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