Journal: Nucleic Acids Research

Article Title: Structural and mechanistic insights into UHRF1-mediated DNMT1 activation in the maintenance DNA methylation

doi: 10.1093/nar/gky104

Figure Lengend Snippet: Molecular determinants of DNMT1 binding to ubiquitin and UBL domain of UHRF1. ( A ) Domain organization of DNMT1. Three DNMT1 fragments were prepared for ubiquitin binding analysis. ( B and C ) 15 N HSQC spectra show that ubiquitin binds to DNMT1 RFTS domain (aa: 351–600) but not DNMT1_621 (aa: 621–1616), as illustrated by strong selective line broadening effect induced by addition of DNMT1 RFTS domain but not DNMT1_621. ( D ) Domain organization of DNMT1 fragments that were prepared for UHRF1 UBL binding. ( E and F ) NMR titration indicates UHRF1 UBL binds to DNMT1 RFTS domain as well as DNMT1_621, as illustrated by strong line broadening of certain resonances induced by the binding of DNMT1 RFTS (E) and DNMT1_621 fragment (F). ( G and H ) ITC confirms the molecular interaction of DNMT1 RFTS domain with ubiquitin and UHRF1 UBL domain. The top panel shows experimental ITC curve of titrating DNMT1 RFTS domain into ubiquitin (G) and UHRF1 UBL (H) respectively. The lower panel shows fitted curves of calorimetric titrations. The measured dissociation constant ( K d ) is shown.

Article Snippet: Myc-DDK-tagged human DNMT1 (RC226414) and UHRF1 (RC214251) plasmids were purchased from OriGene.

Techniques: Binding Assay, Titration

Journal: Nucleic Acids Research

Article Title: Structural and mechanistic insights into UHRF1-mediated DNMT1 activation in the maintenance DNA methylation

doi: 10.1093/nar/gky104

Figure Lengend Snippet: Crystal structure of DNMT1 RFTS domain in complex with ubiquitin. ( A ) 1:2 DNMT1 RFTS domain-ubiquitin contents of the asymmetric unit are shown as ribbon diagrams. DNMT1 RFTS domain is colored in green, two ubiquitin molecules are colored in marine and cyan respectively. ( B and C ) Close-up view of the interactions between the RFTS domain and ubiquitin 1 (Ubq1). ( D and E ) Detailed view of the interaction network of the RFTS domain with ubiquitin 2 (Ubq2). Residues that form the binding interface are depicted as stick models and labeled. Hydrogen bonds and salt bridges are shown in magenta dashes. ( F ) DNMT1 RFTS domain undergoes conformational changes upon ubiquitin binding. The α-helix (aa: 493–518) of RFTS domain adopts as a straight α-helical conformation in DNMT1 (PDB ID: 4WXX) but bends about 32° in the middle of α-helix (aa: 493–518) at the position of Met502 in RFTS/ubiquitin complex. Ubiquitin binding caused the bending of RFTS α-helix (aa: 493–518) that connects the preceding β-barrel (aa: 400–490) to the α-helical bundle, this eventually leads to large changes in the relative orientation of the β-barrel with respect to α-helical bundle.

Article Snippet: Myc-DDK-tagged human DNMT1 (RC226414) and UHRF1 (RC214251) plasmids were purchased from OriGene.

Techniques: Binding Assay, Labeling

Journal: Nucleic Acids Research

Article Title: Structural and mechanistic insights into UHRF1-mediated DNMT1 activation in the maintenance DNA methylation

doi: 10.1093/nar/gky104

Figure Lengend Snippet: Experimental measurement of DNMT1 RFTS domain binding to ubiquitin or UHRF1 UBL domain. ( A ) ITC fitting curves for binding of DNMT1 RFTS domain and its mutants to ubiquitin, the insert lists the calculated dissociation constant ( K d ). ND, not detectable. ( B ) ITC fitting curves for binding of DNMT1 RFTS domain to ubiquitin wild-type and mutants, along with the calculated K d . ( C ) ITC fitting curves for binding of DNMT1 RFTS domain and its mutants with UHRF1 UBL domain with dissociation constant ( K d ) values indicated. ( D and E ) In vitro DNA methylation reactions were performed as a function of time and the concentration of ubiquitin or UHRF1 UBL. (D) DNMT1_351 (aa: 351–1616) or ( E ) DNMT1_621 (aa: 621–1616) was used as the enzyme, and a 30-bp long hemimethylated DNA fragment as the substrate. ( F and G ) In vitro DNA methylation reactions were performed as a function of time and the concentration of ubiquitin, histone H3 or H3ub2. (F) DNMT1_351 (aa: 351–1616) or ( G) DNMT1_621 (aa: 621–1616) was used as the enzyme.

