 0.0098, n = 4) in area CA1 of hippocampus with an increase in total bdnf gene expression as assessed by exon IX mRNA (t₍₅₎ = 3.49, p = 0.0175, n = 6). No significant changes in exon II bdnf mRNA were observed with context alone or context + shock relative to naive control. The solid line across the bars represents normalized naive control levels [one-sample t test, *p < 0.05, **p < 0.01, compared with naive controls; Student's t test, not significant (ns), ^#p < 0.05, ^##p < 0.01, compared with context alone]. Error bars indicate SEM.

Figure 2.

Altered bdnf DNA methylation with contextual fear conditioning. A, The positions of the bdnf CpG islands are indicated relative to the transcription start site of exon I (−658 to −447 bp), exon II (+33 to +304 bp), exon IV (−123 to + 161 bp), and exon VI (−141 to +348 bp). The locations of the methylated PCR primer pairs (M1, M2, M4, and M6) are indicated by the arrows, and primer sequences can be found in supplemental Table 1, available at www.jneurosci.org as supplemental material. To minimize confusion the newest structure of the bdnf gene is depicted in the figure (I, II, IV, VI, IX) with the older nomenclature displayed above (I, II, III, IV, V). In the immediate shock-alone group, we observed no significant changes in methylation at the bdnf CpG islands. With context exposure alone, there were decreases in methylation at exons I and VI (I, t₍₇₎ = 7.53, p = 0.001; IV, t₍₄₎ = 9.04, p = 0.0008, n = 5–8). No significant changes in DNA methylation were observed at exons II and IV after context exposure. With fear conditioning, there were increases in methylation at exon VI (t₍₆₎ = 2.34, p = 0.0362, n = 7), but a decrease in methylation at exons I and IV (II, t₍₄₎ = 3.09, p = 0.0367; IV, t₍₄₎ = 11.57, p = 0.0003, n = 5–6). Context + shock had no effect on promoter 2 methylation. B, Bisulfite sequencing analysis performed on 12 CpG sites near the transcription initiation site of exon IV show percentage of cytosine residues that were methylated with shock alone, context exposure alone, or fear conditioning. A two-way ANOVA revealed a highly significant effect of the behavior group (F = 128.8, p < 0.0001) and region (F = 3.07, p < 0.0009). The solid line across the bars represents normalized naive control levels [one-sample t test, *p < 0.05, **p < 0.01, ***p < 0.001, compared with naive controls; Student's t test; not significant (ns), ^#p < 0.05, ^##p < 0.01, compared with context alone]. Error bars indicate SEM.

Figure 3.

Inhibition of DNMT alters bdnf gene expression and DNA methylation in hippocampus in vivo. A, The diagram represents the histology from animals for which needle tips for intra-CA1 infusions were confirmed before biochemistry studies in B–D. B, Reverse transcriptase quantitative real-time PCR was used to determine the effect of intra-CA1 infusions of zebularine (DNMT inhibitor) on basal bdnf mRNA levels relative to saline control. cDNA products for bdnf exons I, IV, VI, and IX indicate that bdnf mRNA is artificially increased in area CA1 of hippocampus with zebularine treatment in vivo (I, t₍₆₎ = 2.71, p = 0.0352; IV, t₍₆₎ = 3.27 p = 0.0171; VI, t₍₅₎ = 3.07, p = 0.0278; IX, t₍₆₎ = 3.26, p = 0.0173, n = 6–7). Products for exon II mRNA did not change with zebularine. C, Correlative studies from the same animals used in B. Zebularine significantly decreased levels of methylated bdnf exons I, II, IV, and VI DNA in area CA1 of hippocampus (I, t₍₆₎ = 13.33, p = 0.0001; II, t₍₅₎ = 2.73, p = 0.0411; IV, t₍₆₎ = 4.371, p = 0.0047; VI, t₍₆₎ = 2.82, p = 0.0304, n = 6–7). The solid line across the bars represents normalized naive control levels. D, Bisulfite sequencing analysis performed on 12 CpG sites near the transcription initiation site of exon IV show percentage of cytosine residues at specific CpG sites that were demethylated after zebularine treatment (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001, compared with naive controls).

Figure 4.

