EMBO/soda-nel huggingface dataset:
RNF34 functions in immunity and selective mitophagy by targeting MAVS for autophagic degradation (2019)
Figure 5

Figure 5 RNF34 promotes the autophagic degradation of MAVS aggregates

(A) Immunoblot showing the degradation of MAVS in HEK293T cells subjected to VSV infection (MOI = 1.0) for the indicated times. α-Tubulin was used as a loading control.

(B) Immunoblot showing the degradation of MAVS in HEK293T cells infected with VSV (MOI = 1.0) for the indicated times in the presence or absence of NH4Cl (3 mM). α-Tubulin was used as a loading control.

(C) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing Flag-MAVS or Flag-mMAVS together with Myc-RNF34. α-Tubulin was used as a loading control.

(D) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing Flag-MAVS or its 4KR mutant together with increasing amounts of Myc-RNF34. α-Tubulin was used as a loading control.

(E) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing Flag-MAVS with increasing amounts of HA-RNF34 or its H342A mutant. α-Tubulin was used as a loading control.

(F) SDD-AGE analysis of MAVS aggregates in HEK293T cells expressing Flag-MAVS together with WT Myc-RNF34 or its ligase-dead mutant and cultured in the presence or absence of MG132 (10 μM), 3-MA (1 mM) or NH4Cl (3 mM). SDS-PAGE immunoblotting was used as a loading control.

(G) SDD-AGE analysis of MAVS aggregates in siCtrl- or siRNF34-1-transfected cells using an anti-MAVS antibody. SDS-PAGE immunoblotting was used as a loading control.

(H) Immunoblot showing the levels of the MAVS protein in siCtrl- or siRNF34-1-transfected PBMCs. α-Tubulin was used as a loading control.

(I) SDD-AGE analysis of MAVS aggregates in THP-1 cells expressing Flag-MAVS or the K311R mutant together with Myc-RNF34 or Myc-TRIM31. SDS-PAGE immunoblotting was used as a loading control.

Cell-based studies were performed independently at least three times with comparable results.
RNF34 functions in immunity and selective mitophagy by targeting MAVS for autophagic degradation (2019)
Figure 6

Figure 6 RNF34-mediated autophagic degradation of MAVS aggregates is facilitated by NDP52

(A) Immunoprecipitation analysis of HEK293T cells transfected with Flag-SQSTM1 or its I431A mutant together with HA-MAVS. Anti-Flag or IgG agarose immunoprecipitates were analyzed using immunoblotting with an anti-HA or anti-Flag antibody.

(B) Luciferase activity driven by the IFN-β promoter in HEK293T cells transfected with HA-MAVS or its 4KR mutant together with Flag-SQSTM1 or its I431A mutant. Luciferase assays were performed 24 hrs after transfection.

(C) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing HA-MAVS or its 4KR mutant together with Flag-SQSTM1. α-Tubulin was used as a loading control.

(D) Luciferase activity driven by the IFN-β promoter in HEK293T cells transfected with HA-MAVS or its 4KR mutant together with Flag-NDP52 or its D439R/C443K mutant. Luciferase assays were performed 24 hrs after transfection.

(E) Immunoprecipitation analysis of HEK293T cells transfected with Flag-MAVS or its 4KR mutant together with Myc-NDP52. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody.

(F) Immunoprecipitation analysis of the DNP52-RNF34 interaction in cells infected with VSV. Anti-DNP52 immunoprecipitates were analyzed using immunoblotting with an anti-DNP52 or anti-RNF34 antibody. The WCL was immunoblotted with an anti-MAVS or anti- RNF34 antibody.

(G) Immunoprecipitation analysis of the interaction between Flag-NDP52 and HA-RNF34 in WT or MAVS-/- cells. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-HA antibody.

(H) Immunoprecipitation analysis of HEK293T cells transfected with Flag-NDP52 or its D439R/C443K mutant together with HA-MAVS followed by VSV infection. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-HA or anti-Flag antibody.

(I) Immunoblot showing the levels of the MAVS protein in siCtrl- or siNDP52-transfected cells expressing Flag-RNF34 or Flag-V. α-Tubulin was used as a loading control.

(J) Immunoblot showing the levels of the MAVS protein in siCtrl- or siNDP52-transfected cells infected with VSV (MOI = 1, 12 hrs). α-Tubulin was used as a loading control.

