Synaptic control of DNA-methylation involves activity-dependent degradation of DNMT3a1 in the nucleus

DNA-methylation is a crucial epigenetic mark for activity-dependent gene expression in neurons. Very little is known how synaptic signals impact promoter methylation in neuronal nuclei. In this study we show that protein levels of the principal de novo DNA-methyltransferase in neurons, DNMT3a1, are tightly controlled by activation of N-methyl-D-aspartate receptors (NMDAR) containing the GluN2A subunit. Interestingly, synaptic NMDAR drive degradation of the methyltransferase in a neddylation-dependent manner. Inhibition of neddylation, the conjugation of the small ubiquitin-like protein NEDD8 to lysine residues, interrupts degradation of DNMT3a1 and results in deficits of promoter methylation of activity-dependent genes, synaptic plasticity as well as memory formation. In turn, the underlying molecular pathway is triggered by the induction of synaptic plasticity and in response to object location learning. Collectively the data show that GluN2A containing NMDAR control synapse-to-nucleus signaling that links plasticity-relevant signals to activity-dependent DNA-methylation involved in memory formation.


Introduction
It is widely believed that rapid and reversible DNA methylation is essential for the stability of long-term memory but still very little is known how synaptic signals can induce changes in DNA-methylation to elicit enduring alterations in plasticity-related gene expression (Bayraktar & Kreutz, 2018a, b;Campbell & Wood, 2019;Day & Sweatt, 2010;Miller & Sweatt, 2007;Guo et al, 2011). In addition, aberrant DNA methylation has been implicated in neuropsychiatric diseases including schizophrenia, bipolar, and major depression disorders (Bayraktar & Kreutz, 2018;Mill et al, 2008;Murgatroyd et al, 2009). NMDAR signaling to the nucleus is instrumental for learning and memory formation and is altered in schizophrenia as well as other neuropsychiatric disorders (Zhou & Sheng, 2013;Paoletti et al, 2013).
However, a mechanistic link between NMDAR signaling and DNA-methylation is currently missing.
Compelling evidence exists for learning-induced de novo DNA methylation with several studies showing the necessity of active DNA methylation as well as demethylation particularly in the hippocampus during memory consolidation (Bayraktar & Kreutz, 2018;Oliveira, 2016;Kaas et al, 2013;Rudenko et al, 2013).
One of the target genes is the brain-derived neurotrophic factor (BDNF), which undergoes promoter-specific DNA demethylation in the CA1 region of the hippocampus during memory consolidation (Lubin et al, 2005). The underlying signaling machinery is also here not well understood and it is still essentially unclear how synaptic signals conveyed to the nucleus impact mechanisms of DNA methylation and demethylation of the Bdnf promoter. DNMT3a1 is the major de novo DNA methyltransferase in brain and plays a documented role in activity-dependent DNA methylation (Feng et al, 2005;Feng et al, 2010). Along these lines impaired spatial learning and memory and attenuated CA1 long-term potentiation (LTP) have been reported following a forebrain specific DNMT gene knockout in principal neurons (Feng et al, 2010;Morris et al, 2014).
Finally, it is nowadays widely accepted that memory consolidation as well as synaptic plasticity not only rely on de novo protein synthesis but also protein degradation (Jarome & Helmstetter, 2013;Karpova et al, 2006). Proteasomal degradation of proteins in neurons has been studied mainly in the context of ubiquitylation and sumoylation, whereas neddylation -the attachment of the small ubiquitin-like peptide neural precursor cell-expressed developmentally down-regulated gene 8 (NEDD8) -has not been much investigated. Here we show that activation of synaptic GluN2Acontaining NMDARs drives the neddylation-dependent proteasomal degradation of the principal de novo DNA-methyltransferase in the adult brain DNMT3a1.
Collectively the data point to a mechanism that allows for the synaptic control of nuclear DNMT3a1 protein levels thereby creating a time window for reduced de novo DNA-methylation at a subset of target genes. This signaling pathway highlights how synapse-to-nucleus signaling might directly impact on DNA-methylation and memory consolidation.

