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Heterochromatic genome stability requires regulators of histone H3 K9 methylation.

Peng JC, Karpen GH - PLoS Genet. (2009)

Bottom Line: Heterochromatin contains many repetitive DNA elements and few protein-encoding genes, yet it is essential for chromosome organization and inheritance.Similar effects of lower magnitude were observed in animals that lack the RNA interference pathway component Dcr2.These results suggest that the H3K9 methylation and RNAi pathways ensure heterochromatin stability.

View Article: PubMed Central - PubMed

Affiliation: Lawrence Berkeley National Laboratory, Department of Genome and Computational Biology, Berkeley, California, USA.

ABSTRACT
Heterochromatin contains many repetitive DNA elements and few protein-encoding genes, yet it is essential for chromosome organization and inheritance. Here, we show that Drosophila that lack the Su(var)3-9 H3K9 methyltransferase display significantly elevated frequencies of spontaneous DNA damage in heterochromatin, in both somatic and germ-line cells. Accumulated DNA damage in these mutants correlates with chromosomal defects, such as translocations and loss of heterozygosity. DNA repair and mitotic checkpoints are also activated in mutant animals and are required for their viability. Similar effects of lower magnitude were observed in animals that lack the RNA interference pathway component Dcr2. These results suggest that the H3K9 methylation and RNAi pathways ensure heterochromatin stability.

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Related in: MedlinePlus

DNA repair checkpoint and mitotic checkpoint proteins are essential for the viability of Su(var)3-9 mutants.A) The chart lists the viability of the double mutants of Su(var)3-9 with mutations in the DNA damage checkpoint and mitotic checkpoint pathway. Viability was calculated relative to single homozygous checkpoint mutants, which are less viable than Su(var)3-9 single mutants. Progeny counts are in Table S1. P values were calculated by the Chi-square test. B) Cell cycle analysis of wild type, Su(var)3-9, grp; Su(var)3-9 and lok; Su(var)3-9 mutant imaginal discs and brains. The percentages of G1 cells in the two double mutants were higher than wild type and single Su(var)3-9 mutants. The percentages of S phase cells in the two double mutants are lower than wild type, but do not differ from Su(var)3-9. The percentages of G2 cells in the double mutants are lower than wild type and Su(var)3-9. The mitotic indices in the double mutants were lower than Su(var)3-9 and do not significantly differ from the wild type. P<0.05 for all tests that show significant differences between wild type and Su(var)3-9; p values were calculated by Student's t test, and n>1000 cells for each genotype.
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pgen-1000435-g006: DNA repair checkpoint and mitotic checkpoint proteins are essential for the viability of Su(var)3-9 mutants.A) The chart lists the viability of the double mutants of Su(var)3-9 with mutations in the DNA damage checkpoint and mitotic checkpoint pathway. Viability was calculated relative to single homozygous checkpoint mutants, which are less viable than Su(var)3-9 single mutants. Progeny counts are in Table S1. P values were calculated by the Chi-square test. B) Cell cycle analysis of wild type, Su(var)3-9, grp; Su(var)3-9 and lok; Su(var)3-9 mutant imaginal discs and brains. The percentages of G1 cells in the two double mutants were higher than wild type and single Su(var)3-9 mutants. The percentages of S phase cells in the two double mutants are lower than wild type, but do not differ from Su(var)3-9. The percentages of G2 cells in the double mutants are lower than wild type and Su(var)3-9. The mitotic indices in the double mutants were lower than Su(var)3-9 and do not significantly differ from the wild type. P<0.05 for all tests that show significant differences between wild type and Su(var)3-9; p values were calculated by Student's t test, and n>1000 cells for each genotype.

Mentions: Increased proportions of G2 and mitotic cells in Su(var)3-9 animals suggests that the G2 and mitotic cell cycle checkpoints may be activated by the increased frequencies of DSBs in heterochromatin. We thereby hypothesized that compromising the DNA damage checkpoint, using mutations in the checkpoint components, may result in lethality of Su(var)3-9 animals. To test this hypothesis, we analyzed flies homozygous for Su(var)3-9 and homozygous for mutations in DNA damage checkpoint activation (ATR/mei-41, Checkpoint kinase 1 (Chk1)/grp, and Checkpoint kinase 2 (Chk2)/lok). Animals double mutant for Su(var)3-9 and cell cycle checkpoint mutations showed sub-viability ranging from 50% to 64.6% (Figure 6A; viability of double mutants were compared to single checkpoint mutants, which exhibit lower viability than Su(var)3-9 mutants). Su(var)3-9 mutant animals containing both grp and lok mutations are 100% lethal (Figure 6A). This demonstrates that DNA damage checkpoints are essential to the survival of Su(var)3-9 animals. Cell cycle analysis showed that the percentage of grp; Su(var)3-9 or lok; Su(var)3-9 cells in S phase and G2 were lower than in Su(var)3-9 alone, with a corresponding increase in cells in G1 (Figure 6B). Cell cycle characterization combined with the observed genetic interactions between Su(var)3-9 and DNA damage checkpoint mutations demonstrate that the DNA damage checkpoint is activated in Su(var)3-9 mutant animals, and is required for mutant viability.


