Dnmt1 and Dnmt3a maintain DNA methylation and regulate synaptic function in adult forebrain neurons

Abstract

Dnmt1 and Dnmt3a are important DNA methyltransferases that are expressed in postmitotic neurons, but their function in the CNS is unclear. We generated conditional mutant mice that lack Dnmt1, Dnmt3a or both exclusively in forebrain excitatory neurons and found that only double knockout (DKO) mice showed abnormal long-term plasticity in the hippocampal CA1 region together with deficits in learning and memory. Although we found no neuronal loss, hippocampal neurons in DKO mice were smaller than in the wild type; furthermore, DKO neurons showed deregulated expression of genes, including the class I MHC genes and Stat1, that are known to contribute to synaptic plasticity. In addition, we observed a significant decrease in DNA methylation in DKO neurons. We conclude that Dnmt1 and Dnmt3a are required for synaptic plasticity, learning and memory through their overlapping roles in maintaining DNA methylation and modulating neuronal gene expression in adult CNS neurons.

Figure 1

Mice with conditional deletion of Dnmt1 and Dnmt3a show small hippocampus without cell loss. a, b, Real time PCR analysis showed decreased expression of Dnmt1(a) and Dnmt3a(b) in DKO forebrain. Relative expression levels of Dnmt1(a) and Dnmt3a(b) in DKO and wildtype littermate control (Con) were compared at postnatal day 3 (P3), P14, 1 month (mo), 2 mo, 3 mo, and 4 mo. Three pairs of samples were used for each time point. c, Stereology analysis identified reduced volume of hippocampus as well as dentate gyrus in DKO as compared with control mice. d, DKO had similar number of dentate gyrus cells as control mice. e, DKO dentate gyrus neuronal cell body is significantly smaller as compared with control mice. 5 control and 6 DKO male mice at 3 mo were used in c, d and e. Data are presented as means±s.e.m. *indicates P<0.05, **indicates P<0.01.

Figure 2

Impaired neural plasticity in DKO mice. a, LTP was reduced in adult DKO mice (* P<0.05). Field EPSP (fEPSP) slopes in control (square symbols) versus DKO mice (triangle symbols) were recorded before and after tetanic stimulation (100Hz, 1sec). Scale bar, 5 msec/1mV. b, LTD was enhanced in adult DKO mice (* P<0.05). fEPSP slopes were recorded before and after stimulation (1Hz, 15min). Scale bar, 10 msec/0.5mV. Representative recordings are shown in insets (a, b). 28 slices from 9 Con and 13 slices from 7 DKO were used in a. 10 slices from 5 Con and 8 slices from 4 DKO were recorded in b. The basal synaptic transmission from the DKO mice and Con are identical as shown by plotting varying stimulus intensity (10–100μA) against the presynaptic fiber volley amplitudes (c) and postsynaptic fEPSP slope (d). n =33 slice from 8 mice for DKO group and n =28 slice from 8 mice for Con group. **indicates P<0.01. e, Paired-pulse facilitation (PPF) studies across different inter-stimulus intervals (ISIs) revealed no difference between DKO and Con group. n =16 slice from 4 mice for DKO group and n =14 slice from 4 mice for Con group. Slice numbers were used for statistical analysis.

Figure 3

Impaired learning and memory in DKO mice. a-d, Morris water maze test. a, Schematic drawing of the Morris water maze test design. b, Escape latency time to find the hidden platform plotted versus training day. Control mice improved significantly faster than DKO mice. c, Percentage time spent in target quadrant during three probe trials demonstrated that the DKO spent less time in the target quadrant. n = 13 mice for DKO group and n =17 mice for Con group. d, The swimming speeds of the DKO were indistinguishable from controls. 17 control and 13 DKO mice at 3 mo were used in b-d. e-f, Contextual fear conditioning test. e, Schematic drawing of the contextual fear conditioning test design. Mice were trained and tested immdediately (IM/average for 3 min) and 24h later in a conditioning chamber. f, Contexual memory consolidation was impaired in DKO mice as demonstrated by decreased freezing frequency at 24 h. The acquisition was normal in DKO immediately after shock. Baseline, freezing before shock presentation; IM, freezing immediately after shock presentation. n = 21 mice for DKO group and n =13 mice for Con group in figure f. Data are presented as means±s.e.m. * indicates P<0.05. **indicates P<0.01.