Synaptic activity controls levels of DNMT3a1 in neuronal nuclei
DNMT3a1 is the major de novo DNA methyltransferase expressed in the adult brain (Feng et al, 2005). When we first addressed the cellular localization of the enzyme using an antibody that recognizes an N-terminal region (Sakai et al, 2004) specific for DNMT3a1 ( Fig EV1A to D), we found a prominent nuclear localization in hippocampal primary neurons and much fainter staining hardly above background in astrocytes ( Fig 1A), indicating that DNMT3a1 is mainly expressed in neurons. This finding prompted us to asked next whether synaptic activity might regulate nuclear DNMT3a1 protein levels. When we induced burst firing of excitatory synapses in hippocampal primary neurons with the GABA-A receptor antagonist bicuculline (bic) and the potassium channel blocker 4-amino-pyridine (4-AP), we observed a prominent reduction in the nuclear immunofluorescence of DNMT3a1 (Fig 1B and C), a finding that was confirmed by quantitative immunoblot analysis of cell lysates from cortical primary neurons (Fig 1D to F). Enhancing excitatory activity for 10 min with bic/4-AP was sufficient to reduce DNMT3a1 immunofluorescence (Fig 1G and H), while excitotoxicity in hippocampal primary neurons elicited with bath application of 100 µM NMDA for 10 minutes -a protocol that results in extrasynaptic NMDAR activation and delayed cell death (Dieterich et al, 2008) -had no effect (Fig 1G and H).

Synaptic GluN2A containing NMDAR drive degradation of nuclear DNMT3a1
The activity-dependent degradation of DNMT3a1 was blocked in the presence of the competitive NMDAR antagonist 2-amino-5-phosphonopentanoic acid (APV) ( Fig. 2A and B). Interestingly, the application of the antagonist NVP-AAM077 at low doses, that mainly target di-heteromeric GluN2A-containing NMDARs (Auberson et al, 2002), completely prevented degradation (Fig. 2C and D). In addition, shRNAinduced protein knockdown (Fig EV2A and B) of GluN2A confirmed a specific requirement for NMDAR containing this subunit to elicit degradation of DNMT3a1 ( Fig   2E and F), whereas application of the GluN2B antagonist ifenprodil had no effect ( Fig   2G and H). Co-application of the L-type Ca 2+ -channel blocker nifedipine (Fig EV2C and D) or the CaMK inhibitor KN93 to the stimulation buffer also hindered nuclear DNMT3a1 degradation (Fig EV2E and F). Taken together, these experiments indicate that brief activation of synaptic GluN2A containing NMDAR, probably by induction of dendritic action potentials and nuclear calcium waves, is a potent stimulus to control DNMT3a1 protein levels in the nucleus.