Inhibition of DNMT interferes with contextual fear memory. A, Diagram outlines the experimental design used with data presented in B. B, On test day 1, zebularine- or RG108-treated animals exhibited lower amounts of freezing behavior compared with vehicle-treated animals (zebularine, t₍₉₎ = 2.52, p = 0.0328; RG108, t₍₈₎ = 3.72, p = 0.0059, n = 9–10; Student's t test, *p < 0.05, **p < 0.01). Error bars indicate SEM.

Figure 5.

Histone modifications at exon-specific bdnf promoters during memory consolidation. A, At the bdnf gene, PH3/AcH3 and AcH3 levels were increased at promoter 4 during fear memory consolidation relative to naive controls (PH3/AcH3, t₍₂₎ = 4.185, p = 0.05; AcH3, t₍₂₎ = 6.21, p = 0.0250, n = 3). AcH4 levels were significantly reduced at promoter 2 relative to naive controls (t₍₂₎ = 12.26, p = 0.0066, n = 3). The top shows the schematic location of the bdnf promoters preceding each exon (P1, P2, P4, and P6) as indicated by the star. In the graph, the areas with no bars indicate that the anti-histone antibody precipitated negligible levels of the bdnf promoter regions that were not detectable. B, At the bdnf promoter 4, AcH3 levels were increased during memory consolidation and significantly attenuated with DNMT inhibition [zebularine (ZEB)] relative to naive controls (t₍₈₎ = 3.047, p = 0.0318, n = 4–6). C, There were no significant histone modifications (AcH3) around the β-actin promoter during memory consolidation (n = 3; one-way ANOVA, *p < 0.05, compared with naive controls; Student's t test, ^#p < 0.05, compared with context + shock alone). Error bars indicate SEM.

Figure 6.

Inhibition of DNMT activation during memory consolidation prevents bdnf DNA demethylation and mRNA expression. A, Sequence map of bdnf exon IV DNA is shown including the 12 CpG sites (bold). Methylation analysis of the 12 CpG dinucleotides of exon IV show that DNMT inhibition altered the percentage of cytosine residues that were demethylated with fear conditioning. B, At 2 h after fear conditioning, DNMT inhibition with zebularine prevented an increase in bdnf exon IV mRNA (t₍₁₀₎ = 2.493, p = 0.0318, n = 6; one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, compared with naive controls; Student's t test, ^#p < 0.05, compared with context + shock). Error bars indicate SEM.

Figure 7.

Inhibition of NMDA receptor activation prevents bdnf DNA demethylation and mRNA expression. A, Diagram outlines the experimental design used with data presented in B–D. B, At 2 h after fear conditioning, NMDA receptor inactivation with MK801 prevented an increase in bdnf total mRNA (exon IX, t₍₉₎ = 4.18, p = 0.0024, n = 7–8). C, NMDA receptor inactivation prevented the demethylation of exon IV induced by fear conditioning (methylated exon IV, t₍₁₀₎ = 2.99, p = 0.0308; unmethylated exon IV, t₍₁₁₎ = 4.441, p = 0.1108, n = 6–7). D, NDMA receptor inactivation prevented changes in exon IV mRNA (exon IV, t₍₁₀₎ = 6.85, p = 0.0156, n = 6). The solid line across the bars represents normalized naive control levels (one-sample t test, *p < 0.05, **p < 0.01, ***p < 0.001, compared with naive controls; Student's t test, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, compared with context + shock). Error bars indicate SEM.

Figure 8.

Inhibition of NMDA receptor activation (top) alters demethylation of cytosine residues at exon IV bdnf DNA induced by fear conditioning. Sequence map of bdnf exon IV DNA is shown (middle), including the 12 CpG sites (bold). Methylation analysis of the 12 CpG dinucleotides of exon IV (bottom) show that NMDA receptor blockade altered the percentage of cytosine residues that were demethylated with fear conditioning (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001, compared with naive controls). A two-way ANOVA revealed a highly significant effect of behavior group (F = 35.71, p < 0.0001) and region (F = 3.428, p < 0.0002), as well as a significant behavior group × region interaction effect (F = 1.676, p < 1.676). Error bars indicate SEM.