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The histone H3K79 methyltransferase Dot1L is essential for mammalian development and heterochromatin structure.

Jones B, Su H, Bhat A, Lei H, Bajko J, Hevi S, Baltus GA, Kadam S, Zhai H, Valdez R, Gonzalo S, Zhang Y, Li E, Chen T - PLoS Genet. (2008)

Bottom Line: Dot1L-deficient ES cells show global loss of H3K79 methylation as well as reduced levels of heterochromatic marks (H3K9 di-methylation and H4K20 tri-methylation) at centromeres and telomeres.These changes are accompanied by aneuploidy, telomere elongation, and proliferation defects.Taken together, these results indicate that Dot1L and H3K79 methylation play important roles in heterochromatin formation and in embryonic development.

View Article: PubMed Central - PubMed

Affiliation: Epigenetics Program, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, United States of America.

ABSTRACT
Dot1 is an evolutionarily conserved histone methyltransferase specific for lysine 79 of histone H3 (H3K79). In Saccharomyces cerevisiae, Dot1-mediated H3K79 methylation is associated with telomere silencing, meiotic checkpoint control, and DNA damage response. The biological function of H3K79 methylation in mammals, however, remains poorly understood. Using gene targeting, we generated mice deficient for Dot1L, the murine Dot1 homologue. Dot1L-deficient embryos show multiple developmental abnormalities, including growth impairment, angiogenesis defects in the yolk sac, and cardiac dilation, and die between 9.5 and 10.5 days post coitum. To gain insights into the cellular function of Dot1L, we derived embryonic stem (ES) cells from Dot1L mutant blastocysts. Dot1L-deficient ES cells show global loss of H3K79 methylation as well as reduced levels of heterochromatic marks (H3K9 di-methylation and H4K20 tri-methylation) at centromeres and telomeres. These changes are accompanied by aneuploidy, telomere elongation, and proliferation defects. Taken together, these results indicate that Dot1L and H3K79 methylation play important roles in heterochromatin formation and in embryonic development.

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Phenotypic analysis of Dot1L mutant ES cells.(A) Western blot analysis using extracts from ES cell lines of the indicated Dot1L genotypes and antibodies specific for di-, and tri-methylated H3K79. Total histone H3 was used as a loading control. (B) Analysis of H3K79 methylation by mass spectrometry. Quantification of different forms of H3K79 methylation was obtained by comparing the extracted ion chromatogram (EIC) intensity of the ion signals corresponding to the unmodified (Me0), mono-methylated (Me1), di-methylated (Me2), and tri-methylated (Me3) K79-containing peptides. (C) The proliferation of Dot1L+/+, Dot1L1lox/+ and Dot1L1lox/1lox ES cells was determined by doing cell counts every 24 hours for five days. Cells were grown in triplicate, and data shown is representative of three independent experiments. (D) The percentages of apoptotic cells in Dot1L+/+, Dot1L1lox/+ and Dot1L1lox/1lox ES cell cultures. The asterisk indicates P<0.05 (Student t-test). ES cells were stained with propidium iodide (PI) and PE conjugated anti-annexin V antibodies and analyzed by FACS. Apoptotic cells were annexin V positive and PI negative. Cells were grown in triplicate, and data shown are representative of two independent experiments. (E) The percentages of each cell cycle stage in Dot1L+/+, Dot1L1lox/+ and Dot1L1lox/1lox ES cell cultures as determined by PI staining and FACS.
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pgen-1000190-g003: Phenotypic analysis of Dot1L mutant ES cells.(A) Western blot analysis using extracts from ES cell lines of the indicated Dot1L genotypes and antibodies specific for di-, and tri-methylated H3K79. Total histone H3 was used as a loading control. (B) Analysis of H3K79 methylation by mass spectrometry. Quantification of different forms of H3K79 methylation was obtained by comparing the extracted ion chromatogram (EIC) intensity of the ion signals corresponding to the unmodified (Me0), mono-methylated (Me1), di-methylated (Me2), and tri-methylated (Me3) K79-containing peptides. (C) The proliferation of Dot1L+/+, Dot1L1lox/+ and Dot1L1lox/1lox ES cells was determined by doing cell counts every 24 hours for five days. Cells were grown in triplicate, and data shown is representative of three independent experiments. (D) The percentages of apoptotic cells in Dot1L+/+, Dot1L1lox/+ and Dot1L1lox/1lox ES cell cultures. The asterisk indicates P<0.05 (Student t-test). ES cells were stained with propidium iodide (PI) and PE conjugated anti-annexin V antibodies and analyzed by FACS. Apoptotic cells were annexin V positive and PI negative. Cells were grown in triplicate, and data shown are representative of two independent experiments. (E) The percentages of each cell cycle stage in Dot1L+/+, Dot1L1lox/+ and Dot1L1lox/1lox ES cell cultures as determined by PI staining and FACS.

