Journal: Frontiers in Endocrinology

Article Title: Short-Term Hypothermic Holding of Mouse Immature Testicular Tissue Does Not Alter the Expression of DNA Methyltransferases and Global DNA Methylation Level, Post-Organotypic Culture

doi: 10.3389/fendo.2022.854297

Figure Lengend Snippet: mRNA expression of Dnmt1 , Dnmt3a , and Dnmt3b . Real-time q PCR analysis to understand the effect of varying holding duration at 4°C on mRNA levels of (A) Dnmt1 , (B) Dnmt3a , and (C) Dnmt3b . ITT held for 0 h at 4°C cultured for 14 days was used as a control in comparison to 6 and 24 h held cultured ITT in similar conditions. mRNA level of Dnmt1 , Dnmt3a , and Dnmt3b gene was normalized against reference genes Actb and Gapdh. Data are presented as Mean ± SEM (n = 3).

Article Snippet: Dnmt1 (Mm01151063_m), Dnmt3a (Mm00432881_m) and Dnmt3b (Mm01240113_m1) were used. q PCR results were normalized to Actb and Gapdh reference genes.

Techniques: Expressing, Cell Culture, Control, Comparison

Journal: Frontiers in Psychiatry

Article Title: Changes in Expression of DNA-Methyltransferase and Cannabinoid Receptor mRNAs in Blood Lymphocytes After Acute Cannabis Smoking

doi: 10.3389/fpsyt.2022.887700

Figure Lengend Snippet: Taqman primer probes for THC study.

Article Snippet: DNMT1 , Hs00154749_m1 , DNA (cytosine-5-)-methyltransferase 1- regulate gene expression- add methyl group to cytosine- increased with THC.

Techniques: Stable Transfection, Gene Expression

Journal: Frontiers in Psychiatry

Article Title: Changes in Expression of DNA-Methyltransferase and Cannabinoid Receptor mRNAs in Blood Lymphocytes After Acute Cannabis Smoking

doi: 10.3389/fpsyt.2022.887700

Figure Lengend Snippet: Baseline mRNA levels in the three treatment groups.

Article Snippet: DNMT1 , Hs00154749_m1 , DNA (cytosine-5-)-methyltransferase 1- regulate gene expression- add methyl group to cytosine- increased with THC.

Techniques:

Journal: Frontiers in Psychiatry

Article Title: Changes in Expression of DNA-Methyltransferase and Cannabinoid Receptor mRNAs in Blood Lymphocytes After Acute Cannabis Smoking

doi: 10.3389/fpsyt.2022.887700

Figure Lengend Snippet: Differences in mRNA levels from baseline in leucocytes in three treatment groups after smoking marijuana or placebo cigarettes.

Article Snippet: DNMT1 , Hs00154749_m1 , DNA (cytosine-5-)-methyltransferase 1- regulate gene expression- add methyl group to cytosine- increased with THC.

Techniques:

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 4. The effects of milk-derived extracellular vesicles (MEVs) supplementation on baseline homeostatic human microglia clone 3 (HMC3) cells at 12 h post-supplementation. DNMT1 and miR-148-5P transcript levels (a), DNMT1 protein abundance (b) and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels and miR-148-5P levels (d), and DNMT enzymatic activity and DNMT1 protein level (e). * p < 0.05, ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 6. The effects of milk-derived extracellular vesicles (MEVs) on IFN-γ primed human microglia clone 3 (HMC3) cells at 12 h post-supplementation. DNMT1 and miR-148a-5P transcript levels (a), DNMT1 protein abundance (b), and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels and miR-148-5P levels (p < 0.05) (d), and DNMT enzymatic activity and DNMT1 protein level (p < 0.05) (e). P-MEV indicates primed cells that received MEVs. P-Ctrl is primed control cells. ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay, Control

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 7. The effects of milk-derived extracellular vesicles (MEVs) on primed human microglia clone 3 (HMC3) cells 9 h post-supplementation. DNMT1 and miR-148a-5P transcript levels (a), DNMT1 protein abundance (b), and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels relative to miR-148a-5P levels (p ≤ 0.05) (d), and DNMT enzymatic activity relative to DNMT1 protein level (A.U) (e). P-MEV indicates primed cells that received MEVs. P-Ctrl is primed control cells. ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay, 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:

Article Title: Neither DNA hypomethylation nor changes in the kinetics of erythroid differentiation explain 5-azacytidine's ability to induce human fetal hemoglobin

doi: 10.1182/blood-2007-06-093948

Figure Lengend Snippet: siRNA knock-down of DNMT1 produces decreased γ-globin promoter methylation but no increase in γ-globin mRNA or fetal hemoglobin. siRNA for the DNMT1 gene was transiently transfected into cells on day +11 of in vitro erythroid differentiation. Controls for this experiment were cells from the same initial culture that were treated with a nonspecific siRNA (siCTRL) or underwent mock transfection without siRNA (CTRL). (A) The effect of siRNA treatment on DNMT1 mRNA levels during differentiation as determined by quantitative RT-PCR. (B) The effect of siRNA treatment on DNMT3a mRNA levels. (C) The effect of siRNA treatment on DNMT1 protein levels as determined by Western blotting. β-actin and DNMT3a serve as nonspecific controls. (D) The effect of siRNA treatment on global DNA methylation as determined by bisulfite conversion analysis of LINE elements. Note that for the day 15 control sample no error bars are shown. This is because all 5 sequences, which included a total of 42 individual CpGs, were 100% methylated so the standard deviation for this data point was 0. (E) The effect of siRNA treatment on γ-globin promoter DNA methylation. (F) The effect of siRNA treatment on γ-globin mRNA during differentiation. (G) The effect of siRNA treatment on hemoglobin production as assessed by HPLC analysis of cell lysates at the end of in vitro differentiation. CD34+ cells from a single normal donor were used for this experiment. Error bars represent 1 SD. P values were determined by t test.

Article Snippet: 17 DNMT1 siRNA and shRNA siRNA for DNMT1 or a fluorescein-labeled control siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) were transfected into cells using HiPerFect reagent (Qiagen, Valencia, CA) following the manufacturer's suggested protocol for transfection of suspension cells on day +11 of the standard culture procedure.

Techniques: Knockdown, Methylation, Transfection, In Vitro, Quantitative RT-PCR, Western Blot, DNA Methylation Assay, Control, Standard Deviation

Journal:

Article Title: Neither DNA hypomethylation nor changes in the kinetics of erythroid differentiation explain 5-azacytidine's ability to induce human fetal hemoglobin

doi: 10.1182/blood-2007-06-093948

Figure Lengend Snippet: shRNA knock-down of DNMT1 produces decreased γ-globin promoter methylation but no increase in γ-globin mRNA or fetal hemoglobin. Lentiviral vector LL3.7 containing DNMT1 shRNA and GFP sequences was used to transduce differentiating cells on day +5. On day +7 cells expressing GFP were sorted and allowed to differentiate into erythroid cells. Controls for this experiment were cells from the same initial culture transduced with vectors carrying a mismatched version of the DNMT1 shRNA, an empty vector, or cells that were untreated. Effects of DNMT1 shRNA on (A) DNMT1 steady-state mRNA levels during in vitro differentiation, (B) DNMT1 protein levels as determined by Western blot, (C) γ-globin promoter DNA methylation, (D) LINE element DNA methylation, (E) γ-globin steady-state mRNA, and (F) Hb levels as determined by HPLC at the end of differentiation. CD34+ cells from a single healthy donor were used for this experiment. Error bars represent 1 SD. P values were determined by t test.

Article Snippet: 17 DNMT1 siRNA and shRNA siRNA for DNMT1 or a fluorescein-labeled control siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) were transfected into cells using HiPerFect reagent (Qiagen, Valencia, CA) following the manufacturer's suggested protocol for transfection of suspension cells on day +11 of the standard culture procedure.

Techniques: shRNA, Knockdown, Methylation, Plasmid Preparation, Transduction, Expressing, In Vitro, Western Blot, DNA Methylation Assay