Patient information and iPS cell generation

In this study we used in total four control and four iPS cells with reduced EHMT1 function. In contrast to a previous study59 we included patients in this study that present the full spectrum of KS associated symptoms, including ID and ASD. KS 1 and KS 2 originate from two individuals: a female KS patient with a frameshift and a missense mutation in the EHMT1 gene, respectively (patient 25 in ref. 22 and patient 20 in ref. 8). An isogenic pair of control (C MOS ) and KS line (KS MOS ) originated from an individual diagnosed with a mosaic heterozygous 233 kbp deletion of chromosome 9 which includes the EHMT1 gene (deletion starts after exon 4) and CACNA1B gene23. Clones were selected after performing RT-qPCR on genomic DNA with primers spanning Exon 3: F-GAAGCAAAACCACGTCACTG; R-GTAGTCCTCAAGGGCTGTGC. Exon 4: F-CCCAGAGAAGTTCGAGAAGC, R- GGGTAAAAGCTGCTGTCGTC. Exon 5 F-CAGCTGCAGTATCTCGGAAG, R- AACATCTCAATCACCGTCCTC. Exon 6 F- GACTCGGATGAGGACGACTC, R- GGAAGTCCTGCTGTCCTCTG. All iPS cells used in this study were obtained from reprogrammed fibroblasts. For most of the lines reprogramming to iPS cells was induced by retroviral vectors expressing four transcription factors: Oct4, Sox2, Klf4, and cMyc. For control line (C 2 ), which was obtained from Mandegar et al.60, episomal reprogramming was performed, whereas the C CRISPR line was generated by non-integrating Sendai virus. Generated clones (at least two per patient line) were selected and tested for pluripotency and genomic integrity based on SNP arrays. IPS cells were always cultured on Matrigel (Corning, #356237) in E8 flex (Thermo Fisher Scientific) supplemented with primocin (0.1 µg/ml, Invivogen) and low puromycin (0.5 µg/ml) and G418 concentrations (50 µg/ml) at 37 °C/5% CO 2 . Medium was refreshed every 2–3 days and cells were passaged twice per week using an enzyme-free reagent (ReLeSR, Stem Cell Technologies). Collecting patient material and establishing hiPSCs have all been performed according to locally (Radboudumc) IRB protocols.

CRISPR/Cas9 editing of EHMT1

We made use of the CRISPR/Cas9 technology in order to create a heterozygous EHMT1 mutation in Exon 2 in a iPS cell line derived from a healthy 51-year-old male to mimic KS in isogenic cell lines, generated by KULSTEM (Leuven, Belgium). In brief, 1 × 106 iPSCs in single-cell suspension were nucleofected with Cas9 ribonucleoprotein complexes (10 µg/reaction S.p. Cas9 Nuclease 3NLS (IDT 1074181), CRIPSPR crRNAs (IDT) and CRISPR tracrRNA (IDT 1072532) 0.4 nmoles each/reaction) and a donor vector (5 µg/ reaction). The two crRNAs were designed to specifically target EHMT1 (TCTAACAGGCAGTTCCGGCGAGG and TAACAGGCAGTTCCGGCGAGGGG). The donor vector was a piggyback construct containing a hygromycin selection cassette as well as sequences that enable homology-directed repair ensuring the insertion of premature stop codons in Exon 2 of the EHMT1 gene. Cells were nucleofected using the Human Stem Cell Nucleofector® Kit 2 (Lonza, VPH-5022) in combination with the AMAXA-2b nucleofector, program F16. After nucleofection cells were resuspended in E8 flex (Thermo Fisher Scientific) supplemented with Revitacell (Thermo Fisher Scientific). When the iPS cells reached a confluency of about 40% selection was started using 5 µg/ml hygromycin. The antibiotics concentration was increased to up to 200 µg/ml over 2 weeks. Hygromycin resistant colonies were picked and PCR validation was used to ensure heterozygous editing of Exon2. To prove reduced EHMT1 expression in the CRISPR/Cas9 edited clone RT-qPCR and Western Blot were performed to measure EHMT1 protein levels in C Crispr and KS Crispr . DNA fingerprinting profile was performed on both lines by qPCR detection of a reference set of SNP panel using the TaqMan assays from Life Technologies. The genome-edited iPS cell line shows identical SNP profile with the corresponding iPS cell line used for gene targeting. Off-target of genome editing was verified by sequencing the top four off-target sites of each gRNA have been sequenced and no INDELs were detected (Supplementary Fig. 5). The generated iPS cells were validated for pluripotency markers and quantitative analysis of tri-lineage differentiation potential was performed. All generated iPS cell lines have the capacity to differentiate toward all three germ layers (endoderm, mesoderm, ectoderm). To this end embryoid bodies were generated in 24-well Corning low attachment plates. For spontaneous differentiation, the culture was kept for 7 days in E6 medium (Thermo Fisher Scientific). The medium was changed every 2 days. Cells were harvested after 7 days for RNA extraction with the GenElute Mammalian Total RNA kit (Sigma). cDNA synthesis was performed with Superscript III and used for qPCR according to manufacturer’s protocol with TaqMan human iPS cell Scorecard assay (Life Technologies). Data analysis was performed with Scorecard software (online tool Life technologies), comparing with a reference set of pluripotent stem cell lines.