Proteasomal degradation of DNMT3a1 requires neddylation
We next examined which mechanisms might contribute to DNMT3a1 down-regulation and found that the proteasome inhibitors MG132, Lactacystin or Carfilzomib, which all operate via different mechanisms (Fenteany et al, 1995;Lee & Goldberg, 1998;Meng et al, 1999), completely abolished the effect of GluN2A stimulation (Fig EV3A to F). Since we found no concomitant alteration of DNMT3a1 mRNA levels ( Fig   EV3G), these data suggest that proteasomal degradation controls the protein levels of the enzyme in an activity-dependent manner.
Potential mediators of DNMT3a1 degradation are members of the family of Cullin proteins (Sarikas et al, 2011). Cullin family members combine with RING proteins to form Cullin-RING E3 ubiquitin ligases (Petroski & Deshaies, 2005) and neddylation is a prerequisite for their activation in the nucleus. Neddylation has been studied only recently in neurons (Vogl et al., 2015;Scudder & Patrick, 2015) and the subcellular distribution of NEDD8 in neurons has not been determined yet. We found that NEDD8 is abundantly localized in the nucleus of hippocampal primary neurons, whereas immunofluorescence intensity was much weaker at synapses (Fig 3A). We observed heterologous co-immunoprecipitation of DNMT3a1 with CUL4B, CUL1, CUL3, CUL4, CUL7, but not with CUL2 and CUL5 from HEK293T cell lysates ( Fig   EV4A). In addition, we found that poly-ubiquitination of DNMT3a1 was elevated following forced expression of CUL4B in HEK293T cells (Fig EV4B to D), which was chosen as an example because of its high expression in brain. Conversely shRNAbased CUL4B protein knockdown resulted in reduced DNMT3a1 poly-ubiquitination ( Fig EV4E). The neddylation inhibitor MLN4924 selectively inhibits the NEDD8-Activating-Enzyme (NAE) at very low concentrations (Soucy et al, 2009). Accordingly, poly-ubiquitination of immunoprecipitated DNMT3a1 was reduced in cells that were subjected to MLN4924 treatment ( Fig EV4F). Moreover, neddylated CUL4B was coimmunoprecipitated with DNMT3a1 ( Fig EV4G).
We next asked whether neddylation of Cullins might be involved in controlling the activity-dependent DNMT3a1 proteasomal degradation in neurons. Coimmunoprecipitation experiments of endogenous proteins extracted from cortical primary neurons using pars pro toto a CUL4B specific antibody revealed that neuronal CUL4B and DNMT3a1 might be in one complex in vivo ( Fig 3B). Acute treatment of hippocampal primary neurons with concentrations of MLN4924 as low as 5 nM prevented DNMT3a1 degradation (Fig 3C and D). Of note, acute treatment of primary neurons for six hours with either a low (5 nM) or even a high dose of MLN4924 (1 µM) did not alter the total number of spines ( Fig EV5A and B) as it was reported previously with the higher concentration and long-term treatment (Vogl et al., 2015;Scudder & Patrick, 2015). Moreover, we observed that nuclear NEDD8 staining intensity was not altered following bic/4AP treatment (Fig 3E and F

Synaptic plasticity inducing stimuli elicit DNMT3a1 degradation in a GluN2Adependent manner
We next addressed whether induction of NMDAR-dependent long-term potentiation (LTP), a form of plasticity that is considered to be a cellular model of learning and memory, involves synaptic control of nuclear DNMT3a1 protein levels. When we induced LTP in the hippocampus with high-frequency stimulation of Schaffercollaterals ( Fig 4A), we found a significant down-regulation of DNMT3a1 protein levels within six hours in the potentiated CA1 region following tetanization of the slices (Fig 4B and C). Moreover, the expression of late-LTP is neddylation-sensitive and field excitatory postsynaptic potentiation slope values returned to baseline within three hours when the slices were treated with the NEDD8 inhibitor MLN4924 (Fig 4D to F).
We next asked whether following the induction of LTP signaling of GluN2Acontaining NMDAR to the nucleus is crucial in neddylation-dependent degradation of DNMT3a1. Interestingly, DNMT3a1 protein levels were already clearly higher in hippocampal tissue homogenates of GluN2A knockout mice as compared to wildtype controls (Fig 4G and H). GluN2A knockout mice show reportedly impaired hippocampal LTP (Sakimura et al, 1995) but a stronger tetanic stimulation restores the impairment and the saturation level of LTP is unaltered (Kiyama et al, 1998). We could replicate these published findings (