(K) Immunoprecipitation analysis of the MAVS-LC3 interaction in siCtrl- or siNDP52-transfected cells infected with VSV. Anti-MAVS immunoprecipitates were analyzed using immunoblotting with an anti-MAVS or anti-LC3 antibody. NDP52 levels were analyzed by performing immunoblotting with an anti-NDP52 antibody. α-Tubulin was used as a loading control.

Cell-based studies were performed independently at least three times with comparable results. The luciferase reporter and ELISA data are presented as means ± SEM. Two-tailed Student's t-tests were used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.
RNF34 functions in immunity and selective mitophagy by targeting MAVS for autophagic degradation (2019)
Figure 7

Figure 7 RNF34 is required for the clearance of damaged mitochondria upon viral infection

(A) Immunoblot showing MAVS, IRF3-p, LC3, TOM20 and HSP60 levels in siCtrl- or siRNF34-1 cells infected with VSV and cultured in the presence of Z-VAD (50 μM) for the indicated times. α-Tubulin was used as a loading control.

(B) Immunoblot showing MAVS, LC3, TOM20 and HSP60 levels in cells transfected with Flag-RNF34 or its H342A mutant and subsequently infected with VSV for the indicated times.

(C-D) Representative confocal images (C) and quantification (D) of immunofluorescence staining for NDP52 colocalization with MAVS in shCtrl- or shRNF34-1 cells infected with VSV for the indicated times. Red represents MAVS and green represents NDP52. Scale bar, 10 μm.

(E) Transmission electron microscopy images of shCtrl- and shRNF34-1 HEK293T cells cultured in the presence or absence of VSV. Arrows indicate mitochondria engulfed in autophagosomes. Double arrows indicate swollen mitochondria with disrupted cristae. M, mitochondria and N, nucleus. 40000× magnification.

(F) Representative confocal images of the shift in the fluorescence emission of Keima from 458 nm to 543 nm. Scale bar, 10 μm.

Cell-based studies were performed independently at least three times with comparable results.

Expanded View Figure Legends
Defects in mitophagy promote redox-driven metabolic syndrome in the absence of TP53INP1. (2015)
Figure 1

TP53INP1-deficient mice are highly susceptible to HFD-induced obesity owing to their chronic oxidative stress

TP53INP1-KO (−/−) and WT (+/+) male mice were subjected to a high-fat diet (HFD, 60% fat) or a control diet (CTRL) for 16 weeks. Mice drank tap water or tap water supplemented with NAC (10 mg/ml or 1%).

Curves show mice body weight recorded every week. CTRL: P (−/− versus +/+; t = 8w) = 0.047; P (−/− versus +/+; t = 9w) = 0.023. HFD: P (−/− versus +/+; t = 7w) = 0.039; P (−/− versus +/+; t = 8w) = 0.029; P (−/− versus +/+; t = 9w) = 0.021; P (−/− versus +/+; t = 10w) = 0.014; P (−/− versus +/+; t = 11w) = 0.0046; P (−/− versus +/+; t = 12w) = 0.0028; P (−/− versus +/+; t = 13w) = 0.0025; P (−/− versus +/+; t = 14w) = 0.00051; P (−/− versus +/+; t = 15w) = 0.00027; P (−/− versus +/+; t = 16w) = 0.00013.

At the end of protocol, mice were sacrificed; liver and epididymal and renal fat masses were taken and weighed. Histograms show organ weight. Liver: P (−/− versus +/+; HFD) = 0.014; P (HFD versus CTRL; +/+) = 0.00063; P (HFD versus CTRL; −/−) = 0.0010; P (HFD versus CTRL; +/+ NAC) = 0.034; P (HFD versus CTRL; −/− NAC) = 0.027; P (NAC versus no NAC; −/− HFD) = 0.014. Epididymal fat mass: P (−/− versus +/+; HFD) = 0.011; P (HFD versus CTRL; +/+) = 0.028; P (HFD versus CTRL; −/−) = 0.000017; P (HFD versus CTRL; +/+ NAC) = 0.019; P (HFD versus CTRL; −/− NAC) = 0.0054; P (NAC versus no NAC; +/+ CTRL) = 0.037; P (NAC versus no NAC; −/− HFD) = 0.0025. Renal fat mass: P (−/− versus +/+; HFD) = 0.0041; P (HFD versus CTRL; +/+) = 0.028; P (HFD versus CTRL; −/−) = 0.000013; P (HFD versus CTRL; +/+ NAC) = 0.019; P (HFD versus CTRL; −/− NAC) = 0.0078; P (NAC versus no NAC; −/− HFD) = 0.0047.

