dnmt1 cdna  (OriGene)

Journal: Frontiers in Oncology

Article Title: 3,3’-Diindolylmethane Enhances Paclitaxel Sensitivity by Suppressing DNMT1-Mediated KLF4 Methylation in Breast Cancer

doi: 10.3389/fonc.2021.627856

Figure Lengend Snippet: Nucleotide sequences of primers used for qRT-PCR reactions.

Article Snippet: Human DNMT1 cDNA synthesized by Genechem (Shanghai, China) was constructed with CMV-MCS-3FLAG-IRES-EGFP-SV40- neomycin plasmid, the overexpression of DNMT1 was named as DNMT1-OE and the control was named as DNMT1-NC.

Techniques: Methylation

Journal: Frontiers in Oncology

Article Title: 3,3’-Diindolylmethane Enhances Paclitaxel Sensitivity by Suppressing DNMT1-Mediated KLF4 Methylation in Breast Cancer

doi: 10.3389/fonc.2021.627856

Figure Lengend Snippet: DNMT1 expression was negatively correlated with KLF4 expression and PTX sensitivity in breast cancers. (A) Immunohistochemical assessment of KLF4 and DNMT1 levels in breast cancer tissues. (B) Average score of KLF4 and DNMT1 immunostaining in breast cancer tissues. (C) Immunohistochemical assessment of DNMT1 expression in breast cancer and adjacent tissues. (D) Average score of DNMT1 immunostaining in breast cancer and adjacent tissues. (E) Western blotting analyses of DNMT1 protein expression in breast cancer and adjacent tissues. (F) Relative quantity of DNMT1 protein expression in breast cancer and adjacent tissues. (G) Real-time PCR and Western blot analysis of DNMT1 expression level in MCF-7 cells after transfection with the DNMT1 interference plasmid. (H) Methylation level of the KLF4 promoter in MCF-7 cells after transfection with the DNMT1 interference plasmid. (I) The viability of MCF-7 cells treated with PTX (5μM, 25μM, 50μM and 75μM) after transfection with the DNMT1 interference plasmid. (J) Real-time PCR and Western blot analysis of DNMT1 expression in T47D cells after transfection with the DNMT1 interference plasmid. (K) Methylation level of the KLF4 promoter in T47D cells after transfection with the DNMT1 interference plasmid. (L) The viability of T47D cells treated with PTX (5μM, 25μM, 50μM and 75μM) after transfection with the DNMT1 interference plasmid. *P <0.05; **P <0.01 versus control.

Article Snippet: Human DNMT1 cDNA synthesized by Genechem (Shanghai, China) was constructed with CMV-MCS-3FLAG-IRES-EGFP-SV40- neomycin plasmid, the overexpression of DNMT1 was named as DNMT1-OE and the control was named as DNMT1-NC.

Techniques: Expressing, Immunohistochemical staining, Immunostaining, Western Blot, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Methylation, Control

Journal: Frontiers in Oncology

Article Title: 3,3’-Diindolylmethane Enhances Paclitaxel Sensitivity by Suppressing DNMT1-Mediated KLF4 Methylation in Breast Cancer

doi: 10.3389/fonc.2021.627856

Figure Lengend Snippet: DIM enhanced PTX sensitivity by inhibiting DNMT1 and downregulating KLF4 methylation. (A) Real-time PCR and Western blotting analyses of KLF4 and DNMT1 expression in MCF-7 cells treated with or without DIM (50 μM or 80 μM) for 48 h. (B) Real-time PCR and Western blot analyses of DNMT1 expression after transfection with the DNMT1 overexpressing plasmid in MCF-7 cells. (C) Real-time PCR and Western blotting analyses of KLF4 and DNMT1 expression in T47D cells treated with or without DIM (30 μM or 50 μM) for 48 h. (D) Real-time PCR and Western blotting analyses of DNMT1 expression in T47D cells transfected with the DNMT1 overexpressing plasmid. (E) The viability of MCF-7 cells treated with PTX and DIM after overexpressing DNMT1. (F) The viability of T47D cells treated with PTX and DIM after overexpressing DNMT1. (G) Methylation levels of the KLF4 gene in MCF-7 cells treated with or without DIM (50 μM or 80 μM) for 48 h. (H) Schematic model of DIM in enhancing PTX sensitivity by regulating the expression of KLF4 and DNMT1. *P <0.05; **P <0.01 versus control.

