dnmt3a knockout  (Santa Cruz Biotechnology)

Journal: Nucleic Acids Research

Article Title: DNMT3A-dependent DNA methylation shapes the endothelial enhancer landscape

doi: 10.1093/nar/gkaf435

Figure Lengend Snippet: Context-dependent impact of DNA methylation on chromatin accessibility. ( A ) Differential chromatin accessibility in the TSS of genes showing a methylation–transcription correlation in EC inf versus EC yAdu (panel 1) and in EC inf versus EC mAdu (panel 2). The enrichment plot displays the binding motives for DNMT3A (panel 3) and DNMT3B (panel 4). n = 3 for each age group. ( B ) qPCR of pulmonary ECs isolated from infant and young adult mice showing the expression of DNA modifiers. n = 6 per age group. The data are presented as the means ± SDs. P -values were determined by the Mann−Whitney test. *** P <0.001, **** P <0.0001. ( C ) Representative images of infant and young adult lung sections stained for CD31 (vascular surface), DNMT3A (DNA methyltransferase), and Hoechst (nucleus). Scale bar: 20 μm.

Article Snippet: C57BL/6N female mice were obtained from Janvier Labs. Heterozygous B6 Dnmt3a knockout (KO) mice were initially obtained from Jackson Laboratory (#018838) to generate homozygous Dnmt3a KO mice.

Techniques: DNA Methylation Assay, Methylation, Binding Assay, Isolation, Expressing, MANN-WHITNEY, Staining

Journal: Nucleic Acids Research

Article Title: DNMT3A-dependent DNA methylation shapes the endothelial enhancer landscape

doi: 10.1093/nar/gkaf435

Figure Lengend Snippet: Loss of DNMT3A-dependent DNA methylation results in mild gene deregulation. ( A ) Schematic overview showing the sample collection of pulmonary ECs isolated from Dnmt3a WT and Dnmt3a KO infant mice. FACS-sorted pulmonary ECs were further used for methylome, transcriptome, chromatin accessibility, and histone landscape analysis. The scheme was created in BioRender. Augustin, H. (2025) https://BioRender.com/k83b042 . ( B ) Pie diagram displaying the relative number of DMRs upon the loss of Dnmt3a located distal (distance > 5 kb) or local (distance ≤ 5 kb) to the nearest TSS. ( C ) Genomic enrichment of distal DNMT3A-dependent DMRs. ( D ) Genomic enrichment of local DNMT3A-dependent DMRs. ( E ) Heatmap showing DNMT3A-dependent DMRs (left) and 258 associated genes (middle) that were differentially expressed (adjusted P -value <0.05) in Dnmt3a KO ECs compared to WT ECs. The distance of the DMRs to the gene TSS is shown on the right as log 10 () in bp. For both, methylome and transcriptome analyses, three biological replicates were used for each genotype. ( F ) Gene ontology analysis based on the biological functions of 258 differentially expressed genes (DEGs) that were associated with DNMT3A-dependent DNA methylation changes. A heatmap (left) displays the clustering of GO terms based on similarity (range: 0–0.8). The letter size represents the significance of the GO term (right). ( G ) Schematic representation of the sprouting assay. Control and shDNMT3A cell lines were analyzed under baseline conditions and angiogenic stimulation with recombinant human VEGF (rhVEGF) supplementation. The scheme was created in BioRender. Augustin, H. (2025) https://BioRender.com/k83b042 . ( H ) Quantification of DNMT3A knockdown efficacy in HUVECs following lentiviral transduction with shDNMT3A (sh#1, sh#2) or control shRNA ( nsh ). Data represent n = 3 biological replicates per cell line and are presented as mean ± SD. Statistical significance was assessed using an unpaired t -test. ** P <0.01. ( I ) Representative images of spheroids formed by HUVECs transduced with control shRNA or shDNMT3A . The spheroid assay was conducted under baseline conditions or upon angiogenic induction with rhVEGF. ( J ) Quantification of the number of sprouts in spheroids formed by HUVECs transduced with control shRNA or shDNMT3A constructs. Data represent n = 3 biological replicates per cell line and are presented as the mean ± SD. Statistical significance was determined using an unpaired t -test. * P <0.05. ( J ) Quantification of the average sprout length, expressed as fold change, in HUVECs transduced with control shRNA or shDNMT3A constructs. Data represent n = 3 biological replicates per cell line and are presented as the mean ± SD. Statistical significance was determined using an unpaired t -test. * P <0.05.