Data information: Results are expressed as the mean ± SEM and are representative of two independent experiments. * −/− versus +/+; £ HFD versus CTRL; § NAC versus no NAC; 1 character: P < 0.05; 2 characters: P < 0.005; 4 characters: P < 0.00005.
Defects in mitophagy promote redox-driven metabolic syndrome in the absence of TP53INP1. (2015)
Figure 3

The gene encoding TP53INP1 is expressed in pancreatic endocrine cells

A, B (A, B) Immunocytofluorescent staining of TP53INP1 (red) and insulin (green) in mouse pancreatic sections (A) and single human islet beta cell (B). Scale bars represent 50 μm (A) and 10 μm (B).

C–E Quantitative PCR for Tp53inp1 mRNA levels in tissues and cells from rat (C) and C57BL/6J mice fed with a normal diet (5% fat; ND) or an high-fat diet (45% fat; HFD) (D, E). Results are expressed as the mean ± SEM and are representative of two independent experiments. n = 2 for rat liver, islets and mouse spleen; n = 4 for INS-1E cells; n = 5 for mouse exocrine pancreas; n = 6 for Min6 cells and ND and HFD islets; n = 11 for mouse islets. *P = 0.035 for HFD versus ND.
Defects in mitophagy promote redox-driven metabolic syndrome in the absence of TP53INP1. (2015)
Figure 4

Absence of TP53INP1 increases mitochondrial (mt) ROS level and mass due to increased mitochondria number

Immortalized MEFs (MEFi) deficient (−/−) or not (+/+) for TP53INP1 were left untreated (NT) or treated with 3-methyladenine (3-MA, 5 mM) during 4 h.

Histograms show mt ROS level measured by flow cytometry upon MitoSox staining. P (−/− versus +/+; NT) = 0.019; P (−/− versus +/+; 3-MA) = 0.025; P (3-MA versus NT; +/+) = 0.031.

Histograms show mt mass evaluated by flow cytometry in KO or WT MEFi using MitoTracker staining. P (−/− versus +/+; NT) = 0.012; P (3-MA versus NT; +/+) = 0.036.

Histogram shows MitoSox fluorescence normalized with MitoTracker fluorescence. P (−/− versus +/+; NT) = 0.041; P (−/− versus +/+; 3-MA) = 0.030; P (3-MA versus NT; +/+) = 0.045.

After 4 h recovering in normal media, H2O2 (1 h, 100 μM) or non-treated (NT) MEFi deficient (−/−) or not (+/+) for TP53INP1 were observed by transmission electron microscopy (TEM). N = nucleus; white arrow = mitophagic vacuoles. Scale bar represents 0.5 μm.

Mean size of mitochondrion (area), number of mitochondria and mitophagic vacuoles normalized by cytoplasmic surface area were quantified. Size: P (−/− versus +/+; H2O2) = 0.000027; P (H2O2 versus NT; +/+) = 0.0070; P (H2O2 versus NT; −/−) = 0.013. Nb mito.: P (−/− versus +/+; NT) = 0.035; P (−/− versus +/+; H2O2) = 0.016. Nb vacuoles.: P (−/− versus +/+; NT) = 0.047.

Data information: Results are expressed as the mean ± SEM and are representative of three independent experiments. In (A-C): *P < 0.05 for TP53INP1−/− versus TP53INP1 +/+; #P < 0.05 for 3-MA versus NT. In (E): * TP53INP1−/− versus TP53INP1 +/+; # H2O2 versus NT. 1 character: P < 0.05; 2 characters: P < 0.01; 3 characters: P < 0.0005.
Defects in mitophagy promote redox-driven metabolic syndrome in the absence of TP53INP1. (2015)
Figure 5

TP53INP1 deficiency is linked with impaired PINK/PARKIN mitophagy

After 4 h recovering in normal media, TCLs from H2O2- (1 h, 100 μM), NAC- (24 h, 10 mM) or non-treated (NT) MEFi deficient (−/−) or not (+/+) for TP53INP1 were analyzed by immunoblotting for PGC-1α, PINK1, PARKIN, BNIP3, BNIP3L/NIX, VDAC1 and β-tubulin.

