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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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Increased APBs in Dot1L-deficient cells.(A) Confocal microscopy images showing either TRF1 (telomere marker, green), PML (marker for PML bodies, red), or combined fluorescence (yellow if colocalize, indicated by arrows) in wild-type (+/+) and Dot1L-deficient (1lox/1lox) ES cells. Late-passage (p120) Dnmt3a/3b-deficient (3a−/−3b−/−) ES cells were used as a positive control. Circled are nuclei of cells. (B) Quantification of percentage of cells showing colocalization of telomeres with PML bodies. A cell was considered positive when it showed 2 or more colocalization events. An increased frequency of cells showing APBs was observed in Dot11lox/1lox cultures compared to wild-type controls (χ2 test, P<0.001). (C) Quantification of the number of APBs per cell. Dot11lox/1lox cells showed a significant increase in the number of APBs compared to wild-type cells (χ2 test, P<0.001).
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pgen-1000190-g005: Increased APBs in Dot1L-deficient cells.(A) Confocal microscopy images showing either TRF1 (telomere marker, green), PML (marker for PML bodies, red), or combined fluorescence (yellow if colocalize, indicated by arrows) in wild-type (+/+) and Dot1L-deficient (1lox/1lox) ES cells. Late-passage (p120) Dnmt3a/3b-deficient (3a−/−3b−/−) ES cells were used as a positive control. Circled are nuclei of cells. (B) Quantification of percentage of cells showing colocalization of telomeres with PML bodies. A cell was considered positive when it showed 2 or more colocalization events. An increased frequency of cells showing APBs was observed in Dot11lox/1lox cultures compared to wild-type controls (χ2 test, P<0.001). (C) Quantification of the number of APBs per cell. Dot11lox/1lox cells showed a significant increase in the number of APBs compared to wild-type cells (χ2 test, P<0.001).

Mentions: Our data suggested a role for Dot1L in the homeostasis of telomere length. Two main mechanisms have been described for the maintenance of mammalian telomeres: the addition of telomeric repeats by telomerase and the so-called alternative lengthening of telomere (ALT) mechanism that relies on homologous recombination between telomeric sequences [15],[16]. Dot1L mutant ES cells showed increased telomere heterogeneity (Figure 4), which is a hallmark of ALT cells [17]. To determine whether the ALT pathway is activated in Dot1L-deficient cells, we assessed the presence of ALT-associated PML bodies (APBs, colocalization of PML and telomeres), another hallmark of ALT [17]. Dot1L+/+, Dot1L1lox/1lox, as well as Dnmt3a−/−3b−/− (positive control) ES cells were immunostained with antibodies against TRF1 (a telomere-binding protein) and PML. In the absence of Dot1L, both the frequency of cells showing APBs and the number of APBs per cell were significantly increased compared to wild-type cells (Figure 5, A–C, χ2 tests, P<0.001), suggesting that aberrant elongation of telomeres in Dot1L-deficient cells was due, at least in part, to activation of the ALT pathway.


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)

Increased APBs in Dot1L-deficient cells.(A) Confocal microscopy images showing either TRF1 (telomere marker, green), PML (marker for PML bodies, red), or combined fluorescence (yellow if colocalize, indicated by arrows) in wild-type (+/+) and Dot1L-deficient (1lox/1lox) ES cells. Late-passage (p120) Dnmt3a/3b-deficient (3a−/−3b−/−) ES cells were used as a positive control. Circled are nuclei of cells. (B) Quantification of percentage of cells showing colocalization of telomeres with PML bodies. A cell was considered positive when it showed 2 or more colocalization events. An increased frequency of cells showing APBs was observed in Dot11lox/1lox cultures compared to wild-type controls (χ2 test, P<0.001). (C) Quantification of the number of APBs per cell. Dot11lox/1lox cells showed a significant increase in the number of APBs compared to wild-type cells (χ2 test, P<0.001).
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Related In: Results  -  Collection

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pgen-1000190-g005: Increased APBs in Dot1L-deficient cells.(A) Confocal microscopy images showing either TRF1 (telomere marker, green), PML (marker for PML bodies, red), or combined fluorescence (yellow if colocalize, indicated by arrows) in wild-type (+/+) and Dot1L-deficient (1lox/1lox) ES cells. Late-passage (p120) Dnmt3a/3b-deficient (3a−/−3b−/−) ES cells were used as a positive control. Circled are nuclei of cells. (B) Quantification of percentage of cells showing colocalization of telomeres with PML bodies. A cell was considered positive when it showed 2 or more colocalization events. An increased frequency of cells showing APBs was observed in Dot11lox/1lox cultures compared to wild-type controls (χ2 test, P<0.001). (C) Quantification of the number of APBs per cell. Dot11lox/1lox cells showed a significant increase in the number of APBs compared to wild-type cells (χ2 test, P<0.001).
Mentions: Our data suggested a role for Dot1L in the homeostasis of telomere length. Two main mechanisms have been described for the maintenance of mammalian telomeres: the addition of telomeric repeats by telomerase and the so-called alternative lengthening of telomere (ALT) mechanism that relies on homologous recombination between telomeric sequences [15],[16]. Dot1L mutant ES cells showed increased telomere heterogeneity (Figure 4), which is a hallmark of ALT cells [17]. To determine whether the ALT pathway is activated in Dot1L-deficient cells, we assessed the presence of ALT-associated PML bodies (APBs, colocalization of PML and telomeres), another hallmark of ALT [17]. Dot1L+/+, Dot1L1lox/1lox, as well as Dnmt3a−/−3b−/− (positive control) ES cells were immunostained with antibodies against TRF1 (a telomere-binding protein) and PML. In the absence of Dot1L, both the frequency of cells showing APBs and the number of APBs per cell were significantly increased compared to wild-type cells (Figure 5, A–C, χ2 tests, P<0.001), suggesting that aberrant elongation of telomeres in Dot1L-deficient cells was due, at least in part, to activation of the ALT pathway.

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