Article Snippet: C57BL/6N female mice were obtained from Janvier Labs. Heterozygous B6 Dnmt3a knockout (KO) mice were initially obtained from Jackson Laboratory (#018838) to generate homozygous Dnmt3a KO mice.

Techniques: DNA Methylation Assay, Isolation, Control, Recombinant, Knockdown, Transduction, shRNA, Construct

Journal: Nucleic Acids Research

Article Title: DNMT3A-dependent DNA methylation shapes the endothelial enhancer landscape

doi: 10.1093/nar/gkaf435

Figure Lengend Snippet: The absence of DNMT3A-dependent DNA methylation results in enhancer loss. ( A ) Bar plot showing the number of identified regulatory elements and silent chromatin in Dnmt3a WT and KO pulmonary ECs. ( B ) Circos plot showing the transition of all regulatory regions identified in Dnmt3a WT pulmonary ECs compared with Dnmt3a KO ECs. Regions are depicted in bp. ( C ) Focused circos plot of identified active enhancers in Dnmt3a WT pulmonary ECs (23,267,740 bp) and their change into other CRE categories in Dnmt3a KO ECs. This plot only focuses on active enhancers in WT ECs and does not include newly established active enhancers in KO ECs, which were gained due to conversions from non-assigned regions, among others, in WT ECs. Regions are depicted in bp. ( D ) Gene ontology analysis based on lost active enhancer regions in Dnmt3a WT compared to Dnmt3a KO ECs. GO terms were based on similarity, ranging from 0 to 0.8 (panel 3). The letter size represents the significance of the GO term (right). ( E ) Gene ontology analysis based on consistent active enhancer regions identified in both Dnmt3a WT and Dnmt3a KO ECs. GO terms were based on similarity, ranging from 0 to 0.8 (panel 3). The letter size represents the significance of the GO term (right).

Article Snippet: C57BL/6N female mice were obtained from Janvier Labs. Heterozygous B6 Dnmt3a knockout (KO) mice were initially obtained from Jackson Laboratory (#018838) to generate homozygous Dnmt3a KO mice.

Techniques: DNA Methylation Assay

Journal: medRxiv

Article Title: Germline and Somatic Mutations in DNA Methyltransferase 3A (DNMT3A) Predispose to Pulmonary Arterial Hypertension (PAH) in Humans and Mice: Implications for Associated PAH

doi: 10.1101/2023.12.30.23300391

Figure Lengend Snippet: A) DNMT3A mutation rates are shown for the gnomAD and SPARK control databases individually, and for the SPARK and gnomAD databases combined. Mutation rates are also shown for PAH, IPAH and NON-IPAH patients. Mutation rate is shown on the y-axis of the graph (in %) and is displayed for the 3 control cohorts and 3 PAH cohorts . B) Fisher’s exact test was used to compare the mutation rate between PAH, SPARK and gnomAD databases. Fisher’s exact test p-values as well as odds ratios and the associated confidence intervals (95% confidence) are shown for several PAH vs control comparisons. Mutation rates were significantly increased in the PAH Biobank (1%) compared to both SPARK (0.33%; p=<0.0001) and gnomAD (=> 0.43%; p = 0.0002) controls. Mutation rates were also significantly increased in both total PAH patients and non-IPAH patients (1.64%) compared to both control databases combined (0.42%, p=0.0003 and 0.0002, respectively). There was no significant difference between the SPARK and gnomAD control databases (p=0.1133). C) The age groups of the cohorts were more comparable between gnomAD and PAH databases. % of the cohort (y-axis) at each age range (x-axis) is shown for the PAH Biobank as well as both control databases.

Article Snippet: We produced male and female conditional hematopoietic Dnmt3a knockout mice by crossing parental floxed and Vav-iCre mice as described by the Jackson Laboratory ( https://www.jax.org/strain/008610 ) and by Joseph et al. 3334 This knockout simulates the ‘loss of function’ effects of DNMT3A somatic mutations seen in patients with PAH in our U.S. PAH Biobank data.