Article Snippet: Myc-DDK-tagged human DNMT1 (RC226414) and UHRF1 (RC214251) plasmids were purchased from OriGene.

Techniques: Binding Assay, In Vitro, DNA Methylation Assay, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Structural and mechanistic insights into UHRF1-mediated DNMT1 activation in the maintenance DNA methylation

doi: 10.1093/nar/gky104

Figure Lengend Snippet: Assessing nuclear localization and DNA methylation status in DNMT1 −/− mouse embryonic stem cells stably expressing DNMT1 wild-type or mutants. ( A ) Flag-Myc-tagged wild-type DNMT1, DNMT1 E384A, E397A, E384A/E397A and Y399A mutants were stably expressed in DNMT1 −/− mouse ESCs, as analyzed by western blot using antibodies that recognize DNMT1. The expression level of these exogenous proteins similar to endogenous DNMT1 was selected for the subsequent study. β-actin was selected as a loading control. ( B ) Immunofluorescence analysis of DNMT1 focal staining pattern in DNMT1 wild-type or knockout mouse ES cells or DNMT1 −/− ES cells stably expressing DNMT1 wild-type, E384A or E397A or Y339G single point mutant, or E384A/E397A double point mutant respectively. Scale bars, 10 μm. ( C ) Immunostaining using an antibody against 5mC in control and DNMT1 −/− mouse ES cells after genetic complementation with DNMT1 wild-type or various mutants. 5mC fluorescence signals from ∼50 cells were quantified and normalized against the wild-type cells, the mean value with a standard error has been provided. ( D ) The DNA methylation status of LINE1 and IAP was analyzed by bisulfite sequencing in control, DNMT1 −/− ESCs and DNMT1 −/− ESCs stably expressing DNMT1 wild-type, E384A, E397A, E384A/E397A and Y399A mutants. The percentage of 5mC was calculated and shown.

Article Snippet: Myc-DDK-tagged human DNMT1 (RC226414) and UHRF1 (RC214251) plasmids were purchased from OriGene.

Techniques: DNA Methylation Assay, Stable Transfection, Expressing, Western Blot, Immunofluorescence, Staining, Knock-Out, Mutagenesis, Immunostaining, Fluorescence, Methylation Sequencing

Journal: Nucleic Acids Research

Article Title: Structural and mechanistic insights into UHRF1-mediated DNMT1 activation in the maintenance DNA methylation

doi: 10.1093/nar/gky104

Figure Lengend Snippet: Both UHRF1 UBL and RING finger are critical for DNMT1 proper nuclear localization and maintenance DNA methylation in mouse embryonic stem cells. ( A ) UHRF1 −/− mouse ES cell lines stably expressing Flag-Myc-tagged UHRF1 wild-type or various mutants were analyzed by western blot using antibodies that recognize UHRF1. Flag-Myc-tagged UHRF1 or mutants expressed at a level similar to endogenous UHRF1 were selected for the study. β-actin was selected as a loading control. ( B ) Immunofluorescence analysis of DNMT1 focal staining pattern in UHRF1 wild-type, UHRF1 knockout mouse ES cells and UHRF1 −/− ES cells transfected with UHRF1-ΔUBL or UHRF1-ΔRING truncated mutant. Exogenous expression of UHRF1 and mutants was detected by Flag antibodies. Scale bars, 10 μm. ( C ) Immunostaining using an antibody against 5mC in control and UHRF1 −/− mouse ES cells after genetic complementation with wild-type or UHRF1-ΔUBL or UHRF1-ΔRING. The 5mC levels relative to wild-type ESCs were shown. Error bars represent ± s.e.m. ( D ) The DNA methylation status of LINE1 and IAP was analyzed by bisulfite sequencing in wild-type ESCs (as control), UHRF1 −/− ESCs and UHRF1 −/- ESCs stably expressing UHRF1 wild-type, or UHRF1-ΔUBL or UHRF1-ΔRING mutants. The percentage of 5mC was calculated and shown.

