Tet1 is critical for neuronal activity-regulated gene expression and memory extinction

Abstract

The ten-eleven translocation (Tet) family of methylcytosine dioxygenases catalyze oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and promote DNA demethylation. Despite the abundance of 5hmC and Tet proteins in the brain, little is known about the functions of the neuronal Tet enzymes. Here, we analyzed Tet1 knockout mice (Tet1KO) and found downregulation of multiple neuronal activity-regulated genes, including Npas4, c-Fos, and Arc. Furthermore, Tet1KO animals exhibited abnormal hippocampal long-term depression and impaired memory extinction. Analysis of the key regulatory gene, Npas4, indicated that its promoter region, containing multiple CpG dinucleotides, is hypermethylated in both naive Tet1KO mice and after extinction training. Such hypermethylation may account for the diminished expression of Npas4 itself and its downstream targets, impairing transcriptional programs underlying cognitive processes. In summary, we show that neuronal Tet1 regulates normal DNA methylation levels, expression of activity-regulated genes, synaptic plasticity, and memory extinction.

Figure 1. Loss of Tet1 does not affect brain size and morphology but leads to a reduction in 5hmC levels in the cortex and hippocampus

(A,B) Global genomic 5hmC levels are significantly reduced, and 5mC levels are slightly but not significantly increased, in the cortex and hippocampus of Tet1KO mice compared to control Tet1+/+ animals. Graphic representation of 5hmC and 5mC levels in the cortex and hippocampus of Tet1+/+ (black) and Tet1KO (red) mice analyzed by liquid chromatography/mass spectrometry multiple reaction monitoring (LC/MS/MS-MRM) (3 + 3 animals). (ns - not significant; *P<0.05; error bars +/− stdev). (A) Representative images of the cingulate cortex and hippocampus CA1 are shown. (red – 5hmC; green – NeuN). (B) Graphs showing the global genomic levels of 5mC and 5hmC in the cortex and hippocampus of Tet1+/+ (black) and Tet1KO (red) mice. (C-E) Tet1KO mice have normal neuronal number in different brain areas (C,D) and normal total brain weight (E). (blue – Hoechst; green – NeuN; 3 + 3animals; scale bar – 100 μm; ns - not significant; *P<0.05; **P<0.01; error bars +/− sem). See also Figure S1.

Figure 2. Tet1KO mice exhibit normal memory acquisition but impaired memory extinction

(A-B) Tet1KO mice demonstrate normal associative fear memory acquisition in contextual (A) and cued (B) Pavlovian fear conditioning paradigms (auditory cue: 30 s, 20 kHz, 75 db sound pressure level; foot shock: 2 s, 0.8 mA, constant current; 11 + 11 animals). (C) Tet1KO mice have normal nociception measured by hot plate behavior. (D) Tet1KO mice show deficit in fear memory extinction. Graph displays the levels of freezing 24 hr following contextual fear conditioning for Tet1+/+ (black) and Tet1KO (red) animals (before extinction) and 24 hr following massed extinction of contextual freezing (after extinction) (10 + 9 animals). (E-F) Tet1KO mice show normal spatial reference memory acquisition in the Morris water maze during training (E) and probe trial (F) (time spent in the target (T), opposite (O), right (R) and left (L) quadrants during probe trial). (G-H) Tet1KO mice show impaired spatial reference memory extinction. (G) Time spent in the target (T) and opposite (O) quadrants during no-platform trials on days 1 – 3 of extinction training. (H) Number of crossings of the platform area on different days of extinction training (I) Similar swim speed in Tet1+/+ and Tet1KO mice (10 + 9 animals; ns - not significant; *P<0.05; error bars +/− sem). See also Figure S2.

Figure 3. Normal basal synaptic transmission and LTP but abnormally enhanced hippocampal LTD in Tet1KO mice

(A,B) Tet1 mice demonstrate normal basal synaptic transmission. CA1 fEPSPs evoked by Schaffer collateral pathway stimulation in acute hippocampal slices. (A) The input-output curve was obtained by plotting the slopes of fEPSP against fiber valley amplitudes. (B) PPF ratio was plotted against various interstimulus intervals. (C) Similar intact presynaptic excitability of hippocampal neurons in Tet1KO mice (red) compared to controls (black). Presynaptic fiber volley amplitude was plotted against various stimulus intensities (3 + 3 animals; 5 and 6 slices). (D) Normal hippocampal LTP in Tet1KO mice. LTP in CA1 in acute hippocampal slices from 6-week-old Tet1+/+ (black) (4 animals, 7 slices) and littermate Tet1KO (red) (4 animals, 6 slices) mice evoked by 2 x theta-burst stimulation (TBS). fEPSP slope is shown as percentage of baseline. A bar graph shows the average of fEPSP as a percentage of baseline during the last 10 min of recording. (E) Tet1KO mice exhibit abnormally increased hippocampal LTD. LTD was induced in the Shaffer collateral-CA1 synapses by single-pulse low frequency stimulation (900 stimuli, 1 Hz) in hippocampal slices from 4 pairs of behaviorally naive 6-week-old Tet1+/+ (black) and Tet1KO (red) littermate mice (8 slices per group). A bar graph represents the average of fEPSP as a percentage of baseline during the last 10 min of recording (ns - not significant; *P<0.05; **P<0.01; error bars +/− sem.). See also Figure S3.