Techniques: Mutagenesis

Journal: medRxiv

Article Title: Germline and Somatic Mutations in DNA Methyltransferase 3A (DNMT3A) Predispose to Pulmonary Arterial Hypertension (PAH) in Humans and Mice: Implications for Associated PAH

doi: 10.1101/2023.12.30.23300391

Figure Lengend Snippet:

Article Snippet: We produced male and female conditional hematopoietic Dnmt3a knockout mice by crossing parental floxed and Vav-iCre mice as described by the Jackson Laboratory ( https://www.jax.org/strain/008610 ) and by Joseph et al. 3334 This knockout simulates the ‘loss of function’ effects of DNMT3A somatic mutations seen in patients with PAH in our U.S. PAH Biobank data.

Techniques:

Journal: medRxiv

Article Title: Germline and Somatic Mutations in DNA Methyltransferase 3A (DNMT3A) Predispose to Pulmonary Arterial Hypertension (PAH) in Humans and Mice: Implications for Associated PAH

doi: 10.1101/2023.12.30.23300391

Figure Lengend Snippet: A) Data was obtained using deep, targeted panel sequencing. The percentage of individuals with CHIP is shown for PAH patients (n=710) and controls (n=3645). Approximately 15% of PAH patients possessed a CHIP mutation while only 7% of controls were found to have CHIP. B) The number of patients with CHIP mutations in each gene is displayed. Number of patients is shown on the y-axis of the graph while genes are shown on the x-axis. DNMT3A was the most mutated gene (red; 49 patients), followed by TET2 (green, 18 patients). All other mutated genes are shown in purple. The pie chart shows the mutation distribution using the same colours and displays the number of patients with no CHIP mutations in blue. C) The prevalence of all CHIP mutations, DNMT3A CHIP and non- DNMT3A CHIP is shown. 104/710 PAH patients were found to have CHIP mutations (49 being in DNMT3A , and 55 in other genes). The odds ratios, with 95% confidence intervals, are also shown for PAH patients with all CHIP mutations, DNMT3A CHIP and non- DNMT3A CHIP compared to controls. The odds ratios for all CHIP mutations and non- DNMT3A CHIP mutations were statistically significant (p=0.0071, p=0.002, respectively), though DNMT3A CHIP alone did not reach statistical significance (p=0.40).

Article Snippet: We produced male and female conditional hematopoietic Dnmt3a knockout mice by crossing parental floxed and Vav-iCre mice as described by the Jackson Laboratory ( https://www.jax.org/strain/008610 ) and by Joseph et al. 3334 This knockout simulates the ‘loss of function’ effects of DNMT3A somatic mutations seen in patients with PAH in our U.S. PAH Biobank data.

Techniques: Sequencing, Mutagenesis

Journal: medRxiv

Article Title: Germline and Somatic Mutations in DNA Methyltransferase 3A (DNMT3A) Predispose to Pulmonary Arterial Hypertension (PAH) in Humans and Mice: Implications for Associated PAH

doi: 10.1101/2023.12.30.23300391

Figure Lengend Snippet: A) The three DNMT3A transcript variants 2 (NM_153759.3), 3 (NM_022552), and 4 (NM_175630.1), which code for the three protein isoforms DNMT3A2, DNMT3A3, and DNMT3A4, respectively, are shown, in comparison to the original DNMT3A transcript variant 1. B-D) Gene expression of the DNMT3A isoforms 2,3 and 4 was measured in PBMCs. Decreased expression of variant 4 in 88% of SSc-PAH and 93.3% of IPAH patients (IPAH 0.77; SSc-PAH 0.8; p<0.0001), as well as decreased expression of variant 3 in 80% of SSc-PAH and 73.3% of IPAH patients compared to healthy individuals (IPAH 0.92; SSc-PAH 0.93; p<0.05) was found. The gene expression of variant 2 was unaffected. E-G) Receiver operating characteristic (ROC) analysis on a cohort consisting of 41 healthy controls and 80 PAH patients (IPAH/SSc-PAH) was performed for DNMT3A isoforms 2, 3 and 4. Sensitivity % is shown on the y-axis while the x-axis represents 100% subtract the Specificity %. The results indicate that DNMT3A variant 4 (AUC: 0.82; p<0.0001) and variant 3 (AUC: 0.67; p<0.002) could serve as potential predictors of PAH.

Article Snippet: We produced male and female conditional hematopoietic Dnmt3a knockout mice by crossing parental floxed and Vav-iCre mice as described by the Jackson Laboratory ( https://www.jax.org/strain/008610 ) and by Joseph et al. 3334 This knockout simulates the ‘loss of function’ effects of DNMT3A somatic mutations seen in patients with PAH in our U.S. PAH Biobank data.