Western Blot

For Western Blot cell lysates were made from confluent wells in six-well plates of either iPS cells or iNeurons. Medium was always refreshed 4 h beforehand. Protein samples were loaded, separated by sodium dodecyl supfate (SDS) polyacrylamide gel electrophoresis, and transferred to nitrocellulose membrane (BIO-RAD). The membrane was then probed with an EHMT1 antibody (1:1000; Abcam ab41969) or NMDAR1 (1:100; Biolegend 818601). To control for loading differences, we probed with anti-gamma tubulin (1:1000; Sigma T5326) or GAPDH (1:1000; Cell Signaling #2118). For visualization horseradish peroxidase-conjugated secondary antibodies were used (1:20000 for both; goat anti-mouse: Jackson ImmunoResearch Laboratries, 115-035-062. Goat-anti rabbit: Invitrogen, A21245).

Neuronal differentiation

iPS cells were directly derived into, excitatory cortical Layer 2/3 neurons by overexpressing the neuronal determinant Neurogenin 2 (NGN2) upon doxycycline treatment based on Zhang et al.24 and as we described previously25. To support neuronal maturation, freshly prepared rat astrocytes25 were added to the culture in a 1:1 ratio two days after plating. At DIV 3 the medium was changed to Neurobasal medium (Thermo Fisher Scientific) supplemented with B-27 (Thermo Fisher Scientific), glutaMAX (Thermo Fisher Scientific), primocin (0.1 µg/ml), NT3 (10 ng/ml), BDNF (10 ng/ml), and doxycycline (4 µg/ml). Cytosine b-d-arabinofuranoside (Ara-C) (2 µM; Sigma) was added once to remove any proliferating cell from the culture. From DIV 6 onwards half of the medium was refreshed three times a week. The medium was additionally supplemented with 2.5% FBS (Sigma) to support astrocyte viability from DIV 10 onwards. Neuronal cultures were kept through the whole differentiation process at 37 °C/5%CO 2 .

Cortical cultures from mice

Primary cortical neurons were prepared from Ehmt1+/− and Ehmt1+/+ mice from individual E16.5 embryos as previously described16. Since the genotype was unknown at the time of harvest, each embryo was collected and the brains were processed separately. Each whole brain was kept on ice in 1 mL L-15 medium, organized separately in a 24-well plate, and tail clips were collected for genotyping.

Neuronal morphology reconstruction

To examine morphology of neurons cells on coverslips were transfected with plasmids expressing Discosoma species red (dsRED) fluorescent protein one week after plating. DNAin (MTI-GlobalStem) was used according to manual instructions. Medium was refreshed completely the day after DNAin application. After the treatment cells were cultured as previously described.