Article Snippet: Myc-DDK-tagged human DNMT1 (RC226414) and UHRF1 (RC214251) plasmids were purchased from OriGene.

Techniques: DNA Methylation Assay, Stable Transfection, Expressing, Western Blot, Immunofluorescence, Staining, Knock-Out, Transfection, Mutagenesis, Immunostaining, Methylation Sequencing

Journal: Cell Cycle

Article Title: RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

doi: 10.4161/15384101.2014.950886

Figure Lengend Snippet: Deletion of RFTS enhances the oncogenic activity of DNMT1. (A) HBEC3 stable cell lines were established to express full-length and DNMT1 deletion forms near endogenous DNMT1 levels. The levels of DNMT1 were determined by RT-qPCR (left) and western blotting (right). Data were normalized to vector cells. (B) Adherent colony formation. (C) Soft-agar colony formation. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, indicates no significant difference in comparison to vector cells.

Article Snippet: To establish full length or deletion mutant human DNMT1 stable lines, a full-length DNMT1 cDNA clone (SC325419, Origene) was used as template to amplify full-length or deletion mutant DNMT1 fragments.

Techniques: Activity Assay, Stable Transfection, Quantitative RT-PCR, Western Blot, Plasmid Preparation

Journal: Cell Cycle

Article Title: RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

doi: 10.4161/15384101.2014.950886

Figure Lengend Snippet: DNMT1-ΔRFTS promotes increased methylation and silencing of the DAPK and DUOX1 genes. (A) The methylation levels of DAPK (left) and DUOX1 (right) promoter-associated CpG islands were analyzed by qPCR. Methylated DNA was analyzed using the MethylMiner kit and amplified with specific primers. (B) Bisulfite sequencing results for DAPK (left) and DUOX1 (right) promoters. White squares represent unmethylated cytosines and black squares represent methylated cytosines in CpG sites. The percentage of methylated CpG dinucleotides from 8 independent clones is indicated. (C) DNMT1 chromatin occupancy was analyzed using DNMT1 ChIP and qPCR. (D) mRNA levels of DAPK (left) and DUOX1 (right) were analyzed by RT-qPCR and normalized to vector cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001 in comparison to vector cells.

Article Snippet: To establish full length or deletion mutant human DNMT1 stable lines, a full-length DNMT1 cDNA clone (SC325419, Origene) was used as template to amplify full-length or deletion mutant DNMT1 fragments.

Techniques: Methylation, Amplification, Methylation Sequencing, Clone Assay, Quantitative RT-PCR, Plasmid Preparation

Journal: Cell Cycle

Article Title: RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

doi: 10.4161/15384101.2014.950886

Figure Lengend Snippet: DNMT1-ΔRFTS decreases chromatin accessibility at DAPK and DUOX1 promoters. Cells were treated with or without DNA nuclease for 1 hr, prior to detection of promoter DNA by qPCR. The index of chromatin accessibility = 2 ((Ct DNase treated)-(Ct Untreated)). *, p < 0.05; ***, p < 0.001 in comparison to vector cells.

Article Snippet: To establish full length or deletion mutant human DNMT1 stable lines, a full-length DNMT1 cDNA clone (SC325419, Origene) was used as template to amplify full-length or deletion mutant DNMT1 fragments.

Techniques: Plasmid Preparation

Journal: Cell Cycle

Article Title: RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

doi: 10.4161/15384101.2014.950886

Figure Lengend Snippet: 5-aza-dC treatment reactivates TSG expression and suppresses DNMT1-dependent transformation. (A) mRNA levels of DAPK (left) and DUOX1 (right) were analyzed by RT-qPCR after 100nM 5-aza-dC treatment for 5 d and normalized to vector cells treated with DMSO. (B) Soft-agar colony formation after 5-aza-dC treatment. ***, p < 0.001 in comparison to the DMSO treated control.

Article Snippet: To establish full length or deletion mutant human DNMT1 stable lines, a full-length DNMT1 cDNA clone (SC325419, Origene) was used as template to amplify full-length or deletion mutant DNMT1 fragments.