Techniques: Comparison, Variant Assay, Expressing

Journal: medRxiv

Article Title: Germline and Somatic Mutations in DNA Methyltransferase 3A (DNMT3A) Predispose to Pulmonary Arterial Hypertension (PAH) in Humans and Mice: Implications for Associated PAH

doi: 10.1101/2023.12.30.23300391

Figure Lengend Snippet: A) Hematopoietic Dnmt3a -knockout mice spontaneously develop PAH by 9 months of age (n=4-5/group). Right ventricular systolic pressure (RVSP; in mmHg) and mean pulmonary arterial pressure (mPAP; in mmHg) are significantly increased in Dnmt3a -knockout mice (green) compared to controls (white; p=0.0004, p=0.0004, respectively). Pulmonary artery acceleration time (PAAT; in ms), tricuspid annular plane systolic excursion (TAPSE; in mm) and cardiac output (CO; in ul/min) are significantly reduced in Dnmt3a -knockout mice compared to controls (p=0.0003, p=0.012; p=0.006, respectively). Right, ventricular end-diastolic pressure (RVEDP; in mmHg) shows a trend towards being increased in Dnmt3a -knockout mice, though not statistically significant. Right-heart catheterization (RHC) traces of RVSP are shown on the left for both control (top) and knockout (bottom) mice. RVSP, mPAP, and RVEDP were obtained via RHC, while PAAT, TAPSE and CO were obtained via echocardiography. B) Hematopoietic Dnmt3a -knockout mice develop PAH, accelerated by a second hit hypoxia (n=6-9/group). 3-month-old control (white boxes) and Dnmt3a -knockout (orange boxes) mice were either maintained in normoxic (solid colour boxes) conditions for 6 weeks or were exposed to 3 weeks of hypoxia followed by 3 weeks of normoxia (boxes with bricks). Representative RVSP traces obtained via RHC are shown for each group. RVSP, mPAP and RVEDP (trending) are elevated in Dnmt3a -knockout mice that underwent a second hit (hypoxia) compared to hypoxic controls (p=0.0004, p=0.0007, p=0.0724, respectively), and RVSP, mPAP and RVEDP are also significantly increased in Dnmt3a -knockout mice that were kept in normoxia compared to the normoxic controls (p=0.0015, p=0.0015, p=0.0234, respectively). PAAT and TAPSE are significantly reduced in Dnmt3a -knockout mice that underwent a second hit (hypoxia) compared to controls (p=0.0020, p=0.0222, respectively) and are also significantly increased in Dnmt3a -knockout mice that were kept in normoxia, compared to the normoxic controls (PAAT: p=0.0030; TAPSE: p=0.0001). CO was significantly reduced in hypoxic Dnmt3a -knockout mice compared to controls (p=0.0101), while normoxic Dnmt3a -knockout mice showed a trend towards being reduced, though not statistically significant (p=0.2061). * = p< 0.05. Treatment of hypoxic Dnmt3a -knockout mice with Canakinumab (yellow bars with bricks) improved hemodynamic measurements and cardiac function (n=3; RVSP: p=0.0362, mPAP: p=0.0449, PAAT: p=0.0258, TAPSE: p=0.0533, CO: p=0.0299). RVEDP was not significantly reduced (p=0.4629).

Article Snippet: We produced male and female conditional hematopoietic Dnmt3a knockout mice by crossing parental floxed and Vav-iCre mice as described by the Jackson Laboratory ( https://www.jax.org/strain/008610 ) and by Joseph et al. 3334 This knockout simulates the ‘loss of function’ effects of DNMT3A somatic mutations seen in patients with PAH in our U.S. PAH Biobank data.

Techniques: Knock-Out

Journal: medRxiv

Article Title: Germline and Somatic Mutations in DNA Methyltransferase 3A (DNMT3A) Predispose to Pulmonary Arterial Hypertension (PAH) in Humans and Mice: Implications for Associated PAH