Figure 4

Induction of immune genes in DKO mouse brain. a, Neuronal induction of MHC I gene expression in DKO. In situ hybridization of H₂D was post-stained with cresyl violet to reveal the nuclei which appeared purple. Silver grains appeared black. In the cortex and the hippocampus of DKO, MHC I signal is highly concentrated in the neurons whose nuclei are large and lightly stained in cresyl violet. Whereas in the white matter, where most cells are glia and whose nuclei are small and dark stained, there were no significant difference compare to wildtype. Scale bar at bottom right, 100um, applied to all panels. b, Real time PCR analysis confirmed upregulation of immune genes Stat1, B₂M, H₂M₂, H₂Q₇, C3 and C4 within DKO hippocampi of 2-3 months adults. The induction was also found as early as postnatal day 14 (P14) (d) but no significant difference in expression was noticed at P3 hippocampi (c). 3-4 pairs of samples were used for each experiment. e, Real time PCR analysis showed significant induction of Stat1, B₂M, H₂M₂, H₂Q₇ as well as decreased expression of Dnmt3a and Dnmt1 in Dnmt1^2lox/2lox Dnmt3a^2lox/2lox mouse hippocampal neuronal cultures 5 days after adeno-cre virus infection as compared with sham control cultures with adeno-GFP viruses infection. Data are presented as means±s.e.m. *indicates P<0.05. **indicates P<0.01.

Figure 5

Protein increase of Stat1 within DKO mouse brain is associated with promoter demethylation in neuronal cells. a, Immunohistochemistry of phosphorylated Stat1 demonstrated its increased expression within DKO cortex, mainly in cells of neuronal shape. Scale bar at bottom right, 50um, applied to both panels. b, Bisulfite sequencing of Stat1 promoter showed demethylation within DKO as compared with control samples at 3month old. Data as shown were from genomic DNA of wildtype and DKO mouse forebrains. Schematic gene promoter structure is shown on top with arrow pointing out transcription starting site (+1). Each CpG site is marked with a vertical slash. The blue bar highlights the promoter region that underwent methylation analysis with the relative location from the transcription starting site. A summary of methylation frequency at individual CpG sites was shown in a line chart and a summary of mean DNA methylation levels of individual alleles was shown in column chart. c, Bisulfite sequencing of Stat1 promoter showed enhanced DNA demethylation within DKO at P14. d, Stat1 promoter demethylation within DKO mouse brain existed only in neurons. Bisulfite sequencing was performed on DNA samples from FACS sorted NeuN positive and negative nucleus sub-populations of both DKO and control mouse forebrains. Total 30 clones from 3 samples were analyzed. Data are presented as means±s.e.m. Unpaired student t-test. *indicates P<0.05, **indicates P<0.01.

Figure 6

Gene ontology and bisulfite sequencing verification of MeDIP-chip analysis. a, Quantitative analysis of 5-methylcytosine using LC-ESI-MS MS. A representative chromatogram following injection of DNA hydrolysis of DKO and WT forebrain samples showing the MRM response for 5mdC (m/z 242.1-126.0) is shown. 5-Methylcytosine content is expressed as the percentage of 5-methylcytosine in the total cytosine pool. Data are the mean ± s.e.m. from replicates of 4 separate experiments (n = 16). b, Gene ontology classifications of MeDIP-chip result based on categories of biological process and molecular function. The ontology term is on the y-axis and the P value of enrichment significance is on the x-axis. c-f, Four gene loci Dhh (c), Kcne1 (d), Pten region1(e) and Pten region2(f)) were verified for DNA demethylation change. The results are displayed in the same manner as in figure 5. Data as shown were from 2-3 month old wildtype control and DKO mouse forebrains. Data are presented as means±s.e.m. Unpaired student t-test. *indicates P<0.05, **indicates P<0.01.