(A) Hep3B cells were treated with glucose (5 mM or 25 mM) with or without Thiamet-G (TMG). Shown are immunoblots of collected lysates using antibody targeting O-GlcNAc and GAPDH (n = 3). (B) Lysates …
Uncropped blot files of Figure 1A–D.
Hep3B cells were treated with Thiamet-G (TMG) or OSMI-4 (OSMI). Shown are representative immunoblots of treated Hep3B lysates performed with antibodies targeting O-GlcNAc and GAPDH and bar graphs of …
Uncropped blot files of Figure 1—figure supplement 1.
(A) HepG2 cells were treated with Thiamet-G or OSMI. Shown are immunoblots of treated HepG2 lysates performed with immunoblots of immunoprecipitates performed with antibodies targeting O-GlcNAc (n = …
Uncropped blot files of Figure 1—figure supplement 2A and B.
(A) Hep3B cells were treated with sucrose (5 mM or 25 mM). Shown are immunoblots of collected lysates using antibody targeting O-GlcNAc and GAPDH (n = 3). Lysates of Hep3B treated with sucrose were …
Uncropped blot files of Figure 1—figure supplement 3A and B.
(A) OGT activity was measured with low (5 mM, CTRL) or high glucose/Thiamet-G (TMG) (25 mM, O-GlcNAc) using the UDP-Glo Glycosyltransferase activity kit (Promega) (n = 3). (B) OGA activity was …
Pooled PBMCs treated with low (5 mM, CTRL) or high glucose/Thiamet-G (TMG) (25 mM, O-GlcNAc) were immunoprecipitated with antibody targeting DNMT1 and immunoblotted for O-GlcNAc (n = 3). ***p<0.0001 …
Uncropped blot files of Figure 1—figure supplement 5.
(A) Schematic drawing of the DNMT1 O-GlcNAc-modified region enriched from Hep3B cells based on mass spectrometry (MS) data and tandem MS (MS/MS) peaks. FTMS+ p NSI full MS (400.0000–1600.0000). …
Uncropped blot files of Figure 2D.
Myc-DNMT1-WT were transfected into Hep3B cells. Shown are immunoblots of treated DNMT1-WT lysates performed with antibodies targeting Myc, DNMT1, Tubulin, H3, and O-GlcNAc (n = 3). Lysates from …
Uncropped blot files of Figure 2—figure supplement 1.
For (A–D), bar graphs are of relative activity of DNA methyltransferase activity measured as absorbance from a DNMT activity/Inhibition ELISA kit and representative immunoblots of immunoprecipitates …
(A) HepG2 cells were treated with low (5 mM, CTRL) or high glucose/TMG (25 mM, O-GlcNAc). Shown are absorbance of DNA methyltransferase activity performed with DNA methyltransferase activity kit (n …
(A) Each Hep3B and Myc-DNMT1-overexpressed mutant (DNMT1-WT or DNMT1-S878A) was treated with low (5 mM, CTRL) or high glucose/TMG (25 mM, O-GlcNAc), 5-aza or GSK-3484862 (negative control). Shown …
(A) Hep3B cells expressed with DNMT1-WT and DNMT1-S878 cells were treated with low (5 mM, CTRL) or high glucose/TMG (25 mM, O-GlcNAc). (B) HepG2 cells expressed with DNMT1-WT and DNMT1-S878 cells …
Raw fluorescence image files of Figure 3.
(A) Density plot of DNA methylation for DNMT1-WT and DNMT1-S878A cells with either low (5 mM, CTRL) or high glucose/Thiamet-G (TMG) (25 mM, O-GlcNAc). (B) Genome browser screenshot of DNA …
Density plot of DNA methylation for DNMT1-WT and DNMT1-S878A cells with either low (5 mM, CTRL) or high glucose/Thiamet-G (TMG) (25 mM, O-GlcNAc). (A) Partially methylated domain (PMD) regions, (B) …
These are defined 'low FPKM' as containing <25% of FPKM regions per Mb window and 'high FPKM' as containing >75% of FPKM regions per Mb window.