doi: 10.1101/2023.12.30.23300391

Figure Lengend Snippet: Histological assessment via hematoxylin and eosin (H&E) staining was performed on lung tissue slides and small pulmonary arteries were identified. Pulmonary medial wall thickness was compared between control and knockout mice. A) Pulmonary vascular remodelling occurs in the lungs of hematopoietic Dnmt3a -knockout mice by 9 months of age. There is a significant increase in the pulmonary medial wall thickness, defined as the % of the wall consisting of tunica media, in hematopoietic Dnmt3a -knockout mice (green) compared to controls (white; p=0.0005; n=5/group). A t-test was used to assess the difference between control and knockout mice. (B) There is an increase in pulmonary medial wall thickness in the lungs of hematopoietic Dnmt3a -knockout mice exposed to a second-hit hypoxia compared to controls (p<0.0001; n=3/group). 4.5-month-old Dnmt3a -knockout mice also spontaneously developed pulmonary vascular remodeling (p<0.0001; n=3/group). Bars with bricks represent hypoxic exposure while solid bars represent normoxia only. White represents control mice and knockout mice values are shown in orange. (C) Collagen deposition assessment of RV tissue using Picrosirius red staining (n=3/ group) demonstrated increased collagen deposition in the RV tissue of both hypoxic and normoxic Dnmt3a -knockout mice compared to their respective controls.

Article Snippet: We produced male and female conditional hematopoietic Dnmt3a knockout mice by crossing parental floxed and Vav-iCre mice as described by the Jackson Laboratory ( https://www.jax.org/strain/008610 ) and by Joseph et al. 3334 This knockout simulates the ‘loss of function’ effects of DNMT3A somatic mutations seen in patients with PAH in our U.S. PAH Biobank data.

Techniques: Staining, Knock-Out

Journal: medRxiv

Article Title: Germline and Somatic Mutations in DNA Methyltransferase 3A (DNMT3A) Predispose to Pulmonary Arterial Hypertension (PAH) in Humans and Mice: Implications for Associated PAH

doi: 10.1101/2023.12.30.23300391

Figure Lengend Snippet: Immunofluorescence and confocal microscopy were used to measure leukocyte infiltration in the lungs of Dnmt3a -knockout mice with PAH compared to controls. CD45, a marker of leukocytes, is shown in green. DAPI, a nuclei stain, is shown in blue. (A) There is a trend towards an increase in the number of CD45+ cells in the lungs of nine-month-old knockout mice (green), that have developed PAH, compared to controls (white; n=2/group). The graph represents the number of CD45+ cells per 0.02 mm . (B) There is a significant increase in the number of CD45+ cells in the lungs of the knockout mice, that have developed PAH, compared to controls (n=5/group, p=0.0473). Hypoxia appears to exacerbate leukocyte infiltration of the lungs, though there was no significant difference between knockout and control mice in hypoxia. Bars with bricks represent 3 weeks of hypoxic exposure with 3 weeks of normoxic exposure while solid bars represent 6 weeks of normoxia. White bars represent control mice and orange bars represent Dnmt3a -knockout mice. (C) Flow cytometry was used to investigate the subpopulations of leukocytes seen on confocal microscopy. Following a live/dead stain, CD45+ cells (leukocytes) were gated for and were found to be increased on 4.5-month-old Dnmt3a -knockout mice compared to controls (n=5-8/group; p=0.05). Specifically, macrophages are increased in Dnmt3a -knockout mice (n=5-8/group; p=<0.01). An LY6G antibody was used for neutrophil detection, an F4/80 antibody was used for macrophage detection, a CD3 antibody was used for T-cell detection, and a CD19 antibody was used for B-cell detection. On average, approximately 90% of CD45+ cells in the control mice were macrophages, while this was increased to approximately 95% in the Dnmt3a -knockout mice. D) IL-13 was found to be increased in the plasma of Dnmt3a -knockout mice compared to controls (p=0.05). There was also a trend towards an increase in plasma G-CSF in the Dnmt3a -knockout mice compared to controls (p=0.16). Plasma cytokine concentration is shown on the y-axis in picogram per milliliter (pg/ml).

Article Snippet: We produced male and female conditional hematopoietic Dnmt3a knockout mice by crossing parental floxed and Vav-iCre mice as described by the Jackson Laboratory ( https://www.jax.org/strain/008610 ) and by Joseph et al. 3334 This knockout simulates the ‘loss of function’ effects of DNMT3A somatic mutations seen in patients with PAH in our U.S. PAH Biobank data.