HEK293T cells were cotransfected with plasmids encoding TP53INP1α-NTAP or TP53INP1β-NTAP. TP53INP1α- or β-NTAP was precipitated with a streptavidin-containing resin (P), resolved by PAGE and Western blots developed with anti-TP53INP1 (TP53INP1 precipitation control), anti-PINK1, anti-PARKIN, anti-BNIP3 or anti-BNIP3L/NIX antibody. Western blot on TCL (on the right) served as a transfection control.

Three-month-old TP53INP1-deficient and WT male mice were sacrificed and their livers harvested. Mitochondrial lysates (Mito) were purified from total liver lysates (TCL), and both were analyzed by immunoblotting for TP53INP1, PINK1, PARKIN, VDAC1 and β-tubulin.

Data information: Results are representative of three independent experiments. Source data are available online for this figure.
Defects in mitophagy promote redox-driven metabolic syndrome in the absence of TP53INP1. (2015)
Figure 6

TP53INP1-deficient MEFi contain a dysfunctional mitochondrial pool

A–D High-resolution respirometry was performed on permeabilized MEFi TP53INP1 deficient (−/−, black bars) or not (+/+, white bars) using glucose (A), lipid-related substrates (C) or complex IV substrate (D). High-resolution respirometry was also performed on intact MEFi (B). For different respiratory state details (routine, 3, leak, and ETS capacity), please refer to the Materials and Methods section. Results are expressed as the mean ± SEM and are representative of three independent experiments. In (A): P (−/− versus +/+; State 3) = 0.0079; P (−/− versus +/+; ETS) = 0.0079; n = 5 in each group. In (B): P (−/− versus +/+; routine) = 0.0159; P (−/− versus +/+; leak) = 0.0159; P (−/− versus +/+; ETS) = 0.0159; n = 5 in each group. In (C): P (−/− versus +/+; State 3) = 0.029; P (−/− versus +/+; ETS) = 0.0079; n = 5 in each group. In (D): P (−/− versus +/+; ETS) = 0.0259; n = 5 in each group. *TP53INP1−/− versus TP53INP1 +/+; 1 character: P < 0.05; 2 characters: P < 0.01.

E TCLs from MEFi were analyzed by immunoblotting for CPT1A, CPT1B and β-tubulin. Results are representative of three independent experiments.

Source data are available online for this figure.
Defects in mitophagy promote redox-driven metabolic syndrome in the absence of TP53INP1. (2015)
Figure 7

Oxidative stress observed in absence of TP53INP1 is at the origin of presence of lipid droplets (LD) in MEFi

MEFi deficient (−/−) or not (+/+) for TP53INP1 were seeded in media containing or not 10 mM NAC. Forty-eight hours later, MEFi were treated or not (NT) with 100 μM hydrogen peroxide (H2O2) during 1 h in serum free media. Cells were left to recover for 4 h in normal media containing or not NAC (10 mM), GW-9662 (PPARγ inhibitor, 10 μM) or PNU-74654 (Wnt/β-catenin pathway inhibitor, 50 μM) before being harvested. Cells were observed by TEM.

Number of LD normalized by cytoplasmic surface area is shown in both histograms at the bottom of the figure. Scale bar represents 0.5 μm. Results are expressed as the mean ± SEM and are representative of three independent experiments. *P (−/− versus +/+; NT) = 0.00086; #P (H2O2 versus NT; +/+) = 0.013.

Cells were also observed by fluorescence microscopy after Bodipy493/503 staining of LD (green) and DAPI staining of nucleus (blue). L = lipid droplets. Scale bar represents 100 μm. Results are representative of three independent experiments.

TCLs from MEFi were analyzed by immunoblotting for β-catenin, PPARγ, ATGL, MGLL and β-tubulin. Results are representative of three independent experiments.

Source data are available online for this figure.
Deconstruction of O-glycosylation –  GalNAc-T Isoforms Direct Distinct Subsets of the O- Glycoproteome (2015)
Figure 1

Figure 1. The isogenic HepG2 liver cell model system and schematic depiction of the workflow for quantitative identification of isoform specific O-glycosylation.

