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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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Changes of heterochromatin structure in Dot1L-deficient ES cells.(A) Quantitative real-time PCR results using DNA from Dot1L mutant and wild-type ES cells immunoprecipitated with antibodies specific for the indicated histone modifications or without an antibody (No Ab) and normalized using input DNA values. PCR primers specific for major satellite repeats, minor satellite repeats, the subtelomere region of chromosome 1 or the subtelomere region of chromosome 2 were used. (B) Dot blot analysis of ChIP DNA using either a telomere-specific probe or a major satellite repeat-specific probe. Input DNA at 1∶10, 1∶100 and 1∶1000 dilutions was used as a positive control. DNA precipitated from 2.5×106 cells were used for each assay.
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pgen-1000190-g006: Changes of heterochromatin structure in Dot1L-deficient ES cells.(A) Quantitative real-time PCR results using DNA from Dot1L mutant and wild-type ES cells immunoprecipitated with antibodies specific for the indicated histone modifications or without an antibody (No Ab) and normalized using input DNA values. PCR primers specific for major satellite repeats, minor satellite repeats, the subtelomere region of chromosome 1 or the subtelomere region of chromosome 2 were used. (B) Dot blot analysis of ChIP DNA using either a telomere-specific probe or a major satellite repeat-specific probe. Input DNA at 1∶10, 1∶100 and 1∶1000 dilutions was used as a positive control. DNA precipitated from 2.5×106 cells were used for each assay.

Mentions: Aneuploidy and telomere elongation can result from defects in the chromatin structure at centromeres and telomeres, respectively [18]–[23]. To evaluate changes in chromatin structure in Dot1L mutant cells, we used chromatin immunoprecipitation (ChIP) to examine histone modifications at major satellite repeats (present at pericentric regions), minor satellite repeats (present at centromeric regions), telomeric repeats, and subtelomeric regions (Figure 6). H3K79 di-methylation was detected in all these heterochromatin regions in Dot1L+/+ cells (Figure 6, A and B). As expected, this modification was reduced in Dot1L1lox/+ cells and almost absent in Dot1L1lox/1lox cells (Figure 6, A and B), validating our experimental procedures. As further controls, the levels of centromere- and telomere-bound histone H3 were similar in wild-type and Dot1L mutant cells (Figure 6, A and B), and the telomere-binding protein TRF1 associated with telomeric repeats, but not with major satellite repeats (Figure 6B).


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)

Changes of heterochromatin structure in Dot1L-deficient ES cells.(A) Quantitative real-time PCR results using DNA from Dot1L mutant and wild-type ES cells immunoprecipitated with antibodies specific for the indicated histone modifications or without an antibody (No Ab) and normalized using input DNA values. PCR primers specific for major satellite repeats, minor satellite repeats, the subtelomere region of chromosome 1 or the subtelomere region of chromosome 2 were used. (B) Dot blot analysis of ChIP DNA using either a telomere-specific probe or a major satellite repeat-specific probe. Input DNA at 1∶10, 1∶100 and 1∶1000 dilutions was used as a positive control. DNA precipitated from 2.5×106 cells were used for each assay.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2527135&req=5

pgen-1000190-g006: Changes of heterochromatin structure in Dot1L-deficient ES cells.(A) Quantitative real-time PCR results using DNA from Dot1L mutant and wild-type ES cells immunoprecipitated with antibodies specific for the indicated histone modifications or without an antibody (No Ab) and normalized using input DNA values. PCR primers specific for major satellite repeats, minor satellite repeats, the subtelomere region of chromosome 1 or the subtelomere region of chromosome 2 were used. (B) Dot blot analysis of ChIP DNA using either a telomere-specific probe or a major satellite repeat-specific probe. Input DNA at 1∶10, 1∶100 and 1∶1000 dilutions was used as a positive control. DNA precipitated from 2.5×106 cells were used for each assay.
Mentions: Aneuploidy and telomere elongation can result from defects in the chromatin structure at centromeres and telomeres, respectively [18]–[23]. To evaluate changes in chromatin structure in Dot1L mutant cells, we used chromatin immunoprecipitation (ChIP) to examine histone modifications at major satellite repeats (present at pericentric regions), minor satellite repeats (present at centromeric regions), telomeric repeats, and subtelomeric regions (Figure 6). H3K79 di-methylation was detected in all these heterochromatin regions in Dot1L+/+ cells (Figure 6, A and B). As expected, this modification was reduced in Dot1L1lox/+ cells and almost absent in Dot1L1lox/1lox cells (Figure 6, A and B), validating our experimental procedures. As further controls, the levels of centromere- and telomere-bound histone H3 were similar in wild-type and Dot1L mutant cells (Figure 6, A and B), and the telomere-binding protein TRF1 associated with telomeric repeats, but not with major satellite repeats (Figure 6B).

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