The loss of methylation was not observed in S878A mutant cells. (A, B) The distribution of each DNA methylation was divided by DNA replication timing.
(A) Boxplot represents the levels of DNA methylation on the TE regions or non-TE regions of each Myc-DNMT1-overexpressed mutants (DNMT1-WT or DNMT1-S878A) were treated with low (5 mM, CTRL) or high …
Evolutionarily recent TEs are more likely to lose methylation than older elements in a variety of systems. Methylation percentages are shown around (A) LTR12C and (B) HERVH-int regions. The …
Genome browser screenshot of DNA methylation data LTR12C elements (blue) that demonstrate binding with ZFP57 and ZNF605 by glucose concentration.
The evolutionary distance between each group is from Perelman et al., 2011. Boxplot represents the DNA methylation by clades of the human genome (Homo sapiens to Haplorhini). CTRL, control; O-Glc, O-…
Shown are overall CpGs sites that detected with >5× coverage DNA methylation analysis using Nanopore technology PromethION sequencer. Each condition is biologically replicated.
(A) Quantitative fluorescence image of ROS in DNMT1-WT and DNMT1-S878A cells with either low (5 mM, CTRL) or high glucose/Thiamet-G (TMG) (25 mM, O-GlcNAc). (B) Quantitative fluorescence image of …
Raw fluorescence image files of Figure 5A–C.
(A) Whole promoters and (B) differentially methylated promoters.
(A) Gene Ontology of the top 11 pathways of hypomethylated promoter DNA by high glucose treatment. (B) Gene interaction map of hypomethylated promoter DNA of DNMT1-WT by high glucose treatment.
PC, positive control; NC, negative control; 1. cleaved-caspase3; 2, cIAP1; 3, claspin; 4, phospho-p53(S15); 5, survivin. ***p<0.0001 by Student’s t-test; Data are represented as mean ± SD from three …
Uncropped blot files of Figure 5—figure supplement 3.
Shown are absorbance of 8-OHdG performed with DNA damage quantification kit (n = 3). **p<0.0005; ***p<0.0001 by Student’s t-test; ns, not significant; data are represented as mean ± SD from three …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Homo sapiens) | DNMT1 | HUGO Gene Nomenclature Committee | HGNC:2976 | - |
Cell line (H. sapiens) | Hep 3B2.1–7 | ATCC | HB-8064 | - |
Cell line (H. sapiens) | Hep G2 | ATCC | HB-8065 | - |
Transfected construct (H. sapiens) | pcDNA3/Myc-DNMT1 | Addgene | Plasmid #36939 | |
Antibody | Anti-beta-actin (D6A8) (rabbit monoclonal) | Cell Signaling Technology | Cat# 8457 | WB (1:1000) |
Antibody | Anti-alpha-tubulin (11H10) (rabbit monoclonal) | Cell Signaling Technology | Cat# 2125 | WB (1:1000) |
Antibody | Anti-DNMT1 (60B1220.1) (mouse monoclonal) | Novus Biologicals | Cat# NB100-56519 | IP (1:250) WB (1:1000) |
Antibody | Anti-DNMT1 (H-12) (mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-271729 | WB (1:1000) |
Antibody | Anti-gamma H2A.X (rabbit polyclonal) | Abcam | Cat# ab11174 | IF (1:1000) |
Antibody | Anti-GAPDH (rabbit monoclonal) | Abcam | Cat# ab181602 | WB (1:1000) |
Antibody | Anti-H3 (rabbit polyclonal) | Abcam | Cat# ab1791 | WB (1:1000) |