Degradation of DNMT3a1 facilitates Bdnf gene expression
In the adult brain brain-derived neurotrophic factor (BDNF) has principal functions in synaptic plasticity, learning and memory (Karpova, 2014). Expression of the Bdnf gene is controlled by eight promoters (Aid et al, 2007) and among those, particularly promoter IV activity is strongly stimulated by the calcium influx through synaptic NMDARs (Lubin et al, 2005;Zheng et al, 2011). DNA methylation of the Bdnf IV promoter has been studied previously also in the context of neuropsychiatric disorders (Kundakovic et al, 2015;Maynard et al, 2016). We therefore chose Bdnf IV gene expression to test whether activity-dependent degradation of DNMT3a1 might impact DNA methylation of promoters of plasticity-related genes and corresponding gene expression. Quantitative real-time PCR experiments first revealed that Bdnf mRNA expression is increased by enhanced synaptic activity in primary hippocampal neurons and this increase in transcript levels was significantly lower in the presence of MLN4924 (Fig 5A). Comparable results were obtained in acute hippocampal slices following high-frequency stimulation of Schaffer-collaterals ( Fig 5B) and importantly, the LTP-induced increase in Bdnf IV transcript levels was reduced in the presence of the NEDD8-inhibitor ( Fig 5B).
We next addressed whether increased Bdnf IV mRNA production was correlated with the demethylation of the Bdnf IV promoter in acute slices following LTP induction, as predicted by the degradation of DNMT3a1. First, methylation specific restriction enzyme analysis was performed using primers that span the Bdnf IV promoter sequence possessing three different restriction sites ( Fig EV7A). Tetanized CA1 samples revealed a reduction in Bdnf IV promoter methylation, whereas increased promoter methylation was observed for the group that received high-frequency stimulation while being treated with MLN4924 ( Fig 5C). A subsequent series of MeDIP-qPCR experiments were performed using tetanized CA1 tissue samples following the induction of LTP and confirmed the change in promoter methylation.
Less amplicons were generated with primers targeting the Bdnf IV promoter that cover multiple cytosine residues ( Fig EV7B) known to regulate mRNA expression ( Fig 5D). More amplicons were detected in MLN4924-treated slices following the induction of LTP (Fig 5D), indicating increased promoter methylation. Among the different Bdnf promoters that were investigated, activity-dependent DNA methylation is particularly prominent for promoter IV, whereas promoter I methylation was not altered following either LTP induction or NEDD8 inhibition with MLN4924 ( Fig 5E).

DNMT3a1 is degraded in the hippocampus as a result of learning
In the final set of experiments, we investigated whether DNMT3a1 degradation occurs in vivo as a result of CA1-dependent learning and whether this degradation and memory formation is neddylation-sensitive. Formation of a memory for the spatial location of objects in an open field ( Fig 6A) requires synaptic activity of CA1 neurons (Assini et al, 2009;Haettig et al, 2013) and is responsive to changes in the expression of BDNF (Intlekofer et al, 2013;Wang et al, 2017). We observed that DNMT3a1 protein levels were reduced in mice three hours following training ( Fig 6B and C). DNMT3a1 protein levels returned to control values within six hours, which might reflect less intense synaptic activity as compared to tetanization of slices by high-frequency stimulation (Fig 6D and E). Bilateral intra-hippocampal infusion of MLN4924 into CA1 immediately after object location learning ( Fig 6F) resulted in a disturbance of object location memory, as indicated by profoundly reduced discrimination of novel and familiar object locations when compared to mice that received vehicle infusion ( Fig 6G). Interestingly, the learning impairment in MLN4924 treated mice was associated with the prevention of DNMT3a1 degradation ( Fig 6H and I). Protein levels were significantly higher in mice injected with MLN4924 three hours after training compared to vehicle-injected mice (Fig 6H and I). Six hours following training DNMT3a1 protein levels were no longer different between treatment groups and returned to baseline levels ( Fig 6J and K).

