Neocortical Tet3-mediated accumulation of 5-hydroxymethylcytosine promotes rapid behavioral adaptation

Abstract

5-hydroxymethylcytosine (5-hmC) is a novel DNA modification that is highly enriched in the adult brain and dynamically regulated by neural activity. 5-hmC accumulates across the lifespan; however, the functional relevance of this change in 5-hmC and whether it is necessary for behavioral adaptation have not been fully elucidated. Moreover, although the ten-eleven translocation (Tet) family of enzymes is known to be essential for converting methylated DNA to 5-hmC, the role of individual Tet proteins in the adult cortex remains unclear. Using 5-hmC capture together with high-throughput DNA sequencing on individual mice, we show that fear extinction, an important form of reversal learning, leads to a dramatic genome-wide redistribution of 5-hmC within the infralimbic prefrontal cortex. Moreover, extinction learning-induced Tet3-mediated accumulation of 5-hmC is associated with the establishment of epigenetic states that promote gene expression and rapid behavioral adaptation.

Fig. 1.

Tet3-mediated hydroxylation of 5-mC is required for rapid behavioral adaptation. (A) Under KCl-induced depolarization conditions, there was an overall reduction in Tet1 mRNA (n = 3 per group; F_9,29 = 3.78; P < 0.01). (B) In contrast, there was a significant increase in Tet3 mRNA expression 7 and 10 h poststimulation (n = 3 per group; F_9,29 = 3.78; P < 0.01). (C) Although there was no effect of behavioral training on Tet1 mRNA within the ILPFC when measured 2 h after training, (D) there was a selective increase in Tet3 mRNA expression (n = 4 per group; F_2,11 = 10.79; P < 0.01; Tukey’s post hoc analysis FC-No EXT vs. EXT, *P < 0.05). (E) Tet3 is highly expressed within the ILPFC. (F) There was no significant effect of Tet1 shRNA on within-session extinction across (Left) and no effect of Tet1 shRNA on fear extinction memory at test 24 h after training (Right). (G) Although there was no significant effect of Tet3 knockdown on within-session extinction (Left), there was a significant impairment in fear extinction memory when tested 24 h after training (Right, n = 7 per group; F_3,26 = 8.44; P < 0.01; Tukey’s post hoc analysis EXT FG12hH1 vs. EXT Tet3 shRNA, *P < 0.05). Error bars represent standard error of the mean.

Fig. 2.

Experience-dependent redistribution of 5-hmC within the ILPFC. (A) Representative heat map of genome-wide 5-hmC enrichment after behavioral training, derived from 8 biological replicates per treatment group. Note the cluster of candidate genes, which showed enrichment specifically after fear extinction learning. (B) In all significant peaks in which 5-hmC accumulated after extinction training, 35–40% of nucleotides spanning a 100-bp region directly under the summit of the peak contained either CA or CT dinucleotide repeats (n = 8 per group; CA F₃, ₃₁ = 18.02; CT F₃, ₃₁ = 14.23; CG F₃, ₃₁ = 10.38; Tukey’s post hoc analysis for all EXT vs. context (CXT), FC, FC-No EXT, ***P < 0.001). (C) Relative to all other groups, a unique pattern of 5-hmC distribution with an increase in CG-rich regions occurred in response to fear extinction learning. (D) There was an extinction training-induced decrease in 5-hmC peaks detected within intronic and intergenic regions, which was accompanied by increased 5-hmC at gene promoters, 5′-UTR, 3′-UTR, and within CDS. (E) After extinction training, there was a significant overlap between 5-hmC and DNaseI-hypersensitive regions across the genome, which expand from 5–27% of total peaks called. CXT, context only, 24 h; FC, fear conditioned, 24 h; FC-No EXT, fear conditioned and context B exposed without extinction, 2 h; EXT, extinction, 2 h. Error bars represent standard error of the mean.

Fig. 3.

