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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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Telomere elongation and aneuploidy in Dot1L-deficient ES cells.(A) Telomere restriction fragment (TRF) analysis upon MboI digestion of genomic DNA from two independent ES cell clones of each of the genotypes: Dot1L+/+, Dot1L1lox/+ and Dot1L1lox/1lox. Note the presence of high molecular weight TRFs in Dot1L1lox/1lox cells, which correspond to longer telomeres. (B) Representative images generated during the Q-FISH assay showing metaphase spreads from Dot1L+/+ and Dot1L1lox/1lox ES cells labeled with a telomere-specific fluorescent probe. (C) Telomere length distribution of two independent ES cell clones of each of the Dot1L genotypes as determined by Q-FISH. Twenty metaphases of each ES cell clone were analyzed. Note the increase in mean telomere length (Mtl) in both clones of Dot11lox/1lox cells and the intermediate phenotype of Dot11lox/+ lines. The percentages of telomeres below 50 kb and above 100 kb in length are indicated. (D) Scatter plot of the chromosome number of a Dot1L+/+ ES cell line and two Dot1L1lox/1lox cell lines. Chromosome number was determined by manually counting chromosomes in chromosome spreads. Each point represents the chromosome number of a single cell (n represents the number of metaphase cells counted).
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pgen-1000190-g004: Telomere elongation and aneuploidy in Dot1L-deficient ES cells.(A) Telomere restriction fragment (TRF) analysis upon MboI digestion of genomic DNA from two independent ES cell clones of each of the genotypes: Dot1L+/+, Dot1L1lox/+ and Dot1L1lox/1lox. Note the presence of high molecular weight TRFs in Dot1L1lox/1lox cells, which correspond to longer telomeres. (B) Representative images generated during the Q-FISH assay showing metaphase spreads from Dot1L+/+ and Dot1L1lox/1lox ES cells labeled with a telomere-specific fluorescent probe. (C) Telomere length distribution of two independent ES cell clones of each of the Dot1L genotypes as determined by Q-FISH. Twenty metaphases of each ES cell clone were analyzed. Note the increase in mean telomere length (Mtl) in both clones of Dot11lox/1lox cells and the intermediate phenotype of Dot11lox/+ lines. The percentages of telomeres below 50 kb and above 100 kb in length are indicated. (D) Scatter plot of the chromosome number of a Dot1L+/+ ES cell line and two Dot1L1lox/1lox cell lines. Chromosome number was determined by manually counting chromosomes in chromosome spreads. Each point represents the chromosome number of a single cell (n represents the number of metaphase cells counted).

Mentions: Dot1-deficient S. cerevisiae show telomere elongation and defects in telomere silencing [3]. We therefore evaluated the effect of Dot1L inactivation on telomere length. First, we used Southern blot terminal restriction fragment (TRF) analysis to estimate telomere length in two ES cell lines of each Dot1L genotype. Both Dot1L1lox/1lox lines and one of the Dot1L1lox/+ lines showed telomere elongation, as evidenced by the presence of high molecular weight (MW) TRFs and the increase in the lengths of bulk TRFs compared to wild-type controls (Figure 4A). Next, we carried out quantitative fluorescence in situ hybridization (Q-FISH) using a telomere-specific probe to determine the mean telomere length (mtl) and the distribution of telomere lengths for each cell line (Figure 4, B and C). Consistent with the TRF results, both Dot1L1lox/1lox lines had higher mtl, greater percentages of elongated (>100 kb) telomeres, and reduced percentages of short (<50 kb) telomeres compared to Dot1L+/+ lines (Figure 4C). The heterozygous ES cells again showed an intermediate phenotype (Figure 4C).


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)

Telomere elongation and aneuploidy in Dot1L-deficient ES cells.(A) Telomere restriction fragment (TRF) analysis upon MboI digestion of genomic DNA from two independent ES cell clones of each of the genotypes: Dot1L+/+, Dot1L1lox/+ and Dot1L1lox/1lox. Note the presence of high molecular weight TRFs in Dot1L1lox/1lox cells, which correspond to longer telomeres. (B) Representative images generated during the Q-FISH assay showing metaphase spreads from Dot1L+/+ and Dot1L1lox/1lox ES cells labeled with a telomere-specific fluorescent probe. (C) Telomere length distribution of two independent ES cell clones of each of the Dot1L genotypes as determined by Q-FISH. Twenty metaphases of each ES cell clone were analyzed. Note the increase in mean telomere length (Mtl) in both clones of Dot11lox/1lox cells and the intermediate phenotype of Dot11lox/+ lines. The percentages of telomeres below 50 kb and above 100 kb in length are indicated. (D) Scatter plot of the chromosome number of a Dot1L+/+ ES cell line and two Dot1L1lox/1lox cell lines. Chromosome number was determined by manually counting chromosomes in chromosome spreads. Each point represents the chromosome number of a single cell (n represents the number of metaphase cells counted).
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Related In: Results  -  Collection

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pgen-1000190-g004: Telomere elongation and aneuploidy in Dot1L-deficient ES cells.(A) Telomere restriction fragment (TRF) analysis upon MboI digestion of genomic DNA from two independent ES cell clones of each of the genotypes: Dot1L+/+, Dot1L1lox/+ and Dot1L1lox/1lox. Note the presence of high molecular weight TRFs in Dot1L1lox/1lox cells, which correspond to longer telomeres. (B) Representative images generated during the Q-FISH assay showing metaphase spreads from Dot1L+/+ and Dot1L1lox/1lox ES cells labeled with a telomere-specific fluorescent probe. (C) Telomere length distribution of two independent ES cell clones of each of the Dot1L genotypes as determined by Q-FISH. Twenty metaphases of each ES cell clone were analyzed. Note the increase in mean telomere length (Mtl) in both clones of Dot11lox/1lox cells and the intermediate phenotype of Dot11lox/+ lines. The percentages of telomeres below 50 kb and above 100 kb in length are indicated. (D) Scatter plot of the chromosome number of a Dot1L+/+ ES cell line and two Dot1L1lox/1lox cell lines. Chromosome number was determined by manually counting chromosomes in chromosome spreads. Each point represents the chromosome number of a single cell (n represents the number of metaphase cells counted).
Mentions: Dot1-deficient S. cerevisiae show telomere elongation and defects in telomere silencing [3]. We therefore evaluated the effect of Dot1L inactivation on telomere length. First, we used Southern blot terminal restriction fragment (TRF) analysis to estimate telomere length in two ES cell lines of each Dot1L genotype. Both Dot1L1lox/1lox lines and one of the Dot1L1lox/+ lines showed telomere elongation, as evidenced by the presence of high molecular weight (MW) TRFs and the increase in the lengths of bulk TRFs compared to wild-type controls (Figure 4A). Next, we carried out quantitative fluorescence in situ hybridization (Q-FISH) using a telomere-specific probe to determine the mean telomere length (mtl) and the distribution of telomere lengths for each cell line (Figure 4, B and C). Consistent with the TRF results, both Dot1L1lox/1lox lines had higher mtl, greater percentages of elongated (>100 kb) telomeres, and reduced percentages of short (<50 kb) telomeres compared to Dot1L+/+ lines (Figure 4C). The heterozygous ES cells again showed an intermediate phenotype (Figure 4C).

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