Discussion
Compelling evidence exists for the necessity of active DNA methylation as well as demethylation in the hippocampus during memory consolidation (Bayraktar & Kreutz, 2018a, b;Oliveira, 2016;Kaas et al, 2013;Rudenko et al, 2013). However, the underlying signaling machinery is not understood and it is essentially unclear how synaptic signals conveyed to the nucleus impact DNA methylation and demethylation. Here, we show that activation of synaptic GluN2A-containing NMDARs drives the neddylation-dependent proteasomal degradation of the principal de novo DNA-methyltransferase in the adult brain DNMT3a1.
The finding that activation of GluN2A containing NMDARs to the nucleus evokes degradation of DNMT3a1 raises several interesting questions about the underlying mechanism of long-distance signaling and the rationale behind it. GluN2A containing NMDAR are in contrast to those containing GluN2B preferentially found at synaptic sites (Wyllie & Hardingham, 2013) and it is possible if not likely that steep and fast synaptic Ca 2+ -influx through these receptors is necessary to elicit nuclear Ca 2+responses that in turn enhance neddylation of Cullins. Neddylation as such has not been investigated in any detail in neurons yet and not much information is currently available on how NAE activity itself is regulated. The present study therefore provides first evidence that an NMDAR-derived synaptic calcium signal is coupled to neddylation of Cullins in the nucleus. Two previous reports have shown that blocking neddylation for extended periods of time (in contrast to the administration regime in the present study) leads to reductions in spine size and impairment of synapse maturation in neurons (Vogl et al, 2015, Scudder & Patrick, 2015. In addition, neddylation alters synapse function and morphology by directly modifying one of the major synaptic scaffolding proteins PSD95 (Vogl et al, 2015). We found that NEDD8 is most abundant in neuronal nuclei and it is tempting to speculate that activitydependent neddylation might reduce the protein levels not only of DNMT3a1 but also of other nuclear epigenetic modifiers which contribute to object location memory.
Along these lines the contribution of neddylation to object location memory seems to be substantial, considering the near-complete removal of the discrimination by the inhibition of neddylation. This is, however, not reflected in the extent of reduction in the levels of DNMT3a1 induced by the behavioral training. Moreover, neddylation is like other posttranslational modifications reversible, which adds potentially another level of regulation for degradation of this methyltransferase. In addition, different efficiencies of proteasomal degradation in neuronal sub-compartments, the necessity for the integration of signaling pathways in the nucleus as well as the complex formation and the potential nuclear export of ubiquitinated DNMT3a1 might account for the relatively slow decline.
Moreover, in this study we propose a long-distance signaling pathway that can provide a potential link between different observations. Converging evidence suggests that NMDAR function in the dorsal CA1 area is critical for novel object location memory (Assini et al, 2009;Haettig et al, 2013) and increased BDNF expression in the hippocampal CA1 region supports object location learning (Intlekofer et al, 2013;Wang et al, 2017). We have, therefore, chosen BDNF as a paradigmatic example for our studies, which is one of the target genes that undergoes promoter-specific DNA demethylation in the CA1 region of the hippocampus during memory consolidation (Lubin et al, 2005) and impaired spatial learning and memory as well as attenuated CA1-LTP have been reported following a forebrain specific DNMT1 and -3 gene knockout in principal neurons (Feng et al, 2010, Morris et al, 2014. Aberrant DNA methylation has been implicated in a plethora of studies in neuropsychiatric diseases including schizophrenia, bipolar, and major depression disorders (Bayraktar & Kreutz, 2018;Mill et al, 2008;Murgatroyd et al, 2009). One of the hallmarks of schizophrenia is a down-regulation of BDNF expression that is associated with the enrichment of 5-methylcytosine at gene regulatory domains within the Bdnf promoter (Zheleznyakova et al, 2016). Moreover, elevated hippocampal DNMT3a expression has been reported in the postmortem brain of schizophrenia patients (Zhubi et al, 2016).
Collectively our data point to a mechanism that allows for the synaptic control of DNMT3a1 levels and thereby creates a time window for reduced de novo DNAmethylation at a subset of target genes. DNMT3a1-mediated methylation has been largely associated with silencing of promoters, which would in turn attenuate activitydependent gene expression. A shorter splice isoform, DNMT3a2, was shown to associate with transcriptional facilitation of the expression of plasticity-relevant genes presumably via methylation of CpG islands in their promoter and coding regions (Oliveira et al, 2012. Dnmt3a2 is an immediate early gene that is identical to Dnmt3a1 except that it lacks the sequence encoding the N-terminal 219 amino acids of the enzyme, which encompasses the epitope of the antibody that was used in the current study. An intriguing question is whether the increased expression of DNMT3a2 mRNA and down-regulation of DNMT3a1 is induced by the same stimulus, i.e. activation of synaptic GluN2A NMDAR. It is currently unknown whether DNMT3a2 mRNA will be immediately translated, as expected for an immediate early gene. In this case it might not replace DNMT3a1 but independently further facilitate activitydependent gene expression.

