Journal: Journal of Cellular and Molecular Medicine

Article Title: TNF-α inhibits aquaporin 5 expression in human salivary gland acinar cells via suppression of histone H4 acetylation

doi: 10.1111/j.1582-4934.2011.01456.x

Figure Lengend Snippet: Expression and activation levels of DNA methyltransferases in TNF-α-treated NS-SV-AC acinar cells. (A) RT-PCR analysis of the expression of Dnmt1, Dnmt3a and Dnmt3b mRNAs in TNF-α-treated NS-SV-AC acinar cells. NS-SV-AC acinar cells expressed Dnmt1, Dnmt3a and Dnmt3b mRNAs under the basal condition. After exposure to 10 ng/ml TNF-α, the expression levels of the three Dnmt mRNAs showed no significant changes. (B) Western blot analysis of Dnmt1, Dnmt3a and Dnmt3b proteins in TNF-α-treated NS-SV-AC acinar cells. There were no marked increases in the expression levels of these three Dnmts with TNF-α treatment. These results were similar to those observed by RT-PCR analysis. (C) Methylation activity assay of TNF-α-treated NS-SV-AC acinar cells. The methylation activity of TNF-α-treated NS-SV-AC cells was measured by using the EpiQuik™ DNA Methyltransferase Activity Assay Kit. The results are expressed as optical density at a wavelength of 450 nm, and are the means ± S.D. of three separate experiments. The results were analysed using the Mann–Whitney U -test. * P < 0.05 compared with control cells.

Article Snippet: The membranes were blocked with 2% skim milk and incubated with goat anti-human AQP5 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-human Dnmt1 antibody (Santa Cruz Biotechnology), rabbit anti-human Dnmt3a antibody (Santa Cruz Biotechnology), rabbit anti-human Dnmt3b antibody (Santa Cruz Biotechnology) or rabbit anti-human IκBα antibody (Santa Cruz Biotechnology).

Techniques: Expressing, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Methylation, Activity Assay, MANN-WHITNEY, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Epigenetic programming of μ-opioid receptor gene in mouse brain is regulated by MeCP2 and brg1 chromatin remodelling factor

doi: 10.1111/j.1582-4934.2008.00535.x

Figure Lengend Snippet: Interaction of MeCP2 with the chromatin-remodelling factor, Brg1. (A) – Antibody against Brm was used to immunoprecipitate and visualize the interaction with MeCP2 (detected with anti-MeCP2 antibody in the Western blot) in mouse brain nuclear extracts. Conversely, MeCP2 antibody was used to detect interactions with Brm (detected with anti-Brm antibody in the Western blot). Using MOR-positive differentiated P19 cells (AP4d), MeCP2 antibody was used to immunoprecipitate Brg1 chromatin-remodelling factor. The interaction of MeCP2 with corepressor was also analysed using mSin3A to validate the coimmunoprecipitation. SN: protein supernatant after immunoprecipitation; IP Ab: immunoprecipitation antibody; IB Ab: immunoblotting antibody. (B) – The expression of Brg1 was assessed by Western blot analysis using anti-Brg1 antibody in different cell types/conditions and mouse brain (MB). Brg1 proteins are indicated with their molecular weights: full-length (200 kD); isoforms (75 and 70 kD), consistent with Upstate antibody information for anti-Brg1 (Upstate, 07–478). (C) – ChIP analysis by real-time qPCR of Brg1 interaction. Primers specific for PP-MOR (S-408 and AS-285; Table ) and DP-MOR (S-731 and AS-623) were used to amplify by real-time qPCR genomic DNA sequences immunoprecipitated with anti-Brg1 antibody. Five brain regions (OB, STM, HPT, CRB, and PCX) and NS20Y cells were used for the ChIP analysis. (D) – ReChIP analysis by real-time qPCR. The primers specific for PP-MOR, DP-MOR, and β-actin were the same as those used above. Three brain tissues, STM ( i.e. a site of intermediate MOR expression), HPT (a high MOR site), and CRB (a non-MOR expressing site) were used for the ReChIP. Data are normalized against the input and are the mean ± S.E.M. from three independent experiments. ( Top panel ) first antibody: anti-Brg1, second antibody: anti-MeCP2. ( Centre panel ) First antibody: anti-MeCP2, second antibody: anti-Brg1. ( Bottom panel ) first antibody: anti-Dnmt1, second antibody: anti-MeCP2.

Article Snippet: Pre-cleared lysates (100 μl) diluted in immunoprecipitation buffer (Upstate) were immunoprecipitated overnight at 4°C with 2 μg of antibodies against each of the following: anti-AcH3 (06–599), anti-AcH4 (06–866), anti-H3 dm K4 (07–030), anti-H3 dm K9 (07–441) (all from Upstate), anti-Dnmt1 (Imgenex; IMG-261A), anti-Dnmt3b (Imgenex; IMG-184A) and anti-MeCP2.

Techniques: Western Blot, Immunoprecipitation, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Epigenetic programming of μ-opioid receptor gene in mouse brain is regulated by MeCP2 and brg1 chromatin remodelling factor

doi: 10.1111/j.1582-4934.2008.00535.x

Figure Lengend Snippet: A proposed molecular mechanism for MOR gene regulation. MeCP2 and Dnmt1 bind to hypermethylated DNA form a histone-associated repressor complex, silencing the MOR gene in cerebellar tissue. In the cerebellum, hypermethylation of CpGs around the proximal promoter (PP) coincides with increased interactions with MeCP2 and Dnmt1. This might lead to compaction of the chromatin structure after histone modifications, followed by silencing of the MOR gene in these cells. In striatal cells, intermediate methylation of CpGs around the PP begins as MeCP2 start to dissociate and Brg1 is recruited, concurrent with histone modifications, resulting in intermediate levels of MOR expression. In the hypothalamus, nearly complete demethylation of the CpGs around the PP is observed as MeCP2 dissociates and Brg1 is recruited. Hyperacetylation of histones also occur in the promoter, suggesting active transcription of the MOR gene in the hypothalamic tissue. The components for active transcription shown in the figure, i.e. GTF (general transcription factors) and their associated-RNA polymerase II (Pol II), are putative factors for general genes.

Article Snippet: Pre-cleared lysates (100 μl) diluted in immunoprecipitation buffer (Upstate) were immunoprecipitated overnight at 4°C with 2 μg of antibodies against each of the following: anti-AcH3 (06–599), anti-AcH4 (06–866), anti-H3 dm K4 (07–030), anti-H3 dm K9 (07–441) (all from Upstate), anti-Dnmt1 (Imgenex; IMG-261A), anti-Dnmt3b (Imgenex; IMG-184A) and anti-MeCP2.

Techniques: Methylation, Expressing

Journal: Cell death discovery

Article Title: IFNγ regulates ferroptosis in KFs by inhibiting the expression of SPOCD1 through DNMT3A.

doi: 10.1038/s41420-024-02257-z

Figure Lengend Snippet: Fig. 4 IFNγ could regulate the expression of DNMT3A in keloids. A The string of protein-protein interactions. B Immunohistochemical staining for IFNγ in normal skin and keloid. C The western blotting results of IFNγ in normal skin and keloid. D The western blotting results of KFs after treated with oe-DNMT1, oe-DNMT3A and oe-DNMT3B for 24 h. E Gene expression of KFs after treated with oe-DNMT1, oe-DNMT3A and oe-DNMT3B for 24 h. F The western blotting results of KFs after treated with si-DNMT1, si-DNMT3A and si-DNMT3B for 24 h. G Gene expression of KFs after treated with si-DNMT1, si-DNMT3A and si-DNMT3B for 24 h. H The western blotting results of IFNγ and DNMT3A after treated with si-IFNγ or oe-IFNγ. Bar=100 μm. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The primary antibodies included rabbit antibodies against β-actin (proteintech, 1:1,000), SPOCD1 (proteintech, 1:5,000), IFNγ (proteintech, 1:1,000), GPX4 (proteintech, 1:10,000), GSH (proteintech, 1:1,000), SLC7A11 (proteintech, 1:1,000), SLC3A2 (proteintech, 1:1,000), DNMT1 (proteintech, 1:1,000), DNMT3A (proteintech, 1:1,000), and DNMT3B (proteintech, 1:2,000).