Article Snippet: Human DNMT1 cDNA synthesized by Genechem (Shanghai, China) was constructed with CMV-MCS-3FLAG-IRES-EGFP-SV40- neomycin plasmid, the overexpression of DNMT1 was named as DNMT1-OE and the control was named as DNMT1-NC.

Techniques: Methylation, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Plasmid Preparation, Control

Journal: Nature communications

Article Title: DNMT1 reads heterochromatic H4K20me3 to reinforce LINE-1 DNA methylation.

doi: 10.1038/s41467-021-22665-4

Figure Lengend Snippet: Fig. 1 Biochemical and structural analysis of the DNMT1 BAH1–H4K20me3 interaction. a Domain architecture of human DNMT1 (hDNMT1), with individual domains delimited by residues numbers. b Histone peptide array analysis of the interactions between hDNMT1728–1600 and histone peptides. c Peptide pull-down analysis of the interactions of hDNMT1728–1600 with different H4K20me peptides. The experiment was repeated three times with consistent results. d ITC-binding assays for hDNMT1BAH1, titrated with different H4K20me peptides. e Peptide pull-down analysis of the interactions between hDNMT1BAH1 and different tri-methylated histone peptides. The experiment was repeated three times with consistent results. f Crystal structure of bDNMT1BAH1 (light pink) and H414–25K20me3 peptide (yellow), with the H4K20me3-binding pocket shown in the expanded view (boxed). The last two β-strands, which were domain-swapped during crystallization, are colored in salmon. The zinc ion is shown as a purple sphere. g Close-up view of the intermolecular interactions between bDNMT1BAH1 and the H414–25K20me3 peptide. The equivalent pocket residues in hDNMT1BAH1 are labeled in parentheses in (f, g). The hydrogen bonds are shown as dashed lines. *The side chain of H4 D24, except for the Cβ atom, is untraceable. h Kd values of hDNMT1BAH1 mutants and H414–25K20me3 peptides, derived from the ITC binding assays. NDB, no detectable binding. Source data are provided as a Source Data file.

Article Snippet: The DNMT1 cDNA was purchased from Addgene (cat # 24952).

Techniques: Peptide Microarray, Binding Assay, Methylation, Crystallization Assay, Labeling, Derivative Assay

Journal: Nature communications

Article Title: DNMT1 reads heterochromatic H4K20me3 to reinforce LINE-1 DNA methylation.

doi: 10.1038/s41467-021-22665-4

Figure Lengend Snippet: Fig. 2 H4K20me3 binding allosterically stimulates DNMT1 activity. a Ribbon and surface diagram of the structure of hDNMT1351–1600 (PDB 4WXX [10.2210/pdb4WXX/pdb]), with individual segments color coded. The RFTS–CXXC linker helix is denoted as HelixRC. The detailed interactions between the autoinhibitory linker and the BAH1 domain are shown in the expanded view. The hydrogen bonds are shown as dashed lines. b Close-up view of the structural overlay between hDNMT1351–1600 (PDB 4WXX [10.2210/pdb4WXX/pdb]) and the bDNMT1BAH1–H4K20me3 complex, with the autoinhibitory linker and the H4K20me3 peptide colored in slate and yellow, respectively, and the corresponding binding site in hDNMT1351–1600 shown as electrostatic surface. The bDNMT1BAH1 domain was removed for clarity. c ITC binding analysis of the interaction between hDNMT1BAH1, either WT, W796A, or MutYW, and the autoinhibitory linker peptide, in the absence of presence of the H414–25K20me3 peptide. d DNA methylation activity of hDNMT1351-1600, WT, W796A, MutYW, or Mutlinker. Data are mean ± s.d. (n = 3 biological replicates). Statistical analysis for WT vs. mutants used two-tailed Student’s t test. ***p < 0.001. e H4K20me3-dependent DNA methylation activity of hDNTM1351–1600, either WT or W796A. The total concentration of H414–25K20me0 and H414–25K20me3 peptides were maintained at 100 μM to eliminate the substrate-competing effects of the H4 peptides. Data are mean ± s.d. (n = 3 biological replicates). f The RMSD values obtained for the autoinhibitory linker in the structural models of WT (PDB 4WXX [10.2210/pdb4WXX/pdb]) or W796A hDNMT1351–1600 and the hDNMT1351–1600-H4K20me3 complex (complex 1 and complex 2) during 100 ns MD simulations. g Sausage view of the RMSF values of the autoinhibitory linker in peptide-free and H4K20me3-bound hDNMT1351–1600 (complex 2), colored in blue and red, respectively. Source data are provided as a Source Data file.