Primary neuronal culture and drug treatments
Rat cortices and hippocampi were dissected from embryonic day 18 rats (Sprague Cortical neurons were treated with 1 µM tetrodotoxin (TTX, Alomone Labs, Jerusalem, Israel) for 12 h, media was washed-out and bic/4AP were applied for 6 h at 21 DIV.

HEK293T Cells Culture
HEK293T cells were cultured in DMEM media supported by fetal bovine serum.

Experimental Animals
Neurons for primary cell cultures and slices for electrophysiology experiments were  #AGC-002) was applied in cell media for 20 minutes, then cells were fixed, and staining protocol was followed as mentioned above.

Confocal laser scanning microscopy and image analysis
The SP5

Intrahippocampal injections
Mice were anesthetized with 5% isofluorane in O2/N2O mixture. Mice were placed in a stereotaxic frame (World Precision Instruments) and anesthesia was maintained with 1.5% isofluorane using gas anesthesia system (Rothacher Medical GmbH., Switzerland). After craniotomy, 10 µl NanoFil microsyringes (World Precision Instruments) containing 33G injection needles were lowered into the dorsal CA1 area under stereotactic guidance with the coordinates anterioposterior (AP) -2.0 mm, mediolateral (ML) ±1.5 mm from Bregma and dorsoventral (DV) -0.14 mm from brain surface. Each animal received 1.5 µl/hemisphere bilateral infusion of drug (MLN4924) while (saline with respective volume of DMSO) as sham control, at an infusion rate of 0.5 µl/min.

Overexpression and knockdown experiments
The overexpression experiments in HEK-293T cells were performed using the CCCAAGGTCAAGGAGATCA and GCTTCGCGCCGTAGTCTTA, respectively.

Tissue collection and analysis
After the LTP recordings, only the potentiated CA1 region of the Hippocampus was collected from the acute slices. The tissue was either subjected to RNA or DNA extraction for transcription or promoter methylation analysis, respectively, or for total protein extraction for western blotting. For the experiments investigating learningdependent DNMT3A1 degradation, mice were sacrificed 3 or 6 hours after training and CA1 region of the hippocampus was dissected. Bilateral intra-hippocampal MLN4924 (7.5 pmol/site) or saline infusions were performed immediately after training.

Immunoprecipitation experiments
Endogenous IP experiments were performed from cultured rat cortical neurons. 50 µl dynabeads Protein G (ThermoFischer Scientific, Waltham, MA, USA) were blocked using albumin from chicken egg white (Sigma-Aldrich, St. Louis, MO, USA) while rocking for 30 min. Then, the dynabeads protein G were incubated with 2-3 µg of rabbit polyclonal CUL4B antibody (Proteintech, catalog #20882-1-AP) while gently rotating for 2h at 4°C. Nuclear protein extracts were then incubated with antibodybound-protein G dynabeads overnight at 4 °C. The next day, following several washes with ice-cold TBS-T, samples were eluted using 4x Laemmli sample buffer.

Western blot
Total homogenates were prepared from mouse and rat brain or cultured cortical neurons by performing the lysis in 10mM Tris/HCl pH 7.5, 0.5% TritonX, and protease inhibitor cocktail (Roche, catalog #04693116001) containing TBS. 4x Laemmli buffer was added to a final dilution of 1.5x following 10 min-incubation at 95 °C, samples were ready for protein analysis. HEK-293T cells were harvested in TBS, which contains protease inhibitor cocktail. Cells were lysed in 1% Triton-X containing lysis buffer; centrifuged for 30 min at 21000 rpm, the supernatant fraction was collected. Protein estimation was performed either by amidoblack or BCA assay (Thermo Scientific). Western blots were then performed using 4-20% gradient polyacrylamide gels. The following antibodies were used in this study: DNMT3AN Immunoresearch Laboratories, catalog #115035003 and #111035003, respectively).
Quantification of immunoblots was done with ImageJ software (NIH, Maryland, USA).
Integrated density values were evaluated for the analysis. When necessary, experimental data from individual experiments performed at different time points were normalized to respective controls and pooled together with other data set.