Techniques: Immunofluorescence, Confocal Microscopy, Knock-Out, Marker, Staining, Flow Cytometry, Concentration Assay

Journal: Cancers

Article Title: DNMT3A/ miR-129-2-5p /Rac1 Is an Effector Pathway for SNHG1 to Drive Stem-Cell-like and Invasive Behaviors of Advanced Bladder Cancer Cells.

doi: 10.3390/cancers14174159

Figure Lengend Snippet: Figure 4. SNHG1 bound to DNMT3A protein and promoted the interaction of DNMT3A with the miR-129-2 promoter, thereby hyper-methylating and inhibiting the transcription of miR-129-2. (A) T24T(Vector) and T24T(SNHG1) cells were incubated with Act-D for 0, 10, and 20h. Total RNA

Article Snippet: The DNMT3A knockout plasmid and the miR-129-2 expression plasmid were obtained from Addgene.

Techniques: Plasmid Preparation, Incubation

Journal: Cancers

Article Title: DNMT3A/ miR-129-2-5p /Rac1 Is an Effector Pathway for SNHG1 to Drive Stem-Cell-like and Invasive Behaviors of Advanced Bladder Cancer Cells.

doi: 10.3390/cancers14174159

Figure Lengend Snippet: Figure 5. The binding of SNHG1 to DNMT3A protein inhibited miR-129-5p expression and increased Rac1 expression in UMUC3 cells. (A, Left panel) The total protein extracts from indicated cells were subjected to Western blotting to determine the expression of DNMT1, DNMT3A, and DNMT3B. (A, Right panel) Relative protein levels determined by densitometry and expressed as ratios versus β-Actin. (B) An RNA pull-down assay was employed to determine the interaction between SNHG1 and DNMT3A. Antisense RNA sequences of SNHG1 were used as negative controls (NC). (C, Left

Article Snippet: The DNMT3A knockout plasmid and the miR-129-2 expression plasmid were obtained from Addgene.

Techniques: Binding Assay, Expressing, Western Blot, Pull Down Assay

Journal: Cancers

Article Title: DNMT3A/ miR-129-2-5p /Rac1 Is an Effector Pathway for SNHG1 to Drive Stem-Cell-like and Invasive Behaviors of Advanced Bladder Cancer Cells.

doi: 10.3390/cancers14174159

Figure Lengend Snippet: Figure 6. The sphere-forming T24T cells with high expression of SNHG1 and Rac1 and low expression of miR-129-5p were markedly more invasive than the non-sphere-forming T24T cells. (A,B) The isolated sphered T24T cells were subjected to transwell migration and invasion assays, and the results are presented relative to their parental T24T cells. (C–E) The parental T24T and sphered T24T cells were collected, and the proteins were extracted and subjected to either Western blotting to assess the expression of Rac1 (C) or real-time PCR to determine the expression of SNHG1 (D) and miR-129-5p levels (E). The results are presented as means ± SD from triplicate experiments, and asterisks (*) indicate a significant difference (p < 0.01). (F) A schematic model of the SNHG1/DNMT3A/miR- 129-5p/Rac1 signaling pathway in regulating the stem-cell-like and invasive properties of advanced bladder cancer cells. Bars in (A) are equal to 100 µm.

Article Snippet: The DNMT3A knockout plasmid and the miR-129-2 expression plasmid were obtained from Addgene.

Techniques: Expressing, Isolation, Migration, Western Blot, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Endothelial Dnmt3a controls placenta vascularization and function to support fetal growth

doi: 10.1101/2022.07.28.501807

Figure Lengend Snippet: A UMAP of healthy human placenta single-cell data with cell type annotation based on marker gene expression. Endothelial cells are encircled in black. B DNMT3A and DNMT3B expression in different cell types of healthy human placenta. Cell types are sorted descending according to the percentage of cells expressing DNMT3A . C Volcano plot visualizing the gene expression changes (717 genes total) between diseased (= preeclampsia) and healthy placenta EC (red dots indicate significantly differentially expressed genes). Cutoff for the p-value (10e -6 ) is indicated with a dashed horizontal line; fold change cutoff (2) is indicated with dashed vertical lines. D Top decreased functional categories attributed to the genes differentially expressed in diseased vs. healthy placenta EC (Ingenuity Pathway Analysis, QIAGEN). Adj. p-value <0.05. E Scored expression of DNA methylation writers ( DNMTs ) and editors ( TETs ) in EC of normal and diseased placenta. F Expression of de novo DNA methyltransferases in EC of normal and diseased placenta. G Co-staining of CD31 and DNMT3A on healthy human placenta tissue (chorionic villus zone). Scale bar 50µm.

