Fig 1: Overexpression of DNMT3B in T-ALL and Burkitt’s lymphoma is MYC-dependentExpression analysis of DNMT3B in T-ALL and Burkitt’s lymphoma models before and upon MYC inactivation. (A) RT-qPCR of MYC and DNMT3B, (B) Western blot analysis of MYC and DNMT3B, and (C) RT-qPCR of DNMT1 and DNMT3A in T-ALL cells (2833) derived from EµSRa-tTA;tet-o-MYC mice, before and upon inactivation of MYC for 1 and 2 days with 20 ng/mL DOX. Addition of increasing DOX concentrations (0.1-0.5 ng/mL) for 2 days allowed for titration of MYC and DNMT3B levels. (D) RT-qPCR of MYC and DNMT3B, (E) Western blot analysis of MYC and DNMT3B, and (F) RT-qPCR of DNMT1 and DNMT3A in human Burkitt’s lymphoma-like cells (P493-6) expressing a conditional c-MYC allele, before and upon inactivation of MYC for 1 and 2 days with 20 ng/mL DOX. Addition of increasing DOX concentrations (0.1-0.5 ng/mL) for 2 days allowed for titration of MYC and DNMT3B levels. RT-qPCR data was normalized to UBC or RPL13A; TUBULIN serves as loading control for Western blot. Error bars represent mean ± SEM; n = 3; two-tailed Student’s t-test: NS = non-significant; *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 2: Pten loss suppressed cardiac development in vivo. a) A model for the generation of Pten conditional knockout mice. b) Immunohistochemistry experiment showing the expression of DNMT3B in the hearts of WT and Pten-/- mice. Scale bars, 20 µm. c) Quantification of the DNMT3B-positive cells in WT and Pten-/- hearts. Error bars indicate the mean ± SD (n = 5). p values were calculated by the Student's t-test: ***p < 0.001. d) The expression pattern of PTEN/AKT/DNMT3B signaling proteins in WT and Pten-/- hearts. e) Immunohistochemistry experiment showing the expression of IGF2 in the hearts of WT and Pten-/- mice at E12.5. Scale bars, 100 µm and 20 µm. f) Quantification of the IGF2-positive cells in WT and Pten-/- heart at E12.5. Error bars indicate the mean ± SD (n = 5). p values were calculated by the Student's t-test: ***p < 0.001. g) Immunofluorescence experiment showing the expression of NKX2-5 in the hearts of WT and Pten-/- mice at E18.5. Scale bars, 20 µm. h) Immunofluorescence experiment showing the expression of ISL1 and HAND2 in the hearts of WT and Pten-/- mice at E12.5. Scale bars, 50 µm. i–k) Quantification of the NKX2-5-positive, ISL1-positive, and HAND2-positive cells in WT and Pten-/- hearts. Error bars indicate the mean ± SD (n = 5). p values were calculated by the Student's t-test: **p < 0.01. l) A model of how Pten deletion suppresses cardiomyocyte differentiation. During cardiomyocyte differentiation from ESCs, Pten suppresses the expression of the Dnmt3 family; the loss of Pten promotes the non-CG methylation of cardiomyocyte genes and Igf2, which leads to the inhibition of cardiomyocyte differentiation.
Fig 3: Working modelMYC controls DNA methylation in a genome-wide fashion through overexpression of DNMT1 and DNMT3B. Non-malignant state: MYC levels are low, corresponding with low DNMT1 and DNMT3B expression. Tumor maintenance: MYC levels are constitutively high, driving the expression of DNMT1 and DNMT3B. Tumor regression/cell cycle arrest: MYC-inactivation in T-ALL causes tumor regression, associated with low DNMT1 and DNMT3B expression. Knock-down (shRNA) or pharmacologic inhibition (Nanaomycin A) of DNMT3B in tumor cells decreases proliferation. Loss of DNMT3B expression can reverse CpG island/promoter methylation thereby reactivating the corresponding genes. Taken together MYC induces and maintains global DNA methylation through control of a tumor cell-specific DNMT1 and DNMT3B expression.
Fig 4: shRNA-mediated knock-down of DNMT3B decreased tumor cell proliferationshRNA-mediated knock-down of DNMT3B in mouse T-ALL (EµSRa-tTA;tet-o-MYC). T-ALL cells (6780) upon shRNA-mediated knock-down of DNMT3B using two distinct sequences (3B-sh1 and 3B-sh2), were compared to control cells (SCR). (A) RT-qPCR analysis of MYC and DNMT3B. (B) Western blot analysis of MYC and DNMT3B; TUBULIN serves as loading control. (C) RT-qPCR analysis of DNMT1 and DNMT3A. (D) Growth curve comparing viable cell counts. (E) RT-qPCR analysis of cell cycle-dependent kinase inhibitors CDKN2A, CDKN2D, CDKN2B, and CDKN1A. (F) Flow cytometric cell cycle analysis using propidium iodide (PI) staining. (G) Cell cycle distribution (G1, S and G2/M) displayed in percent. (H) Flow cytometric analysis of apoptosis using Annexin V/PI staining. Flow cytometry profile of Annexin V staining (X axis) and PI (Y axis) is shown for representative samples. The lower right quadrant indicates the percentage of early apoptotic cells in each condition; the upper right quadrant indicates the percentage of late apoptotic cells. (I) Apoptotic cells (Annexin V-positive cells) are displayed as the percentage of gated cells. Error bars represent mean ± SEM; n = 3; two-tailed Student’s t-test: *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 5: MYC occupies the promoter of DNMT1 and DNMT3B in T-ALL and Burkitt’s lymphomaChromatin immunoprecipitation (ChIP) analysis indicates high relative enrichment for MYC at the DNMT1 and DNMT3B promoters in mouse T-ALL and human Burkitt’s lymphoma-like cells. (A) Agilent mouse promoter microarrays covering -5.5kb to +2.5kb of the transcription start site were used for ChIP-chip analysis. MYC ChIP-chip data for mouse T-ALL cells (EµSRa-tTA;tet-o-MYC) indicating enrichment for the DNMT1, DNMT3A and DNMT3B locus. (B) MYC ChIP-seq data for Burkitt’s lymphoma-like cells (P493-6) obtained from Sabo et al. [30] indicating enrichment scores for DNMT1, DNMT3A and DNMT3B. Traces for DNMT1, DNMT3A and DNMT3B were generated based on reference genome mm8 and hg19, respectively, using the UCSC Genome Browser. The chromosomal location is indicated in bp, and the scale in kb. MYC binding peaks are displayed as red vertical bars; numbers represent the relative fold enrichment for MYC. E-box sequences and their location are shown in blue for the vicinity of MYC binding peaks. Exons are displayed as black vertical bars, the UTR is represented by a black line, and the transcription start site (TSS) is marked by an arrow indicating the direction of transcription.
Supplier Page from Abcam for Anti-Dnmt3b antibody