Object Location Memory
Object location memory was performed in a square arena (50 x 50 x 50 cm) under mild light conditions, according to Heyward and colleagues (2012), with modifications. Briefly, the task consisted of a habituation session, training and test.
During habituation, animals were allowed to explore the empty arena for 20 minutes.
Twenty-four hours later, training session took place, where animals were free to explore a pair of similar objects (made of plastic mounting bricks), placed in the arena, for 20 minutes. Test session was performed 6 hours after training, where one of the objects was was placed in a new position, and again, animals were free to explore the two objects for 20 minutes. All 3 sessions were video-recorded and behavior was analyzed offline using ANY-maze software ( analysis. Chambers and objects were thoroughly cleaned with 10% ethanol before and after each animal was test.

Statistical Analysis
The data were analyzed by one-way ANOVA and unpaired one/two-tailed Student's t-test. Two-way ANOVA followed by post-hoc Bonferroni test was employed to compare means from multiple groups. Quantitative real-time PCR data were subjected to Grubbs' outlier test and analyzed by either unpaired two-tailed Student's t-test or two-way ANOVA, which was followed by Bonferroni post hoc test, where applicable. The Mann Whitney U-test was used to compare the Averaged field potentials (300-360 min) between two groups of differentially treated slices.
Otherwise stated, error bars present S.E.M. Statistical analyses were performed in GraphPad (GraphPad Software, Inc., La Jolla, USA).
G and H Treatment of neurons with bic/4AP for 10 min or 3 h was equally effective to reduce nuclear DNMT3a1 immunofluoresence. Application of 100 µM NMDA for 10 min had no effect. Students t-test **p<0.01, ***p<0.001.    E MeDIP-qPCR analysis did not show any alteration in Bdnf promoter I methylation.
Two-way ANOVA followed by Bonferroni's post hoc test.   Scale bar is 20 µm. Two-way ANOVA followed by Bonferroni's post hoc test.
***P<0.001, n.s. not significant.  DNMT3A1 was immunoprecipitated from total cell extracts using anti-GFP antibodies coupled to microbeads. Cullin proteins were detected in the immunoprecipitate with a myc-antibody. Same volume of IP and input per sample were loaded.
B HEK-T cells were transfected with expression vectors for GFP-DNMT3A1 and myc-CUL4B or myc-vector. DNMT3A1 was immunoprecipitated from total cell extracts using anti-GFP antibodies coupled to microbeads. CUL4B was detected in the immunoprecipitate with a myc-antibody.
C and D Poly-ubiquitination of DNMT3A1 in the immunoprecipitate is enhanced when DNMT3A1 was co-expressed with CUL4B. Unpaired two-tailed Student's t-test, *p<0.05.
F and G HEK-T cells were transfected with expression vectors for GFP-DNMT3A1, myc-CUL4B (F) and also with active HA-Nedd8 (G). Total cell extracts were immunoprecitated using anti-GFP antibodies. (F) Less DNMT3A1 ubiquitination was  A Averaged fEPSP slopes of the last 30 min LTP showed no significant difference between GluN2A+/+ and GluN2A-/-mice. The Mann Whitney Test was used to compare the Averaged field potentials (150-180min) between two groups.
C Baseline was determined by the second input on the same slice which was induced LTP by the first input.

D Scatterplot and correlation analysis between the normalized DNMT3A1
immunoblotting mean values and the averaged fEPSP slope of the whole LTP recording depicts a negative correlation between the strength of the LTP and the synaptic stimulation-induced DNMT3A1 down-regulation in GluN2A +/+ mice while no significant correlation was observed in slices of GluN2A -/-mice. Unpaired two-tailed Student's t-test, *P<0.05, ns not significant.