After 3 weeks of differentiation cells were fixed in 4% paraformaldehyde/4% sucrose (v/v) in phosphate-buffered saline (PBS) and mounted with DAKO. Transfected neurons were imaged using a Zeiss Axio Imager Z1 and digitally reconstructed using Neurolucida 360 software (MBF–Bioscience, Williston, ND, USA). For large cells multiple 2-dimensional images of these neurons were taken and subsequently stitched together using the stitching plugin of FIJI 2017 software. The three-dimensional reconstructions and quantitative morphometrical analyses focused on the somatodenritic organization of the neurons. We defined origins for individual primary dendrites by identifying emerging neurites with diameters that were less than 50% of the diameter of the soma. Axons, which were excluded from reconstructions and further analysis, we visually identified by their long, thin properties, far reaching projections and numerous directional changes Neurons that had at least two primary dendrites and reached at least the second branching order were considered for analysis. For morphometrical analysis we determined soma size, number of primary dendrites, length and branching points per primary dendrite and total dendritic length. To measure the total span of the dendritic field (receptive area) of a neuron we performed convex hull 3D analysis. Note, that due to the two-dimensional nature of the imaging data, we collapsed the convex hull 3D data to two-dimensions, resulting in a measurement of the receptive area and not the volume of the span of the dendritic field. Furthermore, Sholl analysis was performed to investigate dendritic complexity in dependence form distance to soma. For each distance interval (10 µm each) the number of intersections (the number of dendrites that cross each concentric circle), number of nodes and total dendritic length was measured. Discriminant function analysis with canonical discriminant functions and reclassification of group membership based on parameters describing neuronal morphology were performed in SPSS.

Immunocytochemistry

Cells were fixed with 4% paraformaldehyde/4% sucrose (v/v) for 15 min and permeabilized with 0.2% triton in PBS for 10 min at RT. Nonspecific binding sites were blocked by incubation in blocking buffer (PBS, 5% normal goat serum, 1% bovine serum albumin, 0.2% Triton) for 1 h at RT. Primary antibodies were diluted in blocking buffer incubated overnight at 4 °C. Secondary antibodies, conjugated to Alexa-fluorochromes, were also diluted in blocking buffer and applied for 1 h at RT. Hoechst 33342 (Molecular Probes) was used to stain the nucleus before cells were mounted using DAKO fluorescent mounting medium (DAKO). Neurons were fixed at DIV 21 for synaptic stainings. Used primary antibodies were: mouse anti-MAP2 (1:1000; Sigma M4403); guinea pig anti-MAP2 (1:1000; Synaptic Systems 188004); guinea pig anti-synapsin 1/2 (1:1000; Synaptic Systems 106004); mouse anti-PSD95 (1:50; Thermo Fisher Scientific MA1-045); rabbit anti-GFAP (1:500; Abcam ab7260), mouse anti-pan axon (1:1000; Covance SMI-312R), mouse anti-NMDAR1 (1:1000; ThermoFisher Scientific 54.1). Secondary antibodies that were used are: goat anti-guinea pig Alexa Fluor 647 (1:2000, Invitrogen A-21450); goat anti-rabbit Alexa Fluor 488 (1:2000, Invitrogen A-11034); goat anti-rabbit Alexa Fluor 568 (1:2000, Invitrogen A-11036); goat anti-mouse Alexa Fluor 488 (1:2000, Invitrogen A-11029); goat anti-mouse Alexa Fluor 568 (1:2000, Invitrogen A-11031). We imaged at a 63× magnification using the Zeiss Axio Imager Z1 equipped with apotome. All conditions within a batch were acquired with the same settings in order to compare signal intensities between different experimental conditions. Signals were quantified using FIJI software. The number of synaptic puncta was determined per individual cell via manual counting and divided by the dendritic length of the neuron.

To investigate H3K9me2 iNeurons were fixed at DIV3. Used primary antibodies are: rabbit anti-H3K9me2 antibody (1:500, Millipore 07-441), and guinea pig anti-MAP2 antibody (1:1000, Synaptic Systems 188004). Secondary antibodies used are: goat anti-rabbit Alexa Fluor 488 (1:2000, Invitrogen A-11034) and goat anti-guinea pig Alexa Fluor 647 (1:2000, Invitrogen A-21450). Epifluorescent images were taken with the same exposure time using Zeiss Axio Imager Z1 with apotome. H3K9me2 was assessed based on color intensity measurements by ImageJ analysis tools.

