Fig 1: Mice expressing catalytically inactive Dnmt3b develop ICF-like phenotype.a. (left) Schematic representation of mouse Dnmt3b protein structure. Regulatory domain containing PWWP and ATRX domains and catalytic domain with methyltransferase motifs (I, IV, VI, IX and X) are depicted (right). Nucleotide and amino acid sequences of exon 19 in wild-type (WT) and Dnmt3bCI (CI) allele. Amino acid substitution (P656V and C657D) in CI allele results in inactivation of the CA [13]. Position of genotyping primers is indicated by arrows. F1-R detects wild-type sequence; F2-R detects mutated sequence. b. Micrograph of 6-week-old Dnmt3b+/+ (+/+) and Dnmt3bCI/CI (CI/CI) mice. c. Body weight values of Dnmt3b+/+ and Dnmt3bCI/CI mice at indicated ages (n = 10). p<0.05 by two-tailed Student's t-test. d. Immunoblot of Dnmt1, Dnmt3a and Dnmt3b expression in spleen and thymus of 8-weeks old Dnmt3b+/+ and Dnmt3bCI/CI mice. Dnmt3a−/−;Dnmt3b−/− mouse T-cell lymphoma line (DKO) served as negative control. Hsc70 served as a loading control. e. Gross morphology of skulls of Dnmt3b+/+ and Dnmt3bCI/CI mice. Scale bar represents 2 mm. f. Dimensions of skulls of Dnmt3b+/+ (blue, n = 3) and Dnmt3bCI/CI (green, n = 4) mice. Orientation of α, β, γ and δ dimensions is depicted by orange lines on the image in (e). *p = 0.005 for α; p = 0.003 for β. g. Percentage of 5-methyl-deoxy-cytidines (5mdCs) in DNA of FACS-sorted splenic B-cells (CD19+) and thymocytes (CD4+CD8+) isolated from Dnmt3b+/+ (blue, n = 3) and Dnmt3bCI/CI (green, n = 3) mice as determined by mass spectrometry. *p<0.01. h. Weights of spleen (sp) and thymus (th) of Dnmt3b+/+ (blue, n = 23) and Dnmt3bCI/CI (green, n = 24) mice and normalized to body weight. *p = 0.024. i. Absolute cell counts in spleen (n = 8), thymus (n = 7) and bone marrow (BM, n = 8) in Dnmt3b+/+ (blue) and Dnmt3bCI/CI (green) mice. *p<0.05. j. Cell counts normalized to spleen weight of B-cells (CD19+B220+), mature T-cells (CD4+CD3+ and CD8+CD3+) and myeloid cells (CD11b+CD5-) in spleen of Dnmt3b+/+ (blue) and Dnmt3bCI/CI (green) mice (n = 6). *p<0.05. k. Percentage of total B-cells in spleen of FVB recipient mice reconstituted with BM of Dnmt3b+/+ (blue) and Dnmt3bCI/CI (green) mice (n = 5). *p = 0.0029.All the data in figure are presented as mean ± SEM, p-value was calculated by two-tailed Student's t-test.
Fig 2: MKL-1 affects the expression of DNMT1 and maspin. MDA-MB-231 and MCF-7 after serum starvation (DMEM + 0.5% FBS) for 48 h were transfected with si-control (si-NC) or si-DNMT1 for 48 h. (A–D) The effect of MKL-1 overexpression or knock down in MDA-MB-231 and MCF-7 were detected by Western blot analysis. All experiments were repeated three times in duplicate, and the semiquantitative analysis was calculated by SPSS statistical software. *p < 0.05, **p < 0.01. (E–J) The expression of maspin and DNMT1 in MDA-MB-231 and MCF-7 transfected by MKL-1 overexpression plasmid was detected by Western blot analysis. All experiments were repeated three times in duplicate, and the semiquantitative analysis was calculated by SPSS statistical software. **p < 0.01. (K–P) The expression of maspin and DNMT1 in MDA-MB-231 and MCF-7 transfected by siMKL-1 was detected by Western blot analysis. All experiments were repeated three times in duplicate, and the semiquantitative analysis was calculated by SPSS statistical software. *p < 0.05, **p < 0.01. Data are expressed as mean ± standard deviation (SD). *p < 0.05, **p < 0.01.
Fig 3: DNMT suppresses WAT beiging and Adrb3 expression. a TonEBP, UCP-1 and Adrb3 mRNA levels in 3T3-L1 adipocytes treated with 0–500 μM RG108 as indicated for 24 h (n = 4). b–e Animals were treated with vehicle or RG108 every 2 days from day −4 to 24. All animals were switched from CD to HFD on day 0 (n = 5). Body weight (b), body composition by echo MRI (c), fat pad mass (iWAT and eWAT) (d), and fasting blood glucose levels (e) were measured. f–k Animals were treated as above, except that the experiments were terminated on day 8 (n = 4). VO2 (f), VCO2 (g), heat production (h), and rectal temperature (i) were measured. TonEBP, UCP-1, and Adrb3 mRNA (j) were measured, and H&E and immunostaining for UCP-1 and Adrb3 (k) were performed from iWAT. Scale bars, 100 μm. l TonEBP, UCP-1, and Adrb3 mRNA levels in 3T3-L1 adipocytes transfected with scr siRNA, DNMT1 (1), DNMT3a (3a) or DNMT3b (3b) siRNA (n = 4). m 3T3-L1 adipocytes transfected with siRNA were transfected a second time with WT (left) or ΔTonE (right) Adrb3 promoter-luciferase reporter. Luciferase activity was measured (n = 4). n represents number of independent experiments with triplicate (a, l, m) or biologically independent samples (b–j). All data are presented as mean + s.d. (a, l, m) or s.e.m. (e, g–m). AU arbitrary unit. The p-values are determined by unpaired t-test (c–e, j) or one-way ANOVA (a, b, f–i, l, m). *p < 0.05 vs. 0 (a), vehicle (b–j), or scr siRNA (l, m). Source data are provided as a Source Data file
Fig 4: Adipocyte-specific TonEBP deficiency promotes energy expenditure and beiging of WAT. a Body weight, fasting blood glucose, and food intake by adipocyte-specific TonEBP knockout mice (TonEBPfl/fl, AQ-cre) and their cre-negative littermates (TonEBP fl/fl) fed with HFD for 12 weeks (n = 10). VO2 (b), VCO2 (c), heat production (d), and rectal temperature (e) analyzed in animals fed with HFD (n = 4). mRNA expression of thermogenic genes in iWAT from animals fed with HFD (f) or exposed to cold conditions (g) (n = 4). h Representative images of iWAT sections stained with H&E, UCP-1, and Adrb3 antibody from CD-fed animals exposed to cold (4 °C). Scale bars, 100 μm. i Proposed model for the inhibition of beiging in white adipocytes by TonEBP. TonEBP is induced by excess calorie intake. It binds to the Adrb3 promoter where it recruits DNMT1 leading to DNA methylation and suppression of the promoter. n represents number of biologically independent animals (a–e) or samples (f, g). All data are presented as mean + s.e.m. AU arbitrary unit. The p-values are determined by unpaired t-test (a–e) or one-way ANOVA (b–d, f, g). *p < 0.05 vs. TonEBPfl/fl. Source data are provided as a Source Data file
Fig 5: Keratinocytes of Dnmt1Δ / Δep mice exhibit DNA hypomethylation and derepression of transposon/repeat families ADNA methylation profile of wild‐type keratinocytes at selected upregulated genes upon knockout of Dnmt1. DNA Methylation data were obtained from Chatterjee et al, 2014 and visualized in the IGV browser. The proximal promotor regions show lower methylation levels than surrounding CpGs.BExpression of deregulated genes found by RNA sequencing of cultured primary keratinocytes that were isolated at P3 of control and Dnmt1Δ / Δe p mice (n = 5‐7). Statistical analyses were done using two‐tailed Student's t‐test. Data are mean ± SEM. ***P ≤ 0.001, ****P ≤ 0.0001.CChanges in expression of transposon/repeat families were plotted in a scatter plot of log fold change versus log10(adjusted P‐values).DRelative mRNA expression of transposable elements in the epidermis of control and Dnmt1Δ / Δep mice at P3 (n = 3), P5 (n = 5), and P7 (n = 3). Statistical analyses were done using one‐way ANOVA (Kruskal–Wallis test of multiple comparisons). Data are mean ± SEM. ns, not significant, **P ≤ 0.01.E, FDNA methylation of CD45‐depleted epidermal keratinocytes isolated from newborn wild‐type (n = 3) and Dnmt1Δ / Δe p (n = 3) mice. Methylation status of repetitive elements was analyzed using deep amplicon bisulfite sequencing (IAP‐LTR1a) or deep hairpin‐bisulfite sequencing (major Satellites, mSat1). Mean values of DNA methylation of wild‐type and Dnmt1Δ / Δe p mice are shown in (F), and statistical analysis was done using two‐tailed Student's t‐test (see also Supp. Tab. S4). Data are mean ± SEM. **P ≤ 0.01, ***P ≤ 0.001.
Supplier Page from Abcam for Anti-Dnmt1 antibody [EPR18453]