Fig 2: 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.

Fig 3: Regulation of DNMT1 protein stability by DCAF5. a Isolated DNMT1 protein complexes contain DDB1, DCAF5, and L3MBTL3 proteins (see Supplementary Table 1 for additional information). The Flag-knock-in DNMT1 protein complexes were isolated from HCT116-Flag-DNMT1 cells by anti-Flag M2 antibody affinity resins or from control HCT116 cells without the Flag-DNMT1. The proteins associated with the isolated Flag-DNMT1 complexes were separated in protein gel, excised, trypsinized, and derivative peptides were fractionated by nano-liter liquid chromatography and identified by mass spectrometry. The positions of some proteins associated with the DNMT1 complex are shown. MW: molecular weight markers. b Endogenous DCAF5 and DNMT1 proteins interact with one another. DCAF5 and DNMT1 from HCT116 cells were immunoprecipitated and detected by western blot analyses as indicated. Experiments were repeated three independent times with the same conclusion. c The interaction between DNMT1 and DCAF5 is enhanced by SET7 expression. The SET7 expressing or empty control vectors were transfected into HCT116 cells for 48 h, treated with proteasome inhibitor MG132 (5 μg/ml) for last 6 h, and interactions between DNMT1 and DCAF5 were analyzed by co-immunoprecipitation and western blotting analyses. d Downregulation of DCAF5 prevents DNMT1 degradation triggered by LSD1 deficiency. HCT116 cells were transfected with 50 nM of luciferase siRNA and two independent DCAF5 siRNAs in the presence or absence of LSD1 (LSD1 or L) siRNA. The protein levels of DNMT1, DCAF5, actin, and LSD1 were examined in total cell lysates by western blotting using indicated antibodies and quantified. The P value of LSD1 to control (Luc) siRNAs was calculated by independent Student’s t-test and the P values of double to single knockdowns were calculated by paired Student’s t-test. *P < 0.05, ***P < 0.001. e HCT116 cells were transfected with 50 nM of luciferase and DCAF5 siRNAs in the presence or absence of PHF20L1 or LSD1 siRNAs for 48 h. The protein levels of DNMT1, DCAF5, actin, LSD1 and PHF20L1 were examined and quantified. Student’s t-test was calculated for significant differences. ***P < 0.001. Quantifications for all experiments were represented by bar graph with mean and S.D. for error bars from three replicates, as described in a

Fig 4: CRL4 regulates the protein stability of DNMT1. a Loss of CRL4 core components stabilizes DNMT1. HCT116 cells were transfected with 50 nM siRNAs for each of luciferase, CUL4A + CUL4B (CUL4A/B), and DDB1 for 48 h, in the presence or absence of LSD1 siRNA as indicated. The levels of DNMT1 and CRL4 proteins were analyzed by blotting with indicated antibodies on the left. The protein samples were analyzed with three replicate repeats and the proteins bands were quantified and normalized to the luciferase siRNA control. The quantifications were represented by bar graph with mean and standard deviation (S.D.) for error bars from three replicates. The P value of LDS1 to control (Luc) siRNAs was calculated by independent Student’s t-test and the P values of double to single knockdowns were calculated by paired Student’s t-test (*P < 0.05, **P < 0.01). Experiments were repeated three independent times with the same conclusion. b Loss of DDB1 stabilizes DNMT1 in AKT1 knockdown cells. HCT116 cells were transfected with 50 nM siRNAs for Luc or DDB1, in the presence or absence of AKT1 siRNA as indicated for 48 h. The proteins were analyzed and quantified as in a. Student’s t-test was used for significant differences. *P < 0.05, ***P < 0.001. c The endogenous CRL4 core complexes interact with DNMT1. Endogenous protein complexes were immunoprecipitated (IP) with indicated antibodies and blotted with anti-DNMT1, CUL4A, CUL4B, or CUL4CT (detecting the C termini of CUL4A and CUL4B) antibodies. Non-specific IgG was used as a control. Experiments were repeated four independent times with the same conclusion. d CRL4 binding to DNMT1 is abolished by K142A mutation in DNMT1. HCT116 cells were transiently transfected with the Flag-tagged DNMT1 (WT) or K142A mutant constructs for 48 h. The cells were lysed and proteins were immunoprecipitated by anti-CUL4A/CUL4B antibodies or control IgG. The proteins in the CRL4 complexes were detected by anti-Flag and anti-CUL4A/CUL4B antibodies. Experiments were repeated three independent times with the same conclusion

Fig 5: 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 (19). 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.