Article Snippet: The DNMT1 cDNA was purchased from Addgene (cat # 24952).

Techniques: Binding Assay, Activity Assay, DNA Methylation Assay, Two Tailed Test, Concentration Assay

Journal: Nature communications

Article Title: DNMT1 reads heterochromatic H4K20me3 to reinforce LINE-1 DNA methylation.

doi: 10.1038/s41467-021-22665-4

Figure Lengend Snippet: Fig. 5 RFTS and BAH1 domains crosstalk in DNMT1-mediated DNA methylation. a Close-up view of the structurally overlaid hDNMT1351–1600 (PDB 4WXX [10.2210/pdb4WXX/pdb]), bDNMT1RFTS bound to H3K9me3 and ubiquitin (PDB 6PZV [10.2210/pdb6PZV/pdb]), and the bDNMT1BAH1–H4K20me3 complexes, with the surface of individual DNMT1 domains color coded in the same fashion as in Fig. 2a. hDNMT1 W464, W465, and W796 are colored purple. The autoinhibitory linker colored blue. The H3K9me3 and H4K20me3 peptides are shown as yellow sticks. The ubiquitin molecule is shown in ribbon representation and colored in wheat. The bDNMT1RFTS and bDNMT1BAH1 molecules were removed for clarity. b, c Violin plots showing distribution of absolute methylation levels for CpG sites with >5 coverage at all CpG sites (b) or those within the repeated genomic sequences (c) among the indicated cells samples. (n = 5,040,986 CpG sites in panel (b); n = 812,251 repeats in panel (c)). White dots are the median and box lines are the first and third quartile of the data. d Representative IGV view of CpG methylation at an H3K9me3-marked genomic region located in the chromosome 1 among three replicated 1KO-ESC lines with stable expression of the indicated DNMT1. Cytosines covered by at least ten reads according to eRRBS data (with three replications merged for each group) are shown, with each site designated by a vertical line. e Neutral comet assays, experimental scheme (top) and DNA breaks (bottom). f Quantified DNA breaks after ionizing radiation (IR) treatment of 1KO-ESC cells reconstituted with vector control or the indicated DNMT1. Box-and-whisker plots in panel depict 25–75% in the box, whiskers are 10–90%, and median is indicated. Data are mean ± s.d. from >100 cells (n = 3 biologically independent replicates). One-way ANOVA with post Tukey analysis was used. ****p < 0.0001. The data for vector, WT, W465A, and W464A/W465A were adopted from those published previously29.

Article Snippet: The DNMT1 cDNA was purchased from Addgene (cat # 24952).

Techniques: DNA Methylation Assay, Ubiquitin Proteomics, Methylation, Genomic Sequencing, CpG Methylation Assay, Expressing, Plasmid Preparation, Control, Whisker Assay

Journal: Human Molecular Genetics

Article Title: DNMT1 mutations leading to neurodegeneration paradoxically reflect on mitochondrial metabolism

doi: 10.1093/hmg/ddaa014

Figure Lengend Snippet: Modeling of DNMT1 mutations, methylatransferase activity of human purified DNMT1 mutant proteins and DNMT1 expression in fibroblasts. (A and B) Ribbon diagram of human DNMT1 crystal structure . The numeration of NP_001124295.1 was used and the crystal structure numeration is included between parentheses. The RFTS, CXXC, BAH1, BAH2 and MTase domains are colored in light blue, dark red, light green, dark green and orange, respectively. Amino acid atoms are represented as transparent van der Walls spheres. The Zn(II) ions and the S -adenosyl-l-homocysteine (AdoHcy) ligand are reported as spheres colored accordingly to the atom type. The encircled region is reported in detail in panel (B) , where the residues cited in the text are reported in stick representation (residues involved in the mutations are in bold). Dashed red lines highlight H-bonds, while black dashed lines indicate coordination bonds. The hydrophobic core cited in the text is formed by Ala570(554), Val574(558), Ile601(585), Ala604(588), Val606(590) and Leu608(592). (C) Methyltransferase activity of human wild-type (WT) and mutant DNMT1 proteins expressed in E. coli . DNMT1 activity/mg of protein was obtained using a colorimetric ELISA-like assay. Three biological replicates were analyzed, and data are expressed as % of WT DNMT1 activity (means ± SEM). EV: empty vector. (D) Western blot of DNMT1 , phosphorylated AKT (Ser473) and GSK3-β (Ser9) in fibroblasts; GAPDH, total AKT and GSK3-β were used as loading controls. A representative blot of three independent experiments, analyzing three biological replicates, is shown for each protein. (E) Densitometry of three independent Western blot experiments of DNMT1 content. All values are means ± SEM and are normalized to control cells. (F) DNMT1 gene expression as evaluated by qPCR. TUBB was used as reference gene. Fold-changes are expressed as means ± SEM of three independent experiments, analyzing three biological replicates. (G and H) Densitometry of three independent western blot experiments of phosphorylated AKT and GSK3-β levels, on total AKT and GSK3-β, respectively. All values are means ± SEM and are normalized to control cells. DNMT1 mut represents average of the six mutants. Light grey and dark grey represent ADCA-DN and HSN-IE phenotypes, respectively. Unpaired t test was used for DNMT1 mut vs. controls, and Anova test (Dunnett’s multiple comparisons test) was used for individual mutants vs. controls. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human DNMT1 cDNA in pCMV3-untagged vector (Sino Biological Inc.) was mutagenized through PCR with primers designed with specific point mutations (see list of primer sequences in Supplementary information) and cloned into TOPO® XL PCR Cloning Kit (Life Technologies).

Techniques: Activity Assay, Purification, Mutagenesis, Expressing, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Western Blot

Journal: Human Molecular Genetics

Article Title: DNMT1 mutations leading to neurodegeneration paradoxically reflect on mitochondrial metabolism

doi: 10.1093/hmg/ddaa014

Figure Lengend Snippet: DNMT1 localization in mitochondria and mtDNA methylation analysis. (A and B) Western blot of DNMT1 localization in total lysate (TL), nuclear (NF), cytoplasmic (CF) and mitochondrial (MF) fractions of primary or immortalized fibroblasts (A) or HeLa and SK-N-SH cells (B) . HDAC1 and COXIV were used as markers for nuclear or mitochondrial fractions, respectively. IMM: immortalized fibroblasts. (C) Protease K protection assay on purified mitochondria in HeLa and SK-N-SH. MFN2, TIM23 and CS were used as a marker of outer membrane, inner membrane and matrix. PK: proteinase K (ug); DIG: digitonin. (D and E) Mitochondrial D-loop CpG methylation, assessed by bisulfite-NGS, after linearization of mtDNA with BamHI digestion. Data are expressed as means ± SEM of % of methylated cytosines on total mtDNA molecules sequenced, for each nucleotide position of L-strand (D) and H-strand (E) .

Article Snippet: Human DNMT1 cDNA in pCMV3-untagged vector (Sino Biological Inc.) was mutagenized through PCR with primers designed with specific point mutations (see list of primer sequences in Supplementary information) and cloned into TOPO® XL PCR Cloning Kit (Life Technologies).

Techniques: Methylation, Western Blot, Purification, Marker, CpG Methylation Assay

Journal: Human Molecular Genetics

Article Title: DNMT1 mutations leading to neurodegeneration paradoxically reflect on mitochondrial metabolism

doi: 10.1093/hmg/ddaa014

Figure Lengend Snippet: Assessment of mitochondrial biogenesis. (A) De novo mitochondrial translation assessed by 35 S-methionine incorporation. Coomassie staining was used as loading control. A representative blot of three independent experiments, analyzing three biological replicates, is shown. (B) Densitometry of three independent experiments of newly synthetized mitochondrial proteins. DNMT1 mut and individual mutant values are expressed as means ± SEM and are normalized to control cells. (C) Western blot of OXPHOS subunits; GAPDH was used as a loading control. A representative blot of five independent experiments, analyzing five biological replicates, is shown. (D) Densitometry of five independent western blot experiments of OXPHOS subunits. All values are means ± SEM and are normalized to control cells. (E) mtDNA content evaluation by qPCR. All values are expressed as means ± SEM of four independent experiments and are normalized to control cells. (F) PGC-1α gene expression evaluated by qPCR. TUBB was used as reference gene. Fold-changes are expressed as means ± SEM of three independent experiments, analyzing three biological replicates. (G) Western blot of P53, TIM23, TOM20 and citrate synthase (CS); GAPDH was used as a loading control. A representative blot of independent experiments (six for P53, five for TIM23, three for TOM20 and three for CS), analyzing biological replicates, is shown. (H) Densitometry of six independent western blot experiments of P53 content. All values are means ± SEM and are normalized to control cells. (I) Densitometry of five independent western blot experiments of TIM23 content. All values are means ± SEM and are normalized to control cells. (J) Densitometry of three independent western blot experiments of TOM20 content. All values are means ± SEM and are normalized to control cells. (K) Densitometry of three independent western blot experiments of CS. All values are means ± SEM and are normalized to control cells. Unpaired t -test was used for DNMT1 mut vs. controls, and Anova test (Dunnett’s multiple comparisons test) was used for individual mutants vs. controls. * P ≤ 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human DNMT1 cDNA in pCMV3-untagged vector (Sino Biological Inc.) was mutagenized through PCR with primers designed with specific point mutations (see list of primer sequences in Supplementary information) and cloned into TOPO® XL PCR Cloning Kit (Life Technologies).