Quantification of mRNA by RT-qPCR

RNA samples were isolated from both, iPS cells or iNeurons (DIV28) using Nucleo Spin RNA isolation kit (Machery Nagel, 740955.250) according to the manufacturer’s instructions. RNA samples (200 ng) were converted into cDNA by iScript cDNA synthesis kit (BIO-RAD, 1708891). cDNA products were cleaned up using the Nucleospin Gel and PCR clean-up kit (Machery Nagel, 740609.250). Human-specific primers were designed with Primer3plus and IDT PrimerQuest tools, respectively. Primer sequences are given in supplementary Table 1. qPCRs were performed in the 7500 Fast Real Time PCR System apparatus (Applied Biosystems) with GoTaq qPCR master mix 2× with SYBR Green (Promega, A600A) according to the manufacturer’s protocol. The PCR program was designed as following: After an initial denaturation step at 95 °C for 10 min, PCR amplifications proceeded for 40 cycles of 95 °C for 15 s and 60 °C for 30 s and followed by a melting curve. All samples were analyzed in duplicate in the same run, placed in adjacent wells. Reverse transcriptase-negative controls and no template-controls were included in all runs. The arithmetic mean of the Ct values of the technical replicas was used for calculations. Relative mRNA expression levels were calculated using the 2−ΔΔCt method61 with standardization to housekeeping genes.

Chromatin immunoprecipitation (ChIP)-qPCR

ChIP was performed according to the Blueprint-IHEC (international human epigenome consortium) protocol with slight modifications. Briefly, 5,00,000 cells were used for one immunoprecipitation reaction. Cells were first crosslinked with disuccinimidyl glutarate at a final concentration of 2 mM for 30 min at RT followed by second crosslinking treatment with 1% formaldehyde for 8 min at RT. Crosslinking was stopped by adding glycine to a final concentration of 0.125 M for 10 min. Samples were washed and homogenized in cold lysis buffer (10 mM Tris (pH 8.0), 0.2%NP40, 0.2% TritonX-100, 1 mM EDTA, 0.5 mM EGTA, 5× protease inhibitors EDTA-free, and 10 mM NaCl) for 15 min on ice. Nuclei were lysed with nuclei lysis buffer (1 mM EDTA, 0.5 mM EGTA, 50 mM HEPES) for 20 min rotating at 4 °C. After centrifugation the collected extracts were resuspended in sonication buffer (20 mM HEPES pH = 7.6, 1% SDS, 2.5× protease inhibitors EDTA-free) and sonicated with Bioruptor Power-up (Diagenode) to shear DNA into 150–700 bp fragments. Cleaned-up chromatin was diluted with ChIP dilution buffer (0.15% SDS, 1% TritonX-100, 1.2 mM EDTA, 16.7 mM Tris pH = 8, 167 mM NaCl). Immunoprecipitation was performed with anti-H3K9me2 (5 µg/reaction, Abcam ab1220) and anti-EHMT1 (15 µg/reaction, Abcam ab41969) antibodies overnight rotating at 4 °C. As a negative control, anti-human IgG (10 µg/reaction, Abcam ab2410) was used in place of specific antibodies. Chromatin was enriched by 10 μL of each Protein A (Invitrogen, 10001D) and Protein G Dynabeads (Invitrogen, 10003D) for 2 h rotating at 4 °C. Beads were washed once with wash buffer 1 (2 mM EDTA, 20 mM Tris pH = 8, 1% TritonX-100, 0.1% SDS, 150 mM NaCl), twice with wash buffer 2 (2 mM EDTA, 20 mM Tris pH = 8, 1% TritonX-100, 0.1% SDS, 500 mM NaCl), twice with wash buffer 3 (1 mM EDTA, 10 mM Tris pH = 8) and eluted in elution buffer (1% SDS; 100 mM NaHCO3) by rotating for 20 min at RT. Eluate was de-crosslinked in elution buffer with 40 µg proteinase-K and 200 mM NaCl overnight at 65 °C. DNA material was cleaned with QIAquick MinElute PCR Purification Kit (Qiagen, 28006) according to manufacturer’s instructions. Five microlitreof eluted DNA sample was used for qPCR analysis. qPCRs were performed as described earlier. For primer sequences see Supplementary Table 2. All samples were analyzed in duplicate in the same run and placed in adjacent wells. ChIP-qPCR results are expressed as % input, which was calculated by the following formula: 100 × 2−ΔCt, where ΔCt = Ct[ChIP] − (Ct[input] − Log 2 (input dilution factor)).