Antibody | Anti-Myc [Myc.A7] (mouse monoclonal) | Abcam | Cat# ab18185 | IP (1:250) WB (1:1000) |
Antibody | Anti-O-GlcNAc (RL2) (mouse monoclonal) | Abcam | Cat# ab2739 | WB (1:1000) |
Antibody | Aoat anti-rabbit IgG H&L (HRP) (goat polyclonal) | Abcam | Cat# ab6721 | WB (1:5000) |
Antibody | Goat anti-mouse IgG H&L (HRP) (goat polyclonal) | Abcam | Cat# ab6789 | WB (1:5000) |
Antibody | Goat anti-mouse IgG (H+L), Alexa 488 (goat polyclonal) | Invitrogen | Cat# A32723 | WB (1:1000) |
Sequence-based reagent | DNMT1-T158A | This paper | PCR primers | agccccaggatt CGA aggaaaagcacc |
Sequence-based reagent | DNMT1-T158A | This paper | PCR primers | ggtgcttttcct TCG aatcctggggct |
Sequence-based reagent | DNMT1-T616A | This paper | PCR primers | gacaggggaccc GCG aaagccaccacc |
Sequence-based reagent | DNMT1-T616A | This paper | PCR primers | ggtggtggcttt CGC gggtcccctgtc |
Sequence-based reagent | DNMT1-S878A | This paper | PCR primers | gcgagattcgag GAG cctccaaaaacc |
Sequence-based reagent | DNMT1-S878A | This paper | PCR primers | ggtttttggagg CTC ctcgaatctcgc |
Sequence-based reagent | DNMT1-S878D | This paper | PCR primers | gcgagattcgag GAC cctccaaaaacc |
Sequence-based reagent | DNMT1-S878D | This paper | PCR primers | ggtttttggagg GTC ctcgaatctcgc |
Sequence-based reagent | DNMT1-T882A | This paper | PCR primers | tcccctccaaaa GCC cagccaacagag |
Sequence-based reagent | DNMT1-T882A | This paper | PCR primers | ctctgttggctg GGC ttttggagggga |
Commercial assay or kit | Q5 Site-Directed Mutagenesis kit | NEB | Cat# E0554S | - |
Commercial assay or kit | EpiQuik DNMT Activity/Inhibition ELISA Easy Kit | EpiGentek | Cat# P-3139 | - |
Commercial assay or kit | Global DNA methylation LINE-1 | Active Motif | Cat# 55017 | - |
Commercial assay or kit | EpiQuik 8-OHdG DNA Damage Quantification Direct kit | EpiGentek | Cat# P-6003 | - |
Chemical compound, drug | OSMI-4 | Selleck Chem | Cat# S8910 | - |
Chemical compound, drug | Thiamet-G (TMG) | Cayman Chemical | Cat# 13237 | - |
Software, algorithm | bedGraphToBigWig | Kent et al., 2010 | - | - |
Software, algorithm | Clustal Omega | Sievers et al., 2011 | - | Version 1.2.4 |
Software, algorithm | GraphPad Prism 9 | GraphPad | - | Version: 9.3.1 |
Software, algorithm | Minimap2 | Li and Birol, 2018 | RRID:SCR_018550 | Version: 2.17 |
Software, algorithm | Nanopolish | Loman et al., 2015 | RRID:SCR_016157 | Version: 0.11.1 |
Software, algorithm | Python | Python Core Team | Version: 3.8.2 | |
Software, algorithm | R | R Core Team | - | Version: 3.4.3 |
Software, algorithm | Samtools | Lister et al., 2009 | RRID:SCR_002105 | Version: 1.10 |
Other | UniProt | The UniProt Consortium | - | Database of protein information (https://www.uniprot.org/) |
Other | DAPI stain | Invitrogen | D1306 | 1 µg/ml; marker for nuclear DNA |
Prediction of O-GlcNAcylated sites within DNMT1 using OGTSite.
List of total identified proteins.
List of post-translational modification (PTM) sites of human DNMT1.
List of deposited data in this study.