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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

dcr-2 mutants display increased spontaneous DNA damage in heterochromatin.A) Analysis of the ratios of γH2Av foci to total cell numbers at different cell cycle stages are shown for wild type and dcr-2. γH2Av foci in dcr-2 cells only increased during S phase (p<0.05). P values were calculated by Chi-square test. B) The histograms show cell cycle stage analysis of wild type and dcr-2 cells. The percent of G1 cells in the two groups do not differ significantly (p>0.05). The percentage of S phase cells is not significantly lower in dcr-2 compared to wild type (p>0.05), but the percent of wild-type cells in G2 is significantly lower than in dcr-2 (p<0.05). The mitotic index in dcr-2 cells is 11-fold over wild type (p<0.001), and the percent of apoptotic cells (whole nuclei contain TUNEL signals, instead of foci) in dcr-2 is 18-fold over wild type (p<0.001). P values were calculated by Student's t test, and n>1000 cells for each genotype. C) The chart lists the viability of double mutants of dcr-2 with mutations in the DNA damage checkpoint and mitotic checkpoint pathways. Viability was calculated relative to single homozygous checkpoint mutants, which are less viable than dcr-2 single mutants. Progeny counts are in Table S1. P values were calculated by the Chi-square test.
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pgen-1000435-g007: dcr-2 mutants display increased spontaneous DNA damage in heterochromatin.A) Analysis of the ratios of γH2Av foci to total cell numbers at different cell cycle stages are shown for wild type and dcr-2. γH2Av foci in dcr-2 cells only increased during S phase (p<0.05). P values were calculated by Chi-square test. B) The histograms show cell cycle stage analysis of wild type and dcr-2 cells. The percent of G1 cells in the two groups do not differ significantly (p>0.05). The percentage of S phase cells is not significantly lower in dcr-2 compared to wild type (p>0.05), but the percent of wild-type cells in G2 is significantly lower than in dcr-2 (p<0.05). The mitotic index in dcr-2 cells is 11-fold over wild type (p<0.001), and the percent of apoptotic cells (whole nuclei contain TUNEL signals, instead of foci) in dcr-2 is 18-fold over wild type (p<0.001). P values were calculated by Student's t test, and n>1000 cells for each genotype. C) The chart lists the viability of double mutants of dcr-2 with mutations in the DNA damage checkpoint and mitotic checkpoint pathways. Viability was calculated relative to single homozygous checkpoint mutants, which are less viable than dcr-2 single mutants. Progeny counts are in Table S1. P values were calculated by the Chi-square test.

Mentions: We have investigated whether patterns of DNA damage and repair are affected by loss of Dcr2, as reported here for Su(var)3-9 mutants. Quantitative analyses of gH2Av and Rad51 foci in dcr-2 mutant cells showed significant increases in spontaneous DNA damage and repair (2.1- and 3.5- fold over wild type, respectively; Figures S2A and B). Foci localized to DAPI-bright regions in dcr-2 increased 24-fold over wild type for γH2Av and 33-fold for TUNEL (Figure S2C and D). Foci in DAPI-weak regions do not differ significantly between wild type and dcr-2. Therefore, increased DNA damage in dcr-2 occurs in heterochromatic DNAs of dcr-2 mutant cells. IF analyses showed that γH2Av foci in dcr-2 mutant oocytes increased over wild type by 1.5- (foci number) to 3- fold (volumetric analysis). In both meiotic and mitotic cells, the increases in spontaneous DNA damage were significant, but less severe than in Su(var)3-9 mutants (Figure S3). Su(var)3-9 mutant cells contained elevated frequencies of damage in all interphase cell cycle stages (Figure 5B). However, gH2Av foci enrichment in dcr-2 mutant cells only occurred during S phase (Figure 7A), suggesting that increased DNA breaks are repaired in S phase and do not persist into G2.


Heterochromatic genome stability requires regulators of histone H3 K9 methylation.

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

dcr-2 mutants display increased spontaneous DNA damage in heterochromatin.A) Analysis of the ratios of γH2Av foci to total cell numbers at different cell cycle stages are shown for wild type and dcr-2. γH2Av foci in dcr-2 cells only increased during S phase (p<0.05). P values were calculated by Chi-square test. B) The histograms show cell cycle stage analysis of wild type and dcr-2 cells. The percent of G1 cells in the two groups do not differ significantly (p>0.05). The percentage of S phase cells is not significantly lower in dcr-2 compared to wild type (p>0.05), but the percent of wild-type cells in G2 is significantly lower than in dcr-2 (p<0.05). The mitotic index in dcr-2 cells is 11-fold over wild type (p<0.001), and the percent of apoptotic cells (whole nuclei contain TUNEL signals, instead of foci) in dcr-2 is 18-fold over wild type (p<0.001). P values were calculated by Student's t test, and n>1000 cells for each genotype. C) The chart lists the viability of double mutants of dcr-2 with mutations in the DNA damage checkpoint and mitotic checkpoint pathways. Viability was calculated relative to single homozygous checkpoint mutants, which are less viable than dcr-2 single mutants. