Dnmt3b is a haploinsufficient tumor suppressor gene in Myc-induced lymphomagenesis

Abstract

The drivers of abnormal DNA methylation in human cancers include widespread aberrant splicing of the DNMT3B gene, producing abnormal transcripts that encode truncated proteins that may act as dominant negative isoforms. To test whether reduced Dnmt3b dosage can alter tumorigenesis, we bred Dnmt3b(+/-) mice to Eµ-Myc mice, a mouse model susceptible to B-cell lymphomas. Eµ-Myc/Dnmt3b(+/-) mice showed a dramatic acceleration of lymphomagenesis, greater even than that observed in Eµ-Myc mice that express a truncated DNMT3B isoform found in human tumors, DNMT3B7. This finding indicates that Dnmt3b can act as a haploinsufficient tumor suppressor gene. Although reduction in both Dnmt3b dosage and expression of DNMT3B7 within the Eµ-Myc system had similar effects on tumorigenesis and DNA hypermethylation, different molecular mechanisms appear to underlie these changes. This study offers insight into how de novo DNA methyltransferases function as tumor suppressors and the sensitivity of Myc-induced lymphomas to DNA methylation.

Figure 1

Incidence of mediastinal lymphomas and changes in DNA methylation in Eμ-Myc, Eμ-Myc/DNMT3B7, and Eμ-Myc/Dnmt3b^+/− mice. (A) Eµ-Myc/Dnmt3b^+/− (dotted line) vs Eµ-Myc/DNMT3B7 (dashed line) and Eµ-Myc mice (solid line). Cumulative incidence curves were compared using the K-sample test (Eµ-Myc vs Eµ-Myc/Dnmt3b^+/−: P < .0001; Eµ-Myc vs Eµ-Myc/DNMT3B7: P = .009). (B) Growth curves of cell lines established from mediastinal lymphomas of Eµ-Myc (solid line), Eµ-Myc/DNMT3B7 (dashed line), and Eµ-Myc/Dnmt3b^+/− mice (dotted line). Curves were compared using 2-way analysis of variance (ANOVA) (P = .012). (C) Total 5-methylcytosine levels were quantified by liquid chromatography-electrospray ionization/tandem mass spectometry, n ≥ 4 for each genotype. DNA methylation levels were normalized to wild-type (WT) peripheral blood B cells (gray bar). The 2-tailed Student t test was used to compare global DNA methylation. (D-G) Relative quantification of Dnmt levels, normalized to β-Actin levels. Dnmt1 (D), Dnmt3a (E), Dnmt3b (F), and Dnmt3l (G) levels in WT lymph node (LN) (gray bar) and Eµ-Myc (white bar), Eµ-Myc/DNMT3B7 (hatched bar), and Eµ-Myc/Dnmt3b^+/− (striped bar) cell lines. P values were calculated by ANOVA, using WTLN as the control.

Figure 2

DNA methylation changes and binding of Myc and Dnmt3b at Mycbp promoter elements. (A) DNA methylation changes measured by bisulfite sequencing around the E-box upstream of Mycbp TSS. Schematic diagram for Mycbp is shown with exons represented by vertical rectangles, and the location of the CpG island shown with a horizontal shaded rectangle. The black arrow indicates the TSS. The smaller, black horizontal rectangle indicates the location of CpGs analyzed for changes in DNA methylation. Each row represents DNA methylation in a single lymphoma, indicated to the right. Numbers across the top indicate specific CpG dinucleotides in a region of the CpG island. Changes in DNA methylation are indicated by shaded circles, with the shading indicating average amount of DNA methylation at each CpG; numbers below represent percent methylated cytosine. Average percent methylation is indicated to the right, and P values calculated using the 2-tailed Student t test. Coefficient of variance (CV) was used to calculate the heterogeneity of DNA methylation at each CpG within tumors of a single genotype. The average CV is given as a percentage underneath each CpG position. CV values with a black box around them denote P < .05 for Eµ-Myc/DNMT3B7 or Eµ-Myc/Dnmt3b^+/− tumors relative to Eµ-Myc tumors. (B) DNA methylation changes as measured by bisulfite sequencing in the Mycbp TSS. Detailed description is as outlined in (A). (C) Fold enrichment of Myc in chromatin from at least 3 independent Eµ-Myc, Eµ-Myc/DNMT3B7, and Eµ-Myc/Dnmt3b^+/− cell lines. The plot shows real-time PCR data for the region around the Mycbp E-box (−363 to −99 relative to Mycbp TSS) from Myc-immunoprecipitated or control-immunoprecipitated chromatin normalized to input. (D) Fold enrichment of Dnmt3b in chromatin from at least 3 independent Eµ-Myc, Eµ-Myc/DNMT3B7, and Eµ-Myc/Dnmt3b^+/− cell lines. The plot shows real-time PCR data for the region around the Mycbp TSS (−99 to +195 relative to Mycbp TSS) from Dnmt3b-immunoprecipitated or control-immunoprecipitated chromatin normalized to input. (E) Expression of Mycbp in Eµ-Myc, Eµ-Myc/DNMT3B7, and Eµ-Myc/Dnmt3b^+/− tumors. Relative expression of Mycbp was measured in all Eµ-Myc tumor types and expressed normalized to β-Actin. (F) Proposed model for the increased acceleration of lymphomagenesis induced by hypermethylation in Eµ-Myc/DNMT3B7 mice. The presence of an unmethylated CpG in the Myc-binding E-box promotes Myc binding, which then assembles a complex that includes Dnmt3b and DNMT3B7. The complex locks into position at the Mycbp TSS and leads to hypermethylation around the TSS.