Techniques: Expressing, Protein-Protein interactions, Immunohistochemical staining, Staining, Western Blot, Gene Expression

Journal: Epigenetics

Article Title: Deep proteomic analysis of Dnmt1 mutant/hypomorphic colorectal cancer cells reveals dysregulation of epithelial–mesenchymal transition and subcellular re-localization of Beta-Catenin

doi: 10.1080/15592294.2019.1656154

Figure Lengend Snippet: DNMT1 primers.

Article Snippet: Antibody Company ID Dilution Host species Actin Abcam 8H10D10 1:1000 Rabbit Β-Catenin ThermoFisher MA1-301 1:1000 Mouse δ-catenin Cell signalling 4989S 1:1000 Rabbit Dnmt1 Cell signalling D63A6 1:1000 Rabbit Dnmt1 Cell signalling D59A4 1:1000 Rabbit E-Cadherin BD Transduction Laboratories 610,182 1:1000 Mouse Histone H3 Cell signalling D18C8 1:1000 Rabbit Lamin A/C Cell signalling 2032S 1:1000 Rabbit Lamin B1 US Biological L1220-21A 1:1000 Mouse N-Cadherin Cell signalling D4R1H 1:1000 Rabbit Slug Cell signalling C19G7 1:1000 Rabbit Snail Cell signalling L70G2 1:1000 Mouse Tubulin Cell signalling 2144S 1:1000 Rabbit Twist Cell signalling 46,702 1:1000 Rabbit Uhrf1 Invitrogen PA5-29,884 1:1000 Rabbit Usp7 Santa Cruz Biotechnology Sc-30,164 1:1000 Rabbit Vimentin Cell signalling D21H3 1:1000 Rabbit Zeb1 Santa Cruz biotech Sc-25,388 1:1000 Rabbit Open in a separate window Primary antibodies used in this study.

Techniques: Sequencing

Journal: Epigenetics

Article Title: Deep proteomic analysis of Dnmt1 mutant/hypomorphic colorectal cancer cells reveals dysregulation of epithelial–mesenchymal transition and subcellular re-localization of Beta-Catenin

doi: 10.1080/15592294.2019.1656154

Figure Lengend Snippet: Primary antibodies used in this study.

Article Snippet: Antibody Company ID Dilution Host species Actin Abcam 8H10D10 1:1000 Rabbit Β-Catenin ThermoFisher MA1-301 1:1000 Mouse δ-catenin Cell signalling 4989S 1:1000 Rabbit Dnmt1 Cell signalling D63A6 1:1000 Rabbit Dnmt1 Cell signalling D59A4 1:1000 Rabbit E-Cadherin BD Transduction Laboratories 610,182 1:1000 Mouse Histone H3 Cell signalling D18C8 1:1000 Rabbit Lamin A/C Cell signalling 2032S 1:1000 Rabbit Lamin B1 US Biological L1220-21A 1:1000 Mouse N-Cadherin Cell signalling D4R1H 1:1000 Rabbit Slug Cell signalling C19G7 1:1000 Rabbit Snail Cell signalling L70G2 1:1000 Mouse Tubulin Cell signalling 2144S 1:1000 Rabbit Twist Cell signalling 46,702 1:1000 Rabbit Uhrf1 Invitrogen PA5-29,884 1:1000 Rabbit Usp7 Santa Cruz Biotechnology Sc-30,164 1:1000 Rabbit Vimentin Cell signalling D21H3 1:1000 Rabbit Zeb1 Santa Cruz biotech Sc-25,388 1:1000 Rabbit Open in a separate window Primary antibodies used in this study.

Techniques: Transduction

Journal: Epigenetics

Article Title: Deep proteomic analysis of Dnmt1 mutant/hypomorphic colorectal cancer cells reveals dysregulation of epithelial–mesenchymal transition and subcellular re-localization of Beta-Catenin

doi: 10.1080/15592294.2019.1656154

Figure Lengend Snippet: shRNA containing vectors used to knockdown DNMT1 expression Dnmt1 protein expression (derived from GenBank Accession: {"type":"entrez-nucleotide","attrs":{"text":"NM_001379","term_id":"1677703423","term_text":"NM_001379"}} NM_001379 ).

Article Snippet: Antibody Company ID Dilution Host species Actin Abcam 8H10D10 1:1000 Rabbit Β-Catenin ThermoFisher MA1-301 1:1000 Mouse δ-catenin Cell signalling 4989S 1:1000 Rabbit Dnmt1 Cell signalling D63A6 1:1000 Rabbit Dnmt1 Cell signalling D59A4 1:1000 Rabbit E-Cadherin BD Transduction Laboratories 610,182 1:1000 Mouse Histone H3 Cell signalling D18C8 1:1000 Rabbit Lamin A/C Cell signalling 2032S 1:1000 Rabbit Lamin B1 US Biological L1220-21A 1:1000 Mouse N-Cadherin Cell signalling D4R1H 1:1000 Rabbit Slug Cell signalling C19G7 1:1000 Rabbit Snail Cell signalling L70G2 1:1000 Mouse Tubulin Cell signalling 2144S 1:1000 Rabbit Twist Cell signalling 46,702 1:1000 Rabbit Uhrf1 Invitrogen PA5-29,884 1:1000 Rabbit Usp7 Santa Cruz Biotechnology Sc-30,164 1:1000 Rabbit Vimentin Cell signalling D21H3 1:1000 Rabbit Zeb1 Santa Cruz biotech Sc-25,388 1:1000 Rabbit Open in a separate window Primary antibodies used in this study.

Techniques: shRNA, Expressing, Derivative Assay, Sequencing, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: BAH domains and a histone-like motif in DNA methyltransferase 1 (DNMT1) regulate de novo and maintenance methylation in vivo

doi: 10.1074/jbc.RA118.004612

Figure Lengend Snippet: Domain organization and structure of DNMT1. A, functional domains of DNMT1: nuclear localization sequence (NLS) and RFTS (both from Ref. 6); CXXC domain, a zinc-binding motif that specifically binds to unmethylated CpG dinucleotides in DNA (for a review, see Ref. 52); BAH1 and BAH2 (for a review, see Ref. 18); GK repeats, a stretch of alternating glycine and lysine residues (19); and the methyltransferase domain, the catalytic domain with strong similarities to DNA (cytosine-5)-methyltransferases of prokaryotes and eukaryotes (for a review, see Ref. 2). B, crystal structure of DNMT1 in complex with unmethylated DNA (5). Sequences N-terminal of the asterisk in A have been omitted. The spatial relationship of the functional domains shown in A are depicted by means of a shared color scheme. The GK repeats were not structured in any of the crystal structures of DNMT1; therefore, their position is approximate. C, mode of binding of the autoinhibitory linker via a leucine residue to BAH1 (left) and binding of the BAH2-TRD loop via a phenylalanine (right). D, conservation of the BAH1-CXXC linker sequence among vertebrates.

Article Snippet: The following primary antibodies were used: mouse monoclonal to PCNA (Santa Cruz Biotechnology, sc-56 (PC10); 1:500 dilution) and rabbit polyclonal to DNMT1 (Santa Cruz Biotechnology, sc-20701; 1:500 dilution).

Techniques: Functional Assay, Sequencing, Binding Assay, Residue

Journal: The Journal of Biological Chemistry

Article Title: BAH domains and a histone-like motif in DNA methyltransferase 1 (DNMT1) regulate de novo and maintenance methylation in vivo

doi: 10.1074/jbc.RA118.004612

Figure Lengend Snippet: In vivo deletion of BAH domains eliminates DNA methylation and recruitment of DNMT1 to replication foci. A, Cas9-mediated deletion of exons encoding the BAH domains. Flanking exons undergo an in-frame splicing event; internally deleted protein product is shown below. B, immunoblot analysis shows that BAH domain deletions are stable and expressed at close to WT levels. Two independent clones are shown. C, failure of BAH deletion proteins to associate with replication foci. The top row shows that WT DNMT1 colocalizes with PCNA, a marker of replication foci, whereas the bottom two rows show that BAH-deleted DNMT1 fails to associate with replication foci. D, BAH-deleted DNMT1 fails to maintain genomic methylation patterns. In all cases, resistance to methylation-sensitive restriction enzymes (HpaII and MaeII) is equivalent between Dnmt1−/− ES cells and DNMT1 BAH-deleted ES cells. aa, amino acids; LTR, long terminal repeat.

Article Snippet: The following primary antibodies were used: mouse monoclonal to PCNA (Santa Cruz Biotechnology, sc-56 (PC10); 1:500 dilution) and rabbit polyclonal to DNMT1 (Santa Cruz Biotechnology, sc-20701; 1:500 dilution).

Techniques: In Vivo, DNA Methylation Assay, Western Blot, Clone Assay, Marker, Methylation

Journal: The Journal of Biological Chemistry

Article Title: BAH domains and a histone-like motif in DNA methyltransferase 1 (DNMT1) regulate de novo and maintenance methylation in vivo

doi: 10.1074/jbc.RA118.004612

Figure Lengend Snippet: GK repeats are dispensable for maintenance methylation. GK DNMT1 or GR DNMT1 expression constructs were transfected into Dnmt1−/− ES cells and tested for restoration of genome-wide DNA methylation. A, conservation of GK repeats in DNMT1 homologs from animals, plants, and fungi. B, depiction of sequence change in GR DNMT1. C, immunoblot analysis that shows near WT levels of expression of GR DNMT1. D, restoration of WT levels of DNA methylation globally, at IAP long terminal repeat (LTR), and at minor satellites as established by measurement of resistance to methylation-sensitive restriction endonucleases and Southern blotting. E, quantification of genomic methylation levels by LUMA. Error bars represent S.D.

Article Snippet: The following primary antibodies were used: mouse monoclonal to PCNA (Santa Cruz Biotechnology, sc-56 (PC10); 1:500 dilution) and rabbit polyclonal to DNMT1 (Santa Cruz Biotechnology, sc-20701; 1:500 dilution).

Techniques: Methylation, Expressing, Construct, Transfection, Genome Wide, DNA Methylation Assay, Sequencing, Western Blot, Southern Blot

Journal: The Journal of Biological Chemistry

Article Title: BAH domains and a histone-like motif in DNA methyltransferase 1 (DNMT1) regulate de novo and maintenance methylation in vivo

doi: 10.1074/jbc.RA118.004612

Figure Lengend Snippet: Specific de novo methylation of paternal ICRs by GR DNMT1. The GK DNMT1 and GR DNMT1 ES cell clones shown in Fig. 3 were examined for DNA methylation by bisulfite genomic sequencing at paternal ICRs (A) and maternal ICRs (B). Positions of CpG dinucleotides are shown by dotted lines. C shows that de novo methylation at paternal ICRs was much greater in the presence of GR DNMT1. Chr, chromosome; ND, not detected. Error bars represent S.D.

Article Snippet: The following primary antibodies were used: mouse monoclonal to PCNA (Santa Cruz Biotechnology, sc-56 (PC10); 1:500 dilution) and rabbit polyclonal to DNMT1 (Santa Cruz Biotechnology, sc-20701; 1:500 dilution).

Techniques: Methylation, Clone Assay, DNA Methylation Assay, Genomic Sequencing

Journal: The Journal of Biological Chemistry

Article Title: BAH domains and a histone-like motif in DNA methyltransferase 1 (DNMT1) regulate de novo and maintenance methylation in vivo

doi: 10.1074/jbc.RA118.004612

Figure Lengend Snippet: Functional domains in mouse and B. mori homologs of DNMT1. DNMT1 is the only DNA (cytosine-5)-methyltransferase homolog detected in the Bombyx genome (42) but does not contain any annotated domains not also present in mammalian DNMT1 as assessed by CDD/SPARCLE (43). This indicates that DNMT1 is very likely to be involved in both maintenance and de novo methylation in Bombyx and suggests that the same is likely to be true of mammalian DNMT1.

Article Snippet: The following primary antibodies were used: mouse monoclonal to PCNA (Santa Cruz Biotechnology, sc-56 (PC10); 1:500 dilution) and rabbit polyclonal to DNMT1 (Santa Cruz Biotechnology, sc-20701; 1:500 dilution).

Techniques: Functional Assay, Methylation