heterozygous b6 dnmt3a knockout (ko) mice  (Jackson Laboratory)

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: 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