Fear extinction learning increased the accumulation of 5-hmC and Tet3 surrounding the extinction-related gene, gephyrin. (A) Extinction training led to a persistent increase in 5-hmC within an intron of the gene encoding gephyrin. Shown is the normalized depth of coverage for this peak and its nearby region in each of the conditions. (B) A significant enrichment of 5-hmC within the intronic region of gephyrin occurred after fear extinction training [n = 3–4 per group, 2 h; F_3,14 = 5.29 (P < 0.01); Tukey’s post hoc analysis FC-No EXT 2 h vs. EXT 2 h (*P < 0.05), 24 h; t₆ = 2.24 (P < 0.05). (C) This effect was accompanied by a reduction in 5-mC 24 h after extinction training (n = 4 per group, 24 h, t₆ = 3.03; P < 0.05. (D) Fear extinction led to a transient increase in gephyrin mRNA expression (n = 4–5 per group; F_3,19 = 4.08; P < 0.05; Tukey’s post hoc analysis FC-No EXT vs. EXT, *P < 0.05). (E) There was a trend toward an increase in Tet1 occupancy within the intronic region of the gephyrin gene 24 h after extinction training. (F) Fear extinction learning leads to a transient increase in Tet3 occupancy (n = 3–5 per group; F_3,15 = 17.58; P < 0.001; Tukey’s post hoc analysis FC-No EXT 2 h vs. EXT 2 h, **P < 0.01).

Fig. 4.

Fear extinction learning is associated with an altered chromatin landscape. Fear extinction led to (A) a transient increase in the occupancy of Sp1 (t₇ = 2.54; P < 0.01) with no effect on bivalent chromatin marks H3K4^me3 (B) or H3K27^me3 (C), (D) a transient reduction in the heterochromatin mark H3K9^me3 (t₇ = 3.12; P < 0.05), (E) a delayed increase in H3K27^ac (n= 4–5 per group; t7 = 2.013; P < 0.05), a transient increase in presence of the enhancer-related elements p300 (f; t₇ = 2.74; P < 0.05) and H3K4^me1 (G) (t₆ = 0.85; P < 0.05), and a persistent increase in accumulation of the euchromatic mark, H3R2^me2s (H) [(2 h, t₆ = 2.21 (P < 0.05); 24 h, t₆ = 5.46 (P < 0.001)] within the ILPFC. Error bars represent standard error of the mean.

Fig. 5.

Tet3 is required for extinction training-induced accumulation of 5-hmC and associated effects on the chromatin landscape. (A) Tet3 occupancy at gephyrin locus was reduced in the presence of Tet3 shRNA (n = 4/group; F_3,12 = 5.27; P < 0.05; Dunnett’s post hoc test FC-No EXT FG12hH1 vs. EXT FG12hH1, **P < 0.01). (B) Tet3 knockdown blocked the accumulation of 5-hmC after fear extinction training (n = 4; F_3,12 = 11.63; P < 0.001; Dunnett’s post hoc test FC-No EXT FG12hH1 vs. EXT FG12hH1, ***P < 0.001). (C) There was no effect on 5-mC after extinction training in the presence of Tet3 shRNA (n = 4; F_3,12 = 4.26; P < 0.05; Dunnett’s post hoc test FC-No EXT FG12hH1 vs. EXT FG12hH1, *P < 0.05). (D) There was no significant increase in gephyrin mRNA expression after extinction training in the presence of Tet3 shRNA (n = 4–6 per group; F_3,19 = 6.364; P < 0.01; Dunnett’s FC-No EXT FG12hH1 vs. EXT FG12hH1, *P < 0.05). Knockdown Tet3 mRNA blocked the effect of extinction training on Sp1 (E) (n = 4/group; F_3,12 = 3.76; P < 0.05; Dunnett’s post hoc test FC-No EXT FG12hH1 vs. EXT FG12hH1, *P < 0.05), H3R2me2S (F) (n = 3–4/group; F_3,12 = 3.52; P < 0.05; Dunnett’s post hoc test FC-No EXT FG12hH1 vs. EXT FG12hH1, *P < 0.05), H3K4me1 (G) (n = 4/group; F_3,12 = 23.56; P < 0.0001; Dunnett’s post hoc test FC-No EXT FG12hH1 vs. EXT FG12hH1, **P < 0.01, vs. FC-No EXT Tet3 shRNA, *P < 0.05), and p300 occupancy at the gephyrin locus (H) (n = 4/group; F_3,12 = 11.88; P < 0.001; Dunnett’s post hoc test FC-No EXT FG12hH1 vs. EXT FG12hH1, *P < 0.05), (I) There was no significant effect of Tet3 shRNA on H3K9me3. Error bars represent standard error of the mean.