Article Snippet: Heterozygous Dnmt3a knockout mice were obtained from Jackson Laboratory (# No. 018838) to generate homozygous Dnmt3a knockout mice (approved by the Competent Authorities in Karlsruhe, Germany; permit G-82/19).

Techniques: Marker, Gene Expression, Expressing, Functional Assay, DNA Methylation Assay, Staining

Journal: bioRxiv

Article Title: Endothelial Dnmt3a controls placenta vascularization and function to support fetal growth

doi: 10.1101/2022.07.28.501807

Figure Lengend Snippet: A Dnmt expression kinetics covering E12.5 to E16.5 in total mouse placenta tissue normalized to Actb expression. Dnmt3a (highlighted in red) is significantly upregulated during placenta maturation. n=5-6. B Heatmap depicting the expression of pro-proliferative genes in total placenta tissue. Upon placenta maturation, pro-proliferative genes are downregulated. Data is row normalized. n=5-6. C Co-staining of DNMT3A, CD31 (EC marker) and DAPI (nuclear marker) in the murine mature E16.5 placenta. DNMT3A is detected in the cell nucleus of EC. Placental zones are separated with yellow dashed lines. M: maternal, J: junctional; L: labyrinth. left: Scale bar 200µm. right: Zoom-in of the yellow boxed region. Scale bar 20µm. D Representative image of 5-methylcytosine (5mC) staining in E14.5 placenta tissue. Scale bar 200µm. E Quantification of 5mC intensity in different placental regions. n=4. F Scored expression of Dnmts and Tets in maternal and fetal EC extracted from mouse placenta single-nuclei RNA-seq . G Expression of de novo DNA methyltransferases in fetal and maternal EC. H Gene expression analysis of EC isolated from the decidua or the labyrinth of E16.5 wildtype placenta. n=6. Shown are mean±SD. Statistical significance was measured by Mann-Whitney test (A, H) or unpaired t-test (E). * p<0.05, ** p<0.01.

Article Snippet: Heterozygous Dnmt3a knockout mice were obtained from Jackson Laboratory (# No. 018838) to generate homozygous Dnmt3a knockout mice (approved by the Competent Authorities in Karlsruhe, Germany; permit G-82/19).

Techniques: Expressing, Staining, Marker, RNA Sequencing, Gene Expression, Isolation, MANN-WHITNEY

Journal: bioRxiv

Article Title: Endothelial Dnmt3a controls placenta vascularization and function to support fetal growth

doi: 10.1101/2022.07.28.501807

Figure Lengend Snippet: A Embryo genotype frequency at E16.5 from Dnmt3a +/- mice crossing showing a significant deviation from the expected Mendelian ratio (Chi-square test, χ 2 =9.058, p=0.011). n=138 embryos from 17 pregnant mice. B Representative images of Dnmt3a +/+ and Dnmt3a -/- E16.5 embryos. Scale bar 0.5cm. C Quantification of Dnmt3a +/+ and Dnmt3a -/- embryo size at E16.5. Normalized to wildtype and per litter. n≥7. D Quantification of Dnmt3a +/+ and Dnmt3a -/- embryo weight at E16.5. Normalized to wildtype and per litter. n≥7. E Representative images of E16.5 Dnmt3a +/+ and Dnmt3a -/- placentas stained for the EC marker CD31. Measurement of the labyrinth outgrowth is indicated with white bars. Scale bar 1000µm. F Quantification of labyrinth outgrowth in Dnmt3a +/+ and Dnmt3a -/- E16.5 placentas. n≥7. G Quantification of embryo weight at E16.5 of Dnmt3a fl/fl mice expressing Cdh5 -Cre (Cre+ considered as Dnmt3a KO in EC) or not (Cre- considered as Dnmt3a WT in EC). Normalized to Cre- embryos and per litter. n≥6. H Quantification of labyrinth outgrowth of Cre- and Cre+ Dnmt3a fl/fl mice. n≥12. Shown are mean±SD. Statistical significance was measured by Mann-Whitney test (C, D, F, G, H). * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Heterozygous Dnmt3a knockout mice were obtained from Jackson Laboratory (# No. 018838) to generate homozygous Dnmt3a knockout mice (approved by the Competent Authorities in Karlsruhe, Germany; permit G-82/19).