(A) GalNAc-T expression levels in human liver (The body map data for human liver was kindly provided by the “Gene Expression Applications research group at Illumina”, http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-513/) and HepG2 wild type cells quantified by RNAseq (n=2, bars representing mean with SD). (B) VVA lectin immunoblot of secretomes from HepG2 SC, SCΔT1, SCΔT2 and SCΔT3. (C) Digests of HepG2SC were always labeled with “light” (L) isotope and each of the three HepG2SCΔT1, T2 and +T3 were labeled with “medium” (M) isotope. After labeling, samples were mixed and glycopeptides were enriched on a long VVA lectin column. Labeled glycopeptides were analysed using nLC-MS/MS, the m/z of glycopeptide precursor ions in the labelled samples was measured, relative abundances calculated and glycopeptide ions selected for fragmentation were sequenced thus giving rise to total glycopeptide identification and quantitated glycopeptide identification.
Deconstruction of O-glycosylation –  GalNAc-T Isoforms Direct Distinct Subsets of the O- Glycoproteome (2015)
Figure 3

Figure 3. Differential O-glycoproteomes in HepG2SC.

Histograms showing the distribution of M/L quantitation ratios of monoglycosylated peptides identified in (A) HepG2SCΔT1, (B) ΔT2 and (C) +T3. Glycopeptides with M/L ratio < -1 are colored red and glycopeptides with a M/L ratio > +1 are colored green. (D) Venn diagram showing the distribution of candidates for isoform-specific sites among HepG2SCΔT1, T2 and +T3 applying a log10 (+/-1) cut-off (excluding sites identified in both TCL and SEC for each isoform), and (E) TCL alone and (F) SEC alone.
FoxO3a Directs a Protective Autophagy Program in Hematopoietic Stem Cells (2013)
Figure 1

HSCs induce autophagy following metabolic stress

a, b, Fluorescent microscopy of cultured Gfp-Lc3 GMPs (a) and HSCs (b). c, Electron microscopy images of WT HSCs. Arrowheads indicate autophagic vesicles. d, GFP-LC3 loss in cultured Gfp-Lc3 HSCs (n = 3). Results are expressed as percentage of GFP-LC3 MFI in -BafA compared to +BafA conditions. e, GFP-LC3 levels in HSCs of fed and starved Gfp-Lc3 mice (n = 3). Results are expressed as percent of GFP-LC3 MFI in fed mice. Data are means ± s.d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
FoxO3a Directs a Protective Autophagy Program in Hematopoietic Stem Cells (2013)
Figure 2

Autophagy protects HSCs from starvation-induced apoptosis

a, Apoptosis levels in cultured WT HSCs (n = 3–5). Results are expressed as percent caspase activation compared to +cytokines conditions. b, Apoptosis levels in cultured control (Cnt) and Atg12cKO HSCs and GMPs (n = 3). Results are expressed as percent caspase activation compared to +cytokines Cnt HSCs (100%). c, Absolute number of HSCs in fed and starved Cnt and Atg12cKO mice (n = 4–8 mice per group). Results are expressed as percent of HSCs in fed mice. Data are means ± s.d. Hatching indicates starved conditions. *P ≤ 0.05, **P ≤ 0.01.
FoxO3a Directs a Protective Autophagy Program in Hematopoietic Stem Cells (2013)
Figure 3

FoxO3a poises HSCs for rapid autophagy induction

a, Status of the autophagy machinery in WT HSCs (n = 3). Results are expressed as log2 fold expression compared to WT GMPs (set to 0). b, c, qRT-PCR analyses of pro-autophagic genes in WT (b) and FoxO3a−/− (c) HSCs (n = 3). Results are expressed as log2 fold expression compared to WT GMPs (b) or FoxO3a+/+ HSCs (c) (set to 0). d, Autophagy flux in cultured FoxO3a−/−::Gfp-Lc3 HSCs (n = 3). Results are expressed as percent GFP-LC3 MFI in -BafA vs. +BafA conditions, and normalized to FoxO3a+/+−::Gfp-Lc3 HSCs. Data are means ± s.d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
De novo DNA methylation controls neuronal maturation during adult hippocampal neurogenesis (2021)
Figure 2

Figure 2: De novo DNA methyltransferases control neuronal differentiation from adult hippocampal NPCs in vitro.

A, Schematic representation of the genomic area of Dnmt3a and Dnmt3b in wildtype (WT) mice and conditional knock-out (KO) mice. WT mice were Nestin::Cre-ERT2(‑/‑)/ Dnmt3afl/fl/Dnmt3bfl/fl, while KO mice were Nestin::Cre-ERT2(‑/+)/Dnmt3afl/fl/Dnmt3bfl/fl.

B, Dentate gyri from WT and KO mice were dissociated 10 days after tamoxifen administration and plated for neurosphere formation. NPC monolayer cultures were generated from single neurospheres. Representative pictures of neurosphere (left) and neurosphere-derived adherent monolayer culture (right). Scale bars: 50 µm.

C, Representative images of polymerase chain reaction used for genotyping of NPC lines.

D, Dnmt3a/b-KO did not affect NPC proliferation. Depicted are data points for every sample with genotype means ± standard errors of the mean (SEM).

E, Reduced percentage of dead cells (Annexin V/propidium iodide (PI) double-positive) in KO cultures at 46 h after start of differentiation. Depicted are data points for every sample with genotype means ± SEM.

F-G, NPCs from WT and KO mice differentiated into astrocytes (GFAP-positive) and neurons (Tubb3-positive). Increased numbers of both neurons and astrocytes were generated from KO NPCs. Depicted are data points for every sample with genotype means ± SEM. Scale bar: 100 µm.

H, High magnification fluorescent image of differentiated neurons. Scale bar: 20 µm.

I, Sholl analysis of Tubb3-labeled WT and KO neurons (n = 29 neurons, WT; n = 30 neurons, KO). Reported p-value corresponds to genotype effect from two-way ANOVA. Asterisks highlight data points with p < 0.05 after multiple testing adjustment (Holm method) of repeated t-tests. Depicted are means ± SEM.

J, High magnification fluorescent image of differentiated astrocytes. Scale bar: 20 µm.

K, Astrocytes from KO cultures covered a larger area than WT astrocytes. Data points represent means of individual cultures (n > 30 astrocytes per culture). Depicted are data points per sample with genotype means ± SEM.

Data information: Depicted p-values in panels D, E, F, K are from unpaired t-test. Sample sizes: n = 4 cultures, WT; n = 5 cultures, KO. Further statistical details are reported in Appendix Table S5.
De novo DNA methylation controls neuronal maturation during adult hippocampal neurogenesis (2021)
Figure 3

Figure 3: De novo DNA methyltransferases establish neuronal DNA methylation patterns and facilitate neuronal gene expression.

A, NPCs carrying Dnmt3a/b-KO or WT alleles were differentiated into neurons. Neurons were isolated by FACS and their DNA methylation patterns analyzed using RRBS (n = 3 cultures per genotype).

B, We identified 22,150 CpGs that differed in methylation (q < 0.05 and absolute methylation difference > 20 %) between KO and WT neurons, accounting to 4.55 % of all CpGs covered by RRBS being hypomethylated (hypoCpG; violet) and 0.22 % being hypermethylated (hyperCpG; red) in KO neurons.

C, Hyper- and hypomethylated CpGs were depleted from CpG islands and promoters, but enriched at gene bodies. Hypomethylated but not hypermethylated CpGs were enriched at enhancer regions (* FDR adjusted p < 0.001, p-values determined by linear regression).

D, Hypomethylated CpGs were enriched at neuronal super-enhancers (p < 0.001; hypergeometric test). Depicted are methylation percentages of the 615 hypomethylated CpGs located within neuronal super-enhancers.

E, CpGs that gained methylation during neuronal differentiation (Fig. 1), were hypomethylated in KO neurons compared to WT neurons. Depicted are the 441 CpGs covered by RRBS in both datasets.

F, Genes containing hypomethylated cytosines (6,275 genes) were significantly enriched in pathways related to neuronal function as annotated in the Reactome Pathway Database.

G, Significantly enriched biological processes and cellular components from SYNGO enrichment analysis.

H, Dnmt3a/b-dependent loss of DNA methylation at neuronal gene candidates was associated with reduced transcription in neurons. Depicted are mean CpG methylation levels at specified genomic regions as determined by RRBS (left) and expression fold changes in KO versus WT neurons as determined by qRT-PCR (right; n = 4 cultures per genotype; p-values from Mann-Whitney test; depicted are data points for every sample with genotype means ± SEM).