Techniques: Expressing, Transformation Assay, Quantitative RT-PCR, Plasmid Preparation

Journal: Cell Cycle

Article Title: RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

doi: 10.4161/15384101.2014.950886

Figure Lengend Snippet: DNMT1-ΔRFTS expression enhances global DNMT1 methylation changes. (A) Genome-wide promoter DNA methylation profiles were obtained using the HELP assay. In volcano plots, the x-axis scores probe-specific methylation ratios and the y-axis scores p-values for the confidence of measurements. The plots allow visualization of methylation differences between vector and DNMT1 cells as well as the differences between vector and DNMT1-ΔRFTS cells. Probes sets that showed significant hyper- or hypomethylation (p < 0.05 for methylation changes (log2(HpaII/MspI)) > 2) are shown in cyan. All other probes are shown in red. (B) Heat map illustration of HpaII-enrichment fragments with methylation changes (log2(HapII/MspI)) > 2 between vector and DNMT1-ΔRFTS cells.

Article Snippet: To establish full length or deletion mutant human DNMT1 stable lines, a full-length DNMT1 cDNA clone (SC325419, Origene) was used as template to amplify full-length or deletion mutant DNMT1 fragments.

Techniques: Expressing, Methylation, Genome Wide, DNA Methylation Assay, HELP Assay, Plasmid Preparation

Journal: Cell Cycle

Article Title: RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

doi: 10.4161/15384101.2014.950886

Figure Lengend Snippet: Genomic hypomethylation is found in DNMT1-ΔRFTS cells. (A) 5-methylcytosine (mC) content of the total cytosine pool was determined by HPLC. (B) Bisulfite sequencing of SAT2. White squares represent unmethylated CpGs, black squares represent methylated CpGs, and gray squares represent undetermined sites. Each row is an independent sequencing result. (C) Quantitation of SAT2 bisulfite sequencing. (D) DNMT1 chromatin occupancy was analyzed using DNMT1 ChIP and qPCR. (E) Expression of SAT2 non-coding RNA was analyzed by RT-qPCR and normalized to vector cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001 in comparison to vector cells.

Article Snippet: To establish full length or deletion mutant human DNMT1 stable lines, a full-length DNMT1 cDNA clone (SC325419, Origene) was used as template to amplify full-length or deletion mutant DNMT1 fragments.

Techniques: Methylation Sequencing, Methylation, Sequencing, Quantitation Assay, Expressing, Quantitative RT-PCR, Plasmid Preparation

Journal: Cell Cycle

Article Title: RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

doi: 10.4161/15384101.2014.950886

Figure Lengend Snippet: Dual roles for RFTS domain in DNMT1-dependent DNA methylation. (A) RFTS-targeted DNMT1 associated proteins (RAP) are proposed to relieve inhibition of DNMT1 for access to euchromatin. (B) The RFTS domain mediates association between DNMT1 and pericentromeric heterochromatin. (C) In cancer, overexpression of RAPs or mutation of RFTS is proposed to relieve DNMT1 inhibition, thereby increasing methylation and silencing of TSGs. However, because the RFTS domain is required for association with heterochromatic SAT2 sequences, DNMT1 with mutant RFTS may be less associated with such sequences, accounting for global hypomethylation.

Article Snippet: To establish full length or deletion mutant human DNMT1 stable lines, a full-length DNMT1 cDNA clone (SC325419, Origene) was used as template to amplify full-length or deletion mutant DNMT1 fragments.

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

Journal: Cancer research

Article Title: DAXX Suppresses Tumor-Initiating Cells in Estrogen Receptor-positive Breast Cancer following endocrine therapy