Techniques: Staining, Mutagenesis, Western Blot, Expressing

Journal: Human Molecular Genetics

Article Title: DNMT1 mutations leading to neurodegeneration paradoxically reflect on mitochondrial metabolism

doi: 10.1093/hmg/ddaa014

Figure Lengend Snippet: Assessment of OXPHOS function and mitochondrial oxidative stress. (A) OCR in basal condition and after injection of oligomycin (O), FCCP (F), rotenone (R) and antimycin A (A). All values are means ± SEM of three independent experiments, analyzing three biological replicates. (B) Basal, ATP-linked and maximal respirations. All values are means ± SEM of three independent experiments, analyzing three biological replicates. (C) OCR/ECAR ratio. All values are means ± SEM of three independent experiments, analyzing three biological replicates and normalized on control cells. (D) Cellular ATP quantification. Graph shows the ratio of luminescence signal of mutants on control cells. All values are means ± SEM of three independent experiments, analyzing three biological replicates. (E) ATP synthesis rate evaluated in digitonin-permeabilized cells in the presence of substrates of CI, CII, GPD or CIII and normalized on CS activity. All values are means ± SEM of three independent experiments, analyzing biological replicates and normalized on control cells. (F) Mitochondrial H 2 O 2 levels expressed as MitoP/MitoB ratio. All values are means ± SEM of three independent experiments, analyzing three biological replicates, and normalized on control cells. (G) Western blot of MnSOD; GAPDH was used as a loading control. A representative blot of four independent experiments, analyzing four biological replicates, is shown. Black bar indicates that one lane (one ctrl sample) was deleted on this blot. (H) Densitometry of four independent western blot experiments of MnSOD content. All values are means ± SEM and are normalized to control cells. Unpaired t -test was used for DNMT1 mut vs. controls, and Anova test (Dunnett’s multiple comparisons test) was used for individual mutants vs. controls. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human DNMT1 cDNA in pCMV3-untagged vector (Sino Biological Inc.) was mutagenized through PCR with primers designed with specific point mutations (see list of primer sequences in Supplementary information) and cloned into TOPO® XL PCR Cloning Kit (Life Technologies).

Techniques: Injection, Activity Assay, Western Blot

Journal: Human Molecular Genetics

Article Title: DNMT1 mutations leading to neurodegeneration paradoxically reflect on mitochondrial metabolism

doi: 10.1093/hmg/ddaa014

Figure Lengend Snippet: Altered metabolites of ‘arginine and proline metabolism’ and ‘alanine, aspartate and glutamate metabolism’. Schematic representation of ‘arginine and proline metabolism’ and ‘alanine, aspartate and glutamate metabolism’, quantified by targeted metabolomics. ND: not detected. Concentrations are expressed as pmol/10 6 cells and are shown as means ± SEM. Unpaired t test with Welch correction was used for DNMT1 mut vs. controls, * P ≤ 0.05, ** P < 0.01.

Article Snippet: Human DNMT1 cDNA in pCMV3-untagged vector (Sino Biological Inc.) was mutagenized through PCR with primers designed with specific point mutations (see list of primer sequences in Supplementary information) and cloned into TOPO® XL PCR Cloning Kit (Life Technologies).

Techniques:

Journal: Human Molecular Genetics

Article Title: DNMT1 mutations leading to neurodegeneration paradoxically reflect on mitochondrial metabolism

doi: 10.1093/hmg/ddaa014

Figure Lengend Snippet: Molecular and bioenergetics validation of metabolic alterations. (A) Extracellular xanthine assessed by fluorimetric metric assay in the culture medium of control and DNMT1 mutant fibroblasts. Data are means ± SEM of three independent experiments, analyzing three biological replicates. (B) Extracellular urea assessed by colorimetric assay in the culture medium of control and DNMT1 mutant fibroblasts. Data are means ± SEM of three independent experiments, analyzing three biological replicates. (C) CPS1 gene expression evaluated by dd-PCR. GAPDH was used as reference gene. Data are means ± SEM of three independent experiments, analyzing three biological replicates. (D) Western blot of ASS1; GAPDH was used as a loading control. A representative blot of three independent experiments, analyzing three biological replicates, is shown. (E) Densitometry of three independent western blot experiments of ASS1 content. All values are means ± SEM and are normalized to control cells. (F) PHGDH gene expression evaluated by dd-PCR. GAPDH was used as reference gene. Data are means ± SEM of three independent experiments, analyzing three biological replicates. (G) ECAR traces in basal condition and after injection of glucose (G), oligomycin (O) and 2-deoxyglucose (2-DG). All values are means ± SEM of three independent experiments, analyzing three biological replicates. (H) Glycolysis and glycolytic capacity. All values are means ± SEM of three independent experiments, analyzing three biological replicates. (I) Cellular energy status expressed as AMP/ATP ratio. Values are means ± SEM for CTRLS (n = 4) and DNMT1 mut (n = 6) or single measures for individual mutants. (J) Western blot of phosphorylated RAPTOR (Ser792), phosphorylated S6 protein (Ser204/244), total S6 and DEPTOR; total RAPTOR and GAPDH were used as loading controls. A representative blot of three independent experiments, analyzing three biological replicates, is shown. (K) Densitometry of three independent western blot experiments showing phosphorylated RAPTOR levels on total RAPTOR. All values are means ± SEM and are normalized to control cells. (L) Phosphorylated S6K protein levels assessed by AlphaLisa assay and normalized on μg of protein analyzed. All values are means ± SEM of two independent experiments. (M and N) Densitometry of three independent western blot experiments showing phosphorylated S6 levels on total S6 and DEPTOR content. All values are means ± SEM and are normalized to control cells. Unpaired t test was used for DNMT1 mut vs. controls, and Anova test (Dunnett’s multiple comparisons test) was used for individual mutants vs. controls. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human DNMT1 cDNA in pCMV3-untagged vector (Sino Biological Inc.) was mutagenized through PCR with primers designed with specific point mutations (see list of primer sequences in Supplementary information) and cloned into TOPO® XL PCR Cloning Kit (Life Technologies).

Techniques: Mutagenesis, Colorimetric Assay, Expressing, Western Blot, Injection

Journal: Human Molecular Genetics

Article Title: DNMT1 mutations leading to neurodegeneration paradoxically reflect on mitochondrial metabolism

doi: 10.1093/hmg/ddaa014

Figure Lengend Snippet: A model for the pathogenic mechanism of DNMT1 mutations. DNMT1 mutations lead to accumulation of DNMT1 protein through the activation of AKT and GSK3-β inhibition. As a possible consequence of the altered nuclear DNA methylation, purine degradation is stimulated, with the enhanced production of NH 4 , a neurotoxic by-product. The excess of NH 4 is cleared by the urea cycle, leading to ATP shortage. To compensate, OXPHOS is stimulated to sustain ATP request, triggering H 2 O 2 over-production. Concurrently, the upregulation of PHGDH directs glucose toward serine synthesis, limiting glycolysis and further affecting cellular ATP. Glutamine anaplerosis associated with PHGDH overexpression leads to production of α-ketoglutarate that can directly enter the TCA cycle, which feeds OXPHOS with substrates, thus contributing to mitochondrial hyper-function. In cells carrying the most severe DNMT1 mutations, the low ATP activates AMPK, which in turn inhibits mTORC1 to restrict other ATP-consuming mechanisms. Red arrow: upregulated; blue arrow: downregulated; dashed arrows: intermediate reactions not shown; 3-PG: 3-phosphoglycerate; GLN: glutamine; GLU: glutamate; α-KG: α-ketoglutarate; Ac-CoA: acetyl-CoA.

Article Snippet: Human DNMT1 cDNA in pCMV3-untagged vector (Sino Biological Inc.) was mutagenized through PCR with primers designed with specific point mutations (see list of primer sequences in Supplementary information) and cloned into TOPO® XL PCR Cloning Kit (Life Technologies).

Techniques: Activation Assay, Inhibition, DNA Methylation Assay, Over Expression