Mentions: To investigate the cellular function of Dot1L, we derived Dot1L mutant ES cells from blastocysts produced from intercrosses of Dot1L1lox/+ mice. Two Dot1L1lox/1lox and multiple Dot1L1lox/+ and Dot1L+/+ lines were established. As expected, H3K79 di- and tri-methylation was greatly reduced in Dot1L1lox/1lox cells compared to Dot1L+/+ cells (Figure 3A). Dot1L1lox/+ cells had intermediate levels of H3K79 di- and tri-methylation, indicating haploinsufficiency of Dot1L (Figure 3A). Surprisingly, Western blot analysis using a “mono methyl H3K79” antibody (ab2886, Abcam) detected no change in signal intensity in Dot1L mutant ES cell lines (data not shown). To verify the results, we carried out mass spectrometry. In wild-type ES cells, ∼11% of histone H3 showed K79 methylation, among which mono-, di-, and tri-methylation accounted for ∼70%, ∼30%, and less than 1%, respectively. In Dot1L1lox/1lox ES cells, H3K79 mono- and tri-methylation was absent although trace amount of di-methylation was detected (Figure 3B and Figure S2). We therefore concluded that the Western blot result showing no alteration in H3K79 mono-methylation in the absence of Dot1L was an artifact due to nonspecific recognition of histone H3 by the “mono methyl H3K79” antibody. The low level of H3K79 di-methylation detected in Dot1L1lox/1lox samples could be from feeder cells present in the culture or due to incomplete inactivation of Dot1L. Taken together, these results indicated that Dot1L is most likely the sole H3K79 methyltransferase in mice.


The histone H3K79 methyltransferase Dot1L is essential for mammalian development and heterochromatin structure.

Jones B, Su H, Bhat A, Lei H, Bajko J, Hevi S, Baltus GA, Kadam S, Zhai H, Valdez R, Gonzalo S, Zhang Y, Li E, Chen T - PLoS Genet. (2008)

Phenotypic analysis of Dot1L mutant ES cells.(A) Western blot analysis using extracts from ES cell lines of the indicated Dot1L genotypes and antibodies specific for di-, and tri-methylated H3K79. Total histone H3 was used as a loading control. (B) Analysis of H3K79 methylation by mass spectrometry. Quantification of different forms of H3K79 methylation was obtained by comparing the extracted ion chromatogram (EIC) intensity of the ion signals corresponding to the unmodified (Me0), mono-methylated (Me1), di-methylated (Me2), and tri-methylated (Me3) K79-containing peptides. (C) The proliferation of Dot1L+/+, Dot1L1lox/+ and Dot1L1lox/1lox ES cells was determined by doing cell counts every 24 hours for five days. Cells were grown in triplicate, and data shown is representative of three independent experiments. (D) The percentages of apoptotic cells in Dot1L+/+, Dot1L1lox/+ and Dot1L1lox/1lox ES cell cultures. The asterisk indicates P<0.05 (Student t-test). ES cells were stained with propidium iodide (PI) and PE conjugated anti-annexin V antibodies and analyzed by FACS. Apoptotic cells were annexin V positive and PI negative. Cells were grown in triplicate, and data shown are representative of two independent experiments. (E) The percentages of each cell cycle stage in Dot1L+/+, Dot1L1lox/+ and Dot1L1lox/1lox ES cell cultures as determined by PI staining and FACS.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2527135&req=5

