Histone H3 lysine 79 methyltransferase Dot1 is required for immortalization by MLL oncogenes - PubMed (original) (raw)

Histone H3 lysine 79 methyltransferase Dot1 is required for immortalization by MLL oncogenes

Ming-Jin Chang et al. Cancer Res. 2010.

Abstract

Chimeric oncoproteins resulting from fusion of MLL to a wide variety of partnering proteins cause biologically distinctive and clinically aggressive acute leukemias. However, the mechanism of MLL-mediated leukemic transformation is not fully understood. Dot1, the only known histone H3 lysine 79 (H3K79) methyltransferase, has been shown to interact with multiple MLL fusion partners including AF9, ENL, AF10, and AF17. In this study, we utilize a conditional Dot1l deletion model to investigate the role of Dot1 in hematopoietic progenitor cell immortalization by MLL fusion proteins. Western blot and mass spectrometry show that Dot1-deficient cells are depleted of the global H3K79 methylation mark. We find that loss of Dot1 activity attenuates cell viability and colony formation potential of cells immortalized by MLL oncoproteins but not by the leukemic oncoprotein E2a-Pbx1. Although this effect is most pronounced for MLL-AF9, we find that Dot1 contributes to the viability of cells immortalized by other MLL oncoproteins that are not known to directly recruit Dot1. Cells immortalized by MLL fusions also show increased apoptosis, suggesting the involvement of Dot1 in survival pathways. In summary, our data point to a pivotal requirement for Dot1 in MLL fusion protein-mediated leukemogenesis and implicate Dot1 as a potential therapeutic target.

©2010 AACR.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

The authors have no conflicts of interest to disclose

Figures

Figure 1

Figure 1

Generation and characterization of Dot1l f/f and Dot1l f/Δ mice. A, targeting vector was cut with _Aat_II to release an 11-kb insert with 4.9-kb 5′ arm (away from the 5′ LoxP site) and 2.6-kb 3′ arm (away from the 3′ FRT sites) for recombination. Boxes with numbers inside indicate exons. The relative positions of _Bam_HI (B), AatII (A) sites, 5′ and 3′ probes used for Southern blot (see Supplementary Fig. S2), and primers for ES clone genotyping, as well as the predicted sizes of _Bam_HI fragments are shown. B and C, PCR-based genotyping of correctly targeted ES cells. Shown are agarose gel analyses of PCR products using the indicated primers. ES cells were microinjected into MF-1 blastocysts for generation of chimeras. D, diagram showing the relative positions of the primers used for mouse genotyping. E–G, PCR-based mouse genotyping. Shown are agarose gel analyses of PCR products from mice carrying different Dot1l alleles.

Figure 2

Figure 2

Cre-mediated deletion of Dot1l impairs survival in mouse hematopoietic progenitor cells immortalized by MLL-GAS7, MLL-AFX, and MLL-AF9 but not by E2a-Pbx1. A, experimental scheme to evaluate the effect of Dot1l deletion shows the time points when CFU activity, genotype (by PCR), or Hoxa9 expression (by qRT-PCR) was examined. All experiments were carried out using cells from Dot1l f/f mice unless specified. B, CFUs per 104 cells and representative colony morphologies (20× magnification) of cells immortalized by the indicated fusion oncogenes after Cre-mediated Dot1l deletion. Error bars indicate SD from 3 independent experiments with the exception of E2a-Pbx1, which is from 2 independent experiments. Each independent experiment was conducted in duplicate. Scale bar, 1 mm. C, Wright–Giemsa stain of GFP-sorted cells 6 days after transduction. Representative cells are enlarged in insets to show morphologic details. Magnification is 400 × and scale bars are 40 μm. Arrowheads indicate cells with condensed, fragmented nuclei. D, percentage of GFP+ cells at 3 or 5 days after GFP or Cre-GFP transduction in cells expressing indicated oncogens. Nontransduced cells were used as controls (gray fill) to determine GFP positivity. Cells from _Dot1f/_Δ mice were used in all samples with the exception of the MLL-AFX–immortalized cells, which originated from Dot1lf/f mice. The percentage of GFP+ cells is shown on each histogram. E, Dot1l genomic status was examined by PCR at days 0 and 8 in methylcellulose culture. Arrowhead, floxed allele at 510 bp; open arrowhead, deleted allele at 378 bp. F, relative expression levels of Hoxa9 after Cre-mediated Dot1l deletion. Expression levels are normalized to GAPDH and expressed relative to GFP-transduced cells (set to 100%). Error bars indicate the SD of analyses carried out in triplicate.

Figure 3

Figure 3

Dot1l deletion abolishes histone H3K79 methylation. A, Coomassie-stained SDS-PAGE gel of histone extract with histone H3 band indicated. B, histone H3K79 methylation status in E2a-Pbx1–immortalized cells obtained from Dot1lf/f or Dot1l_Δ/_Δ backgrounds was examined by Western blot using antibodies specific for dimethyl (K79me2) or trimethyl (K79me3) marks. The total histone H3 serves as a loading control. C, SDS-PAGE–purified histone H3 from E2a-Pbx1–immortalized cells with Dot1lf/f or _Dot1lf/_Δ genotype was subjected to LC-MS analysis. The relative abundance of the H3 peptide was manually set to 100. H3 peptide, YRPGTVALR; K79me0, K79me1, and K79me3 represent the unmodified (EIAQDFK), monomethyl and dimethyl (EIAQDFKTDLR) H3K79 peptides, respectively.

Figure 4

Figure 4

Increased Annexin V labeling in Dot1l deleted cells immortalized by MLL-AF9, MLL-GAS7, or MLL-AFX but not by E2a-Pbx1. Immortalized hematopoietic cells expressing the indicated oncogenes were transduced with GFP or Cre-GFP, labeled with Annexin V-PE/7-AAD, and analyzed by flow cytometry 5 days after transduction. The Annexin V-positive/7-AAD-negative cells in the lower right quadrant and the Annexin V–positive/7-AAD-positive cells in the upper right quadrant represent early apoptotic and late apoptotic/necrotic cells, respectively. GFP+ cells are presented as dual parameter contour plots and the percentage of cells in each quadrant is indicated.

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