Figure 4. Tet1 loss causes significant downregulation of multiple neuronal activity-regulated genes in the brain

(A) Microarray analysis of gene expression in the cortex and hippocampus was performed using naive 4-month-old Tet1+/+ and Tet1KO littermate mice (2 + 2 animals). Scatter plot demonstrated that 118 genes were found to be significantly upregulated (red dots) and 120 downregulated (green dots) in the cortex of Tet1KO mice; 52 genes were upregulated (red dots) and 204 downregulated (green dots) in the hippocampus of Tet1KO animals. Set of neural activity-regulated genes including Npas4, c-Fos and Arc was downregulated in both cortex and hippocampus. (B) RT-qPCR was used to validate microarray data (3-5 animals per genotype including those used for microarray analysis, ns - not significant; *P<0.05; error bars +/− stdev). (C) Analysis of c-Fos showed significant downregulation of its protein in both cortex and hippocampus of naïve Tet1KO mice. No differences in c-Fos spatial expression patterns were found between control and Tet1KO animals (White arrowheads point at some of the c-Fos-expressing cells; 3 + 3 animals; ns - not significant; *P<0.05; **P<0.01; error bars +/− s.e.m). (D) Sodium bisulfite sequencing of the promoter-exon 1 junction of Npas4 containing total 40 CpGs showed that Npas4 promoter area is strongly hypermethylated in both cortex and hippocampus (~20% and ~45% respectively) in naïve Tet1KO mice and hypomethylated in controls (~3.5% and ~8% respectively) (2 + 2 animals). Each of the charts in the figure represents one animal. Each column within the chart represents a single CpG position at the promoter-exon1 junction of Npas4. Each row represents a single clone sequenced. A total of 10 - 20 clones were sequenced for each genotype. White boxes represent unmethylated CpG; black boxes represent methylated CpG; grey boxes represent residues undetermined due to a poor sequence quality. Arrowhead denotes position of start codon in exon1. See also Figure S4 and Tables S1 and S2.

Figure 5. Tet1KO mice exhibit downregulation of neuronal activity-regulated genes and hypermethylation of Npas4 promoter following extinction training

(A-C) mRNA and protein levels of several neuronal activity-regulated genes are decreased in the cortex and hippocampus of Tet1KO mice after memory extinction training. Two groups of male littermate Tet1+/+ and Tet1KO mice underwent contextual fear conditioning (context chamber + 2 s, 0.8 mA constant current shock) followed by massed fear memory extinction training (21 min of context exploration). 24 hr later, mice were placed in the same chamber, given 3 min exploration time to induce extinction memory retrieval. Mice were sacrificed 20 min (3 + 3 animals) and 1 hr (3 + 3 animals) after retrieval for mRNA and protein analysis respectively. (A) Relative mRNA levels of Npas4 and c-Fos in the cortex and hippocampus of Tet1+/+ (black) and Tet1KO (red) are shown. (3 animals used per genotype, ns - not significant; *P<0.05;; error bars +/− stdev). (B) Representative images of Npas4 and c-Fos protein expression in the cortex and hippocampus Tet1+/+ and Tet1KO mice are shown. (C) Relative protein levels of Npas4 and c-Fos in the cortex and hippocampus are shown. Note significant downregulation of both mRNA and protein levels of Npas4 and c-Fos in both cortex and hippocampus of Tet1KO mice after memory extinction (3 animals used per genotype, ns - not significant; *P<0.05; error bars +/− stdev). (D) Sodium bisulfite sequencing of the promoter-exon 1 junction of Npas4 showed that this area is hypermethylated in Tet1KO cortex (~24.5% of CpG methylated) and hippocampus (~30.5% of CpGs methylated) compared to control cortex (~8% of CpGs methylated) and hippocampus (~8% CpGs methylated) (2 + 2 animals). Each of the charts in the figure represents one animal. Each column within the chart represents a single CpG position at the promoter-exon1 junction of Npas4. Each row represents a single clone sequenced. A total of 10 - 20 clones were sequenced for each genotype. White boxes represent unmethylated CpG; black boxes represent methylated CpG; grey boxes represent residues undetermined due to a poor sequence quality. Arrowhead denotes position of start codon in exon1. (E) Direct comparison of Npas4 and c-Fos expression levels in the hippocampus of control and Tet1KO mice in various experimental conditions: in naïve animals, following Pavlovian fear conditioning/fear memory acquisition (20 min after fear memory retrieval), and following fear memory extinction training (20 min after extinction memory retrieval). (3 + 3 animals; ns - not significant; *P<0.05; **P<0.01; error bars +/− stdev). See also Figure S5.