pgen-1000190-g003: Phenotypic analysis of Dot1L mutant ES cells.(A) Western blot analysis using extracts from ES cell lines of the indicated Dot1L genotypes and antibodies specific for di-, and tri-methylated H3K79. Total histone H3 was used as a loading control. (B) Analysis of H3K79 methylation by mass spectrometry. Quantification of different forms of H3K79 methylation was obtained by comparing the extracted ion chromatogram (EIC) intensity of the ion signals corresponding to the unmodified (Me0), mono-methylated (Me1), di-methylated (Me2), and tri-methylated (Me3) K79-containing peptides. (C) The proliferation of Dot1L+/+, Dot1L1lox/+ and Dot1L1lox/1lox ES cells was determined by doing cell counts every 24 hours for five days. Cells were grown in triplicate, and data shown is representative of three independent experiments. (D) The percentages of apoptotic cells in Dot1L+/+, Dot1L1lox/+ and Dot1L1lox/1lox ES cell cultures. The asterisk indicates P<0.05 (Student t-test). ES cells were stained with propidium iodide (PI) and PE conjugated anti-annexin V antibodies and analyzed by FACS. Apoptotic cells were annexin V positive and PI negative. Cells were grown in triplicate, and data shown are representative of two independent experiments. (E) The percentages of each cell cycle stage in Dot1L+/+, Dot1L1lox/+ and Dot1L1lox/1lox ES cell cultures as determined by PI staining and FACS.
Mentions: To investigate the cellular function of Dot1L, we derived Dot1L mutant ES cells from blastocysts produced from intercrosses of Dot1L1lox/+ mice. Two Dot1L1lox/1lox and multiple Dot1L1lox/+ and Dot1L+/+ lines were established. As expected, H3K79 di- and tri-methylation was greatly reduced in Dot1L1lox/1lox cells compared to Dot1L+/+ cells (Figure 3A). Dot1L1lox/+ cells had intermediate levels of H3K79 di- and tri-methylation, indicating haploinsufficiency of Dot1L (Figure 3A). Surprisingly, Western blot analysis using a “mono methyl H3K79” antibody (ab2886, Abcam) detected no change in signal intensity in Dot1L mutant ES cell lines (data not shown). To verify the results, we carried out mass spectrometry. In wild-type ES cells, ∼11% of histone H3 showed K79 methylation, among which mono-, di-, and tri-methylation accounted for ∼70%, ∼30%, and less than 1%, respectively. In Dot1L1lox/1lox ES cells, H3K79 mono- and tri-methylation was absent although trace amount of di-methylation was detected (Figure 3B and Figure S2). We therefore concluded that the Western blot result showing no alteration in H3K79 mono-methylation in the absence of Dot1L was an artifact due to nonspecific recognition of histone H3 by the “mono methyl H3K79” antibody. The low level of H3K79 di-methylation detected in Dot1L1lox/1lox samples could be from feeder cells present in the culture or due to incomplete inactivation of Dot1L. Taken together, these results indicated that Dot1L is most likely the sole H3K79 methyltransferase in mice.

Bottom Line: Dot1L-deficient ES cells show global loss of H3K79 methylation as well as reduced levels of heterochromatic marks (H3K9 di-methylation and H4K20 tri-methylation) at centromeres and telomeres.These changes are accompanied by aneuploidy, telomere elongation, and proliferation defects.Taken together, these results indicate that Dot1L and H3K79 methylation play important roles in heterochromatin formation and in embryonic development.

View Article: PubMed Central - PubMed

Affiliation: Epigenetics Program, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, United States of America.

ABSTRACT
Dot1 is an evolutionarily conserved histone methyltransferase specific for lysine 79 of histone H3 (H3K79). In Saccharomyces cerevisiae, Dot1-mediated H3K79 methylation is associated with telomere silencing, meiotic checkpoint control, and DNA damage response. The biological function of H3K79 methylation in mammals, however, remains poorly understood. Using gene targeting, we generated mice deficient for Dot1L, the murine Dot1 homologue. Dot1L-deficient embryos show multiple developmental abnormalities, including growth impairment, angiogenesis defects in the yolk sac, and cardiac dilation, and die between 9.5 and 10.5 days post coitum. To gain insights into the cellular function of Dot1L, we derived embryonic stem (ES) cells from Dot1L mutant blastocysts. Dot1L-deficient ES cells show global loss of H3K79 methylation as well as reduced levels of heterochromatic marks (H3K9 di-methylation and H4K20 tri-methylation) at centromeres and telomeres. These changes are accompanied by aneuploidy, telomere elongation, and proliferation defects. Taken together, these results indicate that Dot1L and H3K79 methylation play important roles in heterochromatin formation and in embryonic development.

Show MeSH
Related in: MedlinePlus