doi: 10.1158/0008-5472.CAN-19-1110

Figure Lengend Snippet: A. MCF-7 and T47D cells were treated with 0 or 5nM E2 for 3 days. DNMT1 and DAXX protein levels were detected by Western blot analysis using Actin as a loading control. B-C. MCF-7 and T47D cells were transfected with a mock vector (EV) or the human DAXX cDNA (DAXX) for 2 days and then re-transfected with the non-specific (SCBi) or DNMT1-specific (DNMT1i) siRNA for an additional day. B. DAXX and DNMT1 proteins were detected by Western blotting. C. After 7 days, mammospheres were imaged, isolated, measured, and %MFE was calculated. Bar graphs show mean ± S.D. %MFE from three independent experiments. Statistical significance was calculated using two-way ANOVA with a Tukey post-hoc test for multiple comparisons. Symbols denote statistical significance between EV and DAXX, SCBi and DNMT1 under DAXX conditions, and 0nM and 5nM E2: #= P< 0.05, @= P< 0.001. D. MCF-7 cells were transfected with a non-specific (SCBi), DAXX-specific (DAXXi), or DNMT1-specific (DNMT1i) siRNA for 2 days. Cells treated with 0 or 5nM E2 for 1 day. Total DNA was isolated and subjected to bisulfite treatment. Bisulfite converted DNA was amplified using SOX2 promoter-specific primers or NOTCH4-CpG island-specific primers that anneal to bisulfite-treated DNA. The PCR product was purified and sent for DNA sequencing. CpG sites that were read as “T” were considered unmethylated and sites read as “C” were considered methylated. Bar graphs show mean (total number of methylated CpG sites)/ (total CpG sites read × 100) ± S.D. from five independent experiments. Statistical significance was calculated using two-way ANOVA with a Tukey post-hoc test for multiple comparisons. Symbol denotes statistical significance between 0nM and 5nM E2 and SCBi and DAXXi/DNMT1i groups: @= P< 0.001.

Article Snippet: DNMT1 siRNA was purchased from Origene (Catalogue # SR301244).

Techniques: Western Blot, Transfection, Plasmid Preparation, Isolation, Amplification, Purification, DNA Sequencing, Methylation

Journal: Clinical Epigenetics

Article Title: Metformin regulates expression of DNA methyltransferases through the miR-148/-152 family in non-small lung cancer cells

doi: 10.1186/s13148-023-01466-0

Figure Lengend Snippet: miR-148b negatively regulates DNMT1 and DNMT3b expression in A549 cells. A Sequences of miR-148/-152 precursor family members targeting DNMT1 3′UTR. B , C miR-148b-3p precursor was cloned into a pEZX-MT04 vector. miR-148b-3p plasmid ( B ) or miR-148b-3p mimic or inhibitor ( C ) was transfected into A549 cells using Lipofectamine® RNAiMAX. Cells were cultured for 24 h after transfection and then treated with 10 mM metformin for an additional 72 h. After 72 h, protein levels of DNMT1 and DNMT3b were determined using western blot, and α-tubulin was used as an internal loading control. NC, scrambled oligonucleotides as a negative control. Data are presented as mean ± standard deviation (n = 3). Statistical analysis: * P < 0.05, ** P < 0.05, *** P < 0.001

Article Snippet: Putative interactions between miR-148a-3p and DNMT1 3'UTR were analyzed through a luciferase reporter assay. miRNA 3′UTR target expression clone for Human DNMT1 (NM_001130823.2) was purchased from GeneCopoeia (Rockville, MD, Cat no. HmiT094835-MT06).

Techniques: Expressing, Clone Assay, Plasmid Preparation, Transfection, Cell Culture, Western Blot, Control, Negative Control, Standard Deviation

Journal: Clinical Epigenetics

Article Title: Metformin regulates expression of DNA methyltransferases through the miR-148/-152 family in non-small lung cancer cells

doi: 10.1186/s13148-023-01466-0

Figure Lengend Snippet: miR-148a suppresses luciferase reporter activity of DNMT1 3′UTR in NSCLC cells. Luciferase reporter assay was performed in A549 ( A – C ) and H1650 cells ( D – F ) co-transfected with an miR-148a mimic or an miR-148a inhibitor at a final concentration of 50 nM in combination with DNMT1-WT/MT-3′UTR reporter in a 96-well plate using Lipofectamine® RNAiMAX following the manufacturer's instructions. Some cells were incubated for 24 h after transfection, and others were subsequently treated with 10 mM metformin for 72 h. Luciferase activities were measured using a Gaussia luciferase assay kit (Promega) according to the manufacturer's protocol. Data represent mean ± standard deviation (SD) from three independent experiments. * P < 0.05, ** P < 0.05, *** P < 0.001

Article Snippet: Putative interactions between miR-148a-3p and DNMT1 3'UTR were analyzed through a luciferase reporter assay. miRNA 3′UTR target expression clone for Human DNMT1 (NM_001130823.2) was purchased from GeneCopoeia (Rockville, MD, Cat no. HmiT094835-MT06).