Techniques: Staining, Marker, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Endothelial Dnmt3a controls placenta vascularization and function to support fetal growth

doi: 10.1101/2022.07.28.501807

Figure Lengend Snippet: A Representative images of postnatal day 7 (P7) retinas of Dnmt3a +/+ and Dnmt3a -/- neonates. Vessels are stained with isolectin B4. Scale bar 1mm. B Quantification of the vascularized area per retina area relative to the wildtype control. n≥7. C Representative images of the vascular front of P7 retinas. Proliferating cells are stained by phospho-histone H3-Ser10. Scale bar 100µm. D Quantification of phospho-H3-Ser10-positive EC at the vascular front. Normalized to wildtype control. n≥5. E Representative images of the vascular front of P7 retinas. Scale bar 100µm. F Quantification of tip EC per leave. Normalized to wildtype control. n≥5. G Representative images of P7 retinas of Dnmt3a WT and Dnmt3a iECKO neonates. Vessels are stained with isolectin B4. Scale bar 1 mm. H Quantification of the vascularized area per retina area relative to wildtype control. n≥4. Shown are mean±SD. Statistical significance was measured by Mann-Whitney test (B, D, F, H). ** p<0.01, *** p<0.001.

Article Snippet: Heterozygous Dnmt3a knockout mice were obtained from Jackson Laboratory (# No. 018838) to generate homozygous Dnmt3a knockout mice (approved by the Competent Authorities in Karlsruhe, Germany; permit G-82/19).

Techniques: Staining, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Endothelial Dnmt3a controls placenta vascularization and function to support fetal growth

doi: 10.1101/2022.07.28.501807

Figure Lengend Snippet: A Global DNA methylation distribution as measured by Tagmentation-based WGBS of Dnmt3a +/+ and Dnmt3a -/- lung EC. n=3. B Heat map of differentially methylated regions (DMRs) (K-means clustering, left) expanded by genomic feature annotation (right). C Genomic features assigned to DMRs (cutoff: 20% methylation difference) upon loss of Dnmt3a compared to the genomic distribution. D Functional enrichment analysis (GREAT) of DMRs using HALLMARK gene sets. E Genome browser view of gene loci encoding for Nrp1 and Jag1 depicting the DNA methylation level and the DMR location.

Article Snippet: Heterozygous Dnmt3a knockout mice were obtained from Jackson Laboratory (# No. 018838) to generate homozygous Dnmt3a knockout mice (approved by the Competent Authorities in Karlsruhe, Germany; permit G-82/19).

Techniques: DNA Methylation Assay, Methylation, Functional Assay

Journal: bioRxiv

Article Title: Endothelial Dnmt3a controls placenta vascularization and function to support fetal growth

doi: 10.1101/2022.07.28.501807

Figure Lengend Snippet: A Relative expression of genes associated with preeclampsia in the murine E16.5 placenta upon loss of Dnmt3a vs. wildtype control. n=7. B Relative expression of genes associated with PE in HUVEC upon transfection with sh Dnmt3a (sh#1, sh#2) or control shRNA (nsh), respectively. n=3. C Representative images of E16.5 Dnmt3a +/+ and Dnmt3a -/- placentas stained for CD31 and LYVE1. Scale bar 200 µm. M=maternal, J= junctional, L=labyrinth compartment. D Quantification of LYVE1 in the CD31-positive labyrinth area. n=8. E Organ to body weight ratio measured in Dnmt3a +/+ and Dnmt3a -/- mice at P9. n≥5. F Analysis of lung tissue at P9 by board-certified pathologist. “non-patho” = no pathological changes, “patho” = immune infiltrates and/or fibrosis. n≥4. Shown are mean±SD. Statistical significance was measured by Mann-Whitney test (A, D, E) or unpaired t-test (B). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: Heterozygous Dnmt3a knockout mice were obtained from Jackson Laboratory (# No. 018838) to generate homozygous Dnmt3a knockout mice (approved by the Competent Authorities in Karlsruhe, Germany; permit G-82/19).

Techniques: Expressing, Control, Transfection, shRNA, Staining, MANN-WHITNEY

Journal: Toxicology research

Article Title: Epigenetic Toxicity of Trichloroethylene: A Single-Molecule Perspective

doi: 10.1039/C5TX00454C

Figure Lengend Snippet: Table 3

Article Snippet: Dnmt3a knockout with CRISPR/Cas9 Endogenous Dnmt3a in HeLa cells was knocked out with Dnmt3a CRISPR/Cas9 KO plasmids (sc-400323,) and UltraCruz transfection reagent (sc-395739, Santa Cruz Biotechnology).

Techniques: Diffusion-based Assay