Heterochromatic genome stability requires regulators of histone H3 K9 methylation.

Peng JC, Karpen GH - PLoS Genet. (2009)

DNA repair checkpoint and mitotic checkpoint proteins are essential for the viability of Su(var)3-9 mutants.A) The chart lists the viability of the double mutants of Su(var)3-9 with mutations in the DNA damage checkpoint and mitotic checkpoint pathway. Viability was calculated relative to single homozygous checkpoint mutants, which are less viable than Su(var)3-9 single mutants. Progeny counts are in Table S1. P values were calculated by the Chi-square test. B) Cell cycle analysis of wild type, Su(var)3-9, grp; Su(var)3-9 and lok; Su(var)3-9 mutant imaginal discs and brains. The percentages of G1 cells in the two double mutants were higher than wild type and single Su(var)3-9 mutants. The percentages of S phase cells in the two double mutants are lower than wild type, but do not differ from Su(var)3-9. The percentages of G2 cells in the double mutants are lower than wild type and Su(var)3-9. The mitotic indices in the double mutants were lower than Su(var)3-9 and do not significantly differ from the wild type. P<0.05 for all tests that show significant differences between wild type and Su(var)3-9; p values were calculated by Student's t test, and n>1000 cells for each genotype.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2654965&req=5

pgen-1000435-g006: DNA repair checkpoint and mitotic checkpoint proteins are essential for the viability of Su(var)3-9 mutants.A) The chart lists the viability of the double mutants of Su(var)3-9 with mutations in the DNA damage checkpoint and mitotic checkpoint pathway. Viability was calculated relative to single homozygous checkpoint mutants, which are less viable than Su(var)3-9 single mutants. Progeny counts are in Table S1. P values were calculated by the Chi-square test. B) Cell cycle analysis of wild type, Su(var)3-9, grp; Su(var)3-9 and lok; Su(var)3-9 mutant imaginal discs and brains. The percentages of G1 cells in the two double mutants were higher than wild type and single Su(var)3-9 mutants. The percentages of S phase cells in the two double mutants are lower than wild type, but do not differ from Su(var)3-9. The percentages of G2 cells in the double mutants are lower than wild type and Su(var)3-9. The mitotic indices in the double mutants were lower than Su(var)3-9 and do not significantly differ from the wild type. P<0.05 for all tests that show significant differences between wild type and Su(var)3-9; p values were calculated by Student's t test, and n>1000 cells for each genotype.
Mentions: Increased proportions of G2 and mitotic cells in Su(var)3-9 animals suggests that the G2 and mitotic cell cycle checkpoints may be activated by the increased frequencies of DSBs in heterochromatin. We thereby hypothesized that compromising the DNA damage checkpoint, using mutations in the checkpoint components, may result in lethality of Su(var)3-9 animals. To test this hypothesis, we analyzed flies homozygous for Su(var)3-9 and homozygous for mutations in DNA damage checkpoint activation (ATR/mei-41, Checkpoint kinase 1 (Chk1)/grp, and Checkpoint kinase 2 (Chk2)/lok). Animals double mutant for Su(var)3-9 and cell cycle checkpoint mutations showed sub-viability ranging from 50% to 64.6% (Figure 6A; viability of double mutants were compared to single checkpoint mutants, which exhibit lower viability than Su(var)3-9 mutants). Su(var)3-9 mutant animals containing both grp and lok mutations are 100% lethal (Figure 6A). This demonstrates that DNA damage checkpoints are essential to the survival of Su(var)3-9 animals. Cell cycle analysis showed that the percentage of grp; Su(var)3-9 or lok; Su(var)3-9 cells in S phase and G2 were lower than in Su(var)3-9 alone, with a corresponding increase in cells in G1 (Figure 6B). Cell cycle characterization combined with the observed genetic interactions between Su(var)3-9 and DNA damage checkpoint mutations demonstrate that the DNA damage checkpoint is activated in Su(var)3-9 mutant animals, and is required for mutant viability.

Bottom Line: Heterochromatin contains many repetitive DNA elements and few protein-encoding genes, yet it is essential for chromosome organization and inheritance.Similar effects of lower magnitude were observed in animals that lack the RNA interference pathway component Dcr2.These results suggest that the H3K9 methylation and RNAi pathways ensure heterochromatin stability.

View Article: PubMed Central - PubMed

Affiliation: Lawrence Berkeley National Laboratory, Department of Genome and Computational Biology, Berkeley, California, USA.

ABSTRACT
Heterochromatin contains many repetitive DNA elements and few protein-encoding genes, yet it is essential for chromosome organization and inheritance. Here, we show that Drosophila that lack the Su(var)3-9 H3K9 methyltransferase display significantly elevated frequencies of spontaneous DNA damage in heterochromatin, in both somatic and germ-line cells. Accumulated DNA damage in these mutants correlates with chromosomal defects, such as translocations and loss of heterozygosity. DNA repair and mitotic checkpoints are also activated in mutant animals and are required for their viability. Similar effects of lower magnitude were observed in animals that lack the RNA interference pathway component Dcr2. These results suggest that the H3K9 methylation and RNAi pathways ensure heterochromatin stability.

Show MeSH
Related in: MedlinePlus