Techniques: Luciferase, Activity Assay, Reporter Assay, Transfection, Concentration Assay, Incubation, Standard Deviation

Journal: bioRxiv

Article Title: Targeted phosphoproteomics of the Ras signaling network reveal regulatory mechanisms mediated by oncogenic KRAS

doi: 10.1101/695460

Figure Lengend Snippet: Oncogenic KRAS promotes phosphorylation of DNMT1 S714 to epigenetically silence cell cycle inhibitors. ( A ) mRNA levels of several DNMT1 target genes were measured by qPCR in asynchronously growing H358 cells stably expressing GFP or wild-type, S714A or S714D DNMT1. p -values (unpaired t-test) between GFP and S714A lines are shown. n.s. = p > 0.05; * = p < 0.01; ** = p < 0.001. ( B ) GFP or myc-tagged wild-type or mutant DNMT1 was stably expressed in H358, H2030 and H522 NSCLC lines. Arrows point to endogenous (lower) and myc-tagged (upper) DNMT1. ( C ) CDKN1A or CCND2 were upregulated upon KRAS knockdown in H358 or H2030 cells, respectively (black striped bars). The S714A mutant mimicked this upregulation in KRAS replete conditions (red bars) and the S714D mutant slightly suppressed this upregulation in KRAS knockdown conditions (blue bars). p21 protein levels are shown with actin-normalized quantitation (ImageJ). Cyclin D2 protein levels were not consistently detectable. ( D ) In KRAS wild-type H522 cells, KRAS knockdown did not modulate CDKN1A or CCND2 levels. ( E ) Effects on cell proliferation by DNMT1 mutants were most prominent under high confluence. Cells were plated at 75% confluence and proliferation was monitored over 96h. KRAS knockdown significantly inhibited growth in KRAS mutant lines, but not in the KRAS wild-type line. S714A expression was not sufficient to mimic this inhibition in KRAS replete conditions, but S714D expression partially rescued growth inhibition in KRAS knockdown conditions. p -values (unpaired t-test) between WT siKRAS and S714D siKRAS at 96h are shown. ( F ) Model of DNMT1-dependent transcriptional silencing of tumor suppressor genes by oncogenic KRAS signaling.

Article Snippet: A TrueORF clone of DNMT1 cDNA, transcript variant 1, was obtained from OriGene (RC226414L1).

Techniques: Stable Transfection, Expressing, Mutagenesis, Quantitation Assay, Inhibition

Journal: bioRxiv

Article Title: Targeted phosphoproteomics of the Ras signaling network reveal regulatory mechanisms mediated by oncogenic KRAS

doi: 10.1101/695460

Figure Lengend Snippet: MEK inhibition does not mimic KRAS knock­ down in the regulation of DNMT1 target genes. ( A ) Statistical significance of gene expression between H358 cells expressing GFP and DNMT1 WT, S714A or S714D mutants. Experiments were run in technical triplicates, and p-values from unpaired t-tests are shown. ( B ) Growth curves were generated for KRAS mutant and KRAS wild-type lines, treated with negative control siRNA+DMSO, siKRAS or S00nM AZD6244 in 10% or 0% serum. AZD6244 treatment only moderately affected proliferation in H2030 cells, and neither KRAS knockdown nor AZD6244 affected H522 cells. ( C ) AZD6244 treatment did not induce CDKN1A levels in H358 cells, consis­ tent with no change in cell proliferation. CCND2 levels were only moderately elevated in H2030 and H522 cells. Experiments were run in techni­ cal triplicates, and p-values from unpaired t-tests are shown. ( D ) Effects on proliferation by DNMT1 mutants were moderate under AZD6244 treatment compared to siKRAS. n.s. = p ≥0.05.; * = p ≤0.01; ** = p ≤0.001

Article Snippet: A TrueORF clone of DNMT1 cDNA, transcript variant 1, was obtained from OriGene (RC226414L1).

Techniques: Inhibition, Expressing, Generated, Mutagenesis, Negative Control

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