MEA recordings and data analysis

All recordings were performed using the 24-well MEA system (Multichannel Systems, MCS GmbH, Reutlingen, Germany). MEA devices are composed by 24 independent wells with embedded microelectrodes (i.e., 12 electrodes/well, 80 µm in diameter and spaced 300 µm apart). Spontaneous electrophysiological activity of iPS cell-derived neuronal network grown on MEAs was recorded for 20 min. During the recording, the temperature was maintained constant at 37 °C, and the evaporation and pH changes of the medium was prevented by inflating a constant, slow flow of humidified gas (5% CO 2 and 95% O 2 ) onto the MEA plate (with lid on). The signal was sampled at 10 kHz, filtered with a high-pass filter (i.e., Butterworth, 100 Hz cutoff frequency) and the noise threshold was set at ±4.5 standard deviations.

Data analysis was performed off-line by using Multiwell Analyzer (i.e., software from the 24-well MEA system that allows the extraction of the spike trains) and a custom software package named SPYCODE developed in MATLAB (The Mathworks, Natick, MA, USA) that allows the extraction of parameters describing the network activity62.

The mean firing rate (MFR) of the network was obtained by computing the firing rate of each channel averaged among all the active electrodes of the MEA. Burst detection: bursts were detected using a Burst Detection algorithm. The algorithm is based on the computation of the logarithmic inter-spike interval histogram in which inter-burst activity (i.e., between bursts and/or outside bursts) and intra-burst activity (i.e., within burst) for each recording channel can be easily identified, and then a threshold for detecting spikes belonging to the same burst is automatically defined. From the burst detection, the number of bursting channels (above threshold 0.4 burst/s and at least 5 spikes in burst with a minimal inter-spike-interval of 100 ms) was determined. Network burst detection: synchronous events were detected by looking for sequences of closely spaced single-channels bursts. A network burst was defined as burst that occurs in >80% of the network active channels. The distributions of NBDʼs and network inter-burst interval (NIBI, interval between two consecutive network bursts) were computed using bins of 100 ms and 1 s respectively. Network burst irregularity: irregularity was estimated by computing the CV of the NIBI, which is the standard deviation divided by the mean of the NIBI. Discriminant function analysis with canonical discriminant functions and reclassification of group membership based on parameters describing neuronal network activity were performed in SPSS.

Chemicals

All reagents were prepared fresh into concentrated stocks as indicated below, and stored frozen at −20 °C. The following compounds were used in pharmacological experiments: 1-Naphthyl acetyl spermine trihydrochloride (“Naspm”, 100 mM in PBS, Tocris Cat No 2766); (+)-MK 801 maleate (“MK-801”, 100 mM in DMSO, Tocris Cat No 0924); 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (“NBQX”, 100 mM in DMSO, Tocris Cat No 0373); D-2-amino-5-phosphonovalerate (“D-AP5”, 50 mM in PBS, Tocris Cat No 0106); Retigabine (100 mM in DMSO, Tocris Cat No. 6233). For all experiments on MEAs, and immediately before adding a compound to the cells, an aliquot of the concentrated stock was first diluted 1:10 in room temperature DPBS and vortexed briefly. Then, the appropriate amount of working dilution was added directly to wells on the MEA and mixing was primarily through diffusion into the (500 µl) cell culture medium. Of note, where relevant the DMSO concentration in the medium was always ≤0.05% v/v.

Pharmacological experiments

Control and KS patient neuronal networks were treated acutely with D-AP5 (60 µM), NBQX (50 µM), MK-801 (1 µM), Naspm (10 µM), or Retigabine (10 µM) at DIV 28 after a 20-min recording of spontaneous activity. Then, the recording was paused, the compounds were added to the MEA, and the recording was restarted after 5 min. We recorded neuronal network activity for 60 min with D-AP5 or NBQX, 90 min with MK-801, 100 min with Retigabine, and for 20 min after the addition of Naspm. In experiments where we examined the effect of chronic NMDAR blockade, control and KS patient neuronal networks were treated with 1 µM MK-801 starting at DIV 28 and lasting 7 days total. MK-801 was replenished every two days, where it was freshly diluted to 1 µM in complete Neurobasal medium for the routine half-medium change. All experiments were performed at 37 °C.

Single-cell electrophysiology

For single-cell recordings we used neurons derived from C 1 , C 2 , C MOS , C CRISPR , and KS 1 , KS 2 , KS MOS , and KS CRIPSR after 3 weeks of differentiation. Experiments were performed in a recording chamber on the stage of an Olympus BX51WI upright microscope (Olympus Life Science, PA, USA) equipped with infrared differential interference contrast optics, an Olympus LUMPlanFL N 40× water-immersion objective (Olympus Life Science, PA, USA) and kappa MXC 200 camera system (Kappa optronics GmbH, Gleichen, Germany) for visualization. Recordings were made using a SEC-05× amplifier (NPI Electronic GmbH, Tamm, Germany), low-pass filtered at 3 kHz and digitized at 10 kHz using a 1401 acquisition board and recorded with Signal (CED, Cambridge, UK). Recordings were not corrected for liquid junction potential (±10 mV).

We performed the recordings of neurons cultured on cover slips under continuous perfusion with oxygenated (95% O 2 /5% CO 2 ) artificial cerebrospinal fluid (ACSF) at 30 °C containing (in mM) 124 NaCl, 1.25 NaH 2 PO 4 , 3 KCl, 26 NaHCO 3 , 11 Glucose, 2 CaCl 2 , 1 MgCl 2 (adjusted to pH 7.4). Patch pipettes (6–8 MΩ) were pulled from borosilicate glass with filament and fire-polished ends (Science Products GmbH, Hofheim, Germany) using the PMP-102 micropipette puller (MicroData Instrument, NJ, USA). For current clamp recordings of the intrinsic electrophysiological properties, we filled pipettes with a potassium-based solution containing 130 mM K-Gluconate, 5 mM KCl, 10 mM HEPES, 2.5 mM MgCl 2 , 4 mM Na2-ATP, 0.4 mM Na3-ATP, 10 mM Na-phosphocreatine, 0.6 mM EGTA (adjusted to pH 7.25 and osmolarity 290 mosmol). We determined the resting membrane potential (V rmp ) after achieving whole-cell configuration and only considered neurons with V rmp of −55 mV and lower for further testing and analyses. Recordings were not corrected for liquid junction potentials of ca. −10 mV. All further electrophysiological characterizations we performed at a holding potential of −60 mV. We determined the passive membrane properties via a 0.5 s hyperpolarizing current of −25 pA. Active intrinsic properties, i.e., action potential (AP) firing characteristics we derived from the first AP elicited by a 0.5 s depolarizing current injection just sufficient to reach AP threshold. For voltage clamp recordings we filled pipettes with a cesium-based solution containing 115 mM cesium methanesulfonate, 20 mM CsCl, 10 mM HEPES, 2.5 mM MgCl 2 , 4 mM Na2ATP, 0.4 mM Na3GTP, 10 mM sodium phosphocreatine, and 0.6 mM EGTA adjusted to a pH of 7.4. Spontaneous AP-sEPSC were recorded in drug-free ACSF. Bursts were detected when at least 5 events with a minimal inter-event-interval of 100 ms were exhibited by a cell. Burst detection, burst frequency and duration were computed with software developed in MATLAB (The Mathworks, Natick, MA, USA). mEPSC were measured in voltage clamp at a holding potential of −60 mV in the presence of 1 μM TTX (Tocris, Bristol, UK) and 100 μM picrotoxin (Tocris, Bristol, UK). Evoked responses (AMPAR and NMDAR) were measured by infecting iNeurons at DIV 7 with 0.5 µl AAV-mCherry-Chr2 (UNC Vector Core, USA). Uninfected cell were recorded in voltage clamp at a holding potential of −70 mV (AMPAR) or +40 mV (NMDAR). Light-evoked synaptic responses were induced by exciting nearby infected cells (mCherry positive) with blue light (COOLLED pE-200; 10 ms, 470 nm). Stimulus strength, location of stimulus were adjusted to 30–50 pA postsynaptic AMPAR-mediated responses at −70 mV holding voltage, and 30 sweeps were averaged for the final trace. AMPA amplitudes were quantified as the peak amplitude at −70 mV holding voltage, and the NMDA amplitudes were quantified as the average of 5 ms of the trace 65 ms after the stimulus to prevent contamination of AMPAR-mediated response at +40 mV. NMDA/AMPA ratios were calculated by averaging responses in Clampfit (vs. 10.7, Molecular Devices, CA, USA). Intrinsic electrophysiological properties were analyzed using Signal and MatLab (MathWorks, MA, USA), while mEPSCs were analyzed using MiniAnalysis 6.0.2 (Synaptosoft Inc, Decatur, GA, USA) as described earlier16. Discriminant function analysis with canonical discriminant functions and reclassification of group membership based on neuronal intrinsic properties were performed in SPSS.

Acute slice electrophysiology

We used litter-matched Ehmt1+/− and Ehmt1+/+ mice of adolescent age (postnatal day 21–24). Acute slices were prepared as described earlier16. In brief, animals were deeply anesthetized with isoflurane, then quickly decapitated. 350-µm-thick coronal slices were cut using a microtome (HM650V, Thermo Scientific) in ice-cold “cutting and storage” ACSF containing 87 mM NaCl, 11 mM glucose, 75 mM sucrose, 2.5 mM KCl, 1.25 mM NaH 2 PO 4 , 0.5 mM CaCl 2 , 7 mM MgCl 2 , and 26 mM NaHCO 3 , continuously oxygenated with 95% O 2 /5% CO 2 . Slices were incubated for 1 h at 32 °C, after which they were allowed to cool down to room temperature. The slices were then transferred to a recording setup as described above, and incubated in recording ACSF (124 mM NaCl, 10 mM glucose, 3 mM KCl, 1.25 mM NaH 2 PO 4 , 2 mM CaCl 2 , 1 mM MgCl 2 , and 26 mM NaHCO 3 ) at 30 °C with added 100 mM Picrotoxin to block GABAergic transmission. A bipolar electrode (CE2C55, FHC) coupled to an SD9 stimulator (Grass Instruments, RI, USA) was inserted into layer 4 of the auditory cortex, and pyramidal cells were patched in layer 2/3 above the bipolar electrode (<200 µm lateral distance) using 3–6 MΩ borosilicate pipettes filled with a Cesium-based intracellular solution containing 115 mM CsMeSO 3 , 20 mM CsCl 2 , 10 mM HEPES, 2.5 mM MgCl 2 , 4 mM Na2ATP, 0.4 mM NaGTP, 10 mM Na-Phosphocreatine, 0.6 mM EGTA, and 5 mM QX-314. The cells were held in voltage-Clamp mode controlled by an SEC 05-LX amplifier (NPI, Tamm, Germany), low-pass filtered at 3 kHz and sampled at 20 kHz with a Power-1401 acquisition board and Signal software (CED, Cambridge, UK). Data were analyzed in Clampfit 10.7 (Molecular Devices). The AMPA trace was measured at −70 mV holding potential; stimulus strength was adjusted to 50–100 pA postsynaptic response, and 60 sweeps were averaged for the final trace. NMDA was measured at +40 mV holding potential. Cells were discarded if the measured AMPA response contained multiple peaks, indicating multisynaptic input, or if the averaged amplitude was below 25 pA. AMPA amplitudes were quantified as the peak amplitude at −70 mV holding voltage, and the NMDA amplitudes were quantified as the average of 5 ms of the trace 65 ms after the stimulus artifact.

Animals

For the animal experiments presented in this study, mice heterozygous for a targeted loss-of-function mutation in the Ehmt1 gene (Ehmt1+/− mice) and their WT littermates on C57BL/6 background were used, as previously described11. Animal experiments were conducted in conformity with the Animal Care Committee of the Radboud University Nijmegen Medical Centre, The Netherlands, and conform to the guidelines of the Dutch Council for Animal Care and the European Communities Council Directive 2010/63/EU.

Statistics

The statistical analysis for all experiments was performed using GraphPad Prism 5 (GraphPad Software, Inc., CA, USA). We ensured normal distribution using a Kolmogorov–Smirnov normality test. To determine statistical significance for the different experimental conditions p values < 0.05 were considered to be significant. Statistical analysis between lines were performed with two-ways ANOVA and Post hoc Bonferroni correction. We analyzed significance between Control and Kleefstra groups by means of the Mann–Whitney U test. Data are presented as mean ± standard error of the mean (SE). Details about statistics are reported in Supplementary Data 1 (Excel file Statistics).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.