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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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Related in: MedlinePlus

Generation of mutant Dot1L alleles in mice.(A) Schematic depiction of the strategy used to generate the Dot1L3lox and Dot1L1lox alleles. The exons are numbered. The locations of the Southern probe and PCR primers (DF1, DR1, and DR2) used for genotyping, as well as the sizes of the diagnostic fragments recognized by the Southern probe, are indicated (E, EcoRI). loxP sites are shown as triangles. (B) Southern blot analysis of EcoRI-digested genomic DNA probed with an 860-bp 5′ probe external to the targeting vector. The presence of the 8.3-kb band confirms homologous recombination. (C) PCR genotyping of DNA from ES cells. WT, 485 bp; 1lox, 233 bp.
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pgen-1000190-g001: Generation of mutant Dot1L alleles in mice.(A) Schematic depiction of the strategy used to generate the Dot1L3lox and Dot1L1lox alleles. The exons are numbered. The locations of the Southern probe and PCR primers (DF1, DR1, and DR2) used for genotyping, as well as the sizes of the diagnostic fragments recognized by the Southern probe, are indicated (E, EcoRI). loxP sites are shown as triangles. (B) Southern blot analysis of EcoRI-digested genomic DNA probed with an 860-bp 5′ probe external to the targeting vector. The presence of the 8.3-kb band confirms homologous recombination. (C) PCR genotyping of DNA from ES cells. WT, 485 bp; 1lox, 233 bp.

Mentions: To target the Dot1L gene, we constructed a targeting vector in which a 2.3-kb genomic region containing exons 5 and 6 and a promoterless β-geo selection cassette were flanked, respectively, by three loxP sites (Figure 1A). Exons 5 and 6 encode 108 amino acids that form several conserved motifs in the Dot1L catalytic domain, including the SAM-binding motif and motifs X, I, and II [6]. Since mutations of conserved residues within motif I abolish the methyltransferase activity of Dot1L [4], we predicted that deletion of exons 5 and 6 would inactivate Dot1L.


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)

Generation of mutant Dot1L alleles in mice.(A) Schematic depiction of the strategy used to generate the Dot1L3lox and Dot1L1lox alleles. The exons are numbered. The locations of the Southern probe and PCR primers (DF1, DR1, and DR2) used for genotyping, as well as the sizes of the diagnostic fragments recognized by the Southern probe, are indicated (E, EcoRI). loxP sites are shown as triangles. (B) Southern blot analysis of EcoRI-digested genomic DNA probed with an 860-bp 5′ probe external to the targeting vector. The presence of the 8.3-kb band confirms homologous recombination. (C) PCR genotyping of DNA from ES cells. WT, 485 bp; 1lox, 233 bp.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000190-g001: Generation of mutant Dot1L alleles in mice.(A) Schematic depiction of the strategy used to generate the Dot1L3lox and Dot1L1lox alleles. The exons are numbered. The locations of the Southern probe and PCR primers (DF1, DR1, and DR2) used for genotyping, as well as the sizes of the diagnostic fragments recognized by the Southern probe, are indicated (E, EcoRI). loxP sites are shown as triangles. (B) Southern blot analysis of EcoRI-digested genomic DNA probed with an 860-bp 5′ probe external to the targeting vector. The presence of the 8.3-kb band confirms homologous recombination. (C) PCR genotyping of DNA from ES cells. WT, 485 bp; 1lox, 233 bp.
Mentions: To target the Dot1L gene, we constructed a targeting vector in which a 2.3-kb genomic region containing exons 5 and 6 and a promoterless β-geo selection cassette were flanked, respectively, by three loxP sites (Figure 1A). Exons 5 and 6 encode 108 amino acids that form several conserved motifs in the Dot1L catalytic domain, including the SAM-binding motif and motifs X, I, and II [6]. Since mutations of conserved residues within motif I abolish the methyltransferase activity of Dot1L [4], we predicted that deletion of exons 5 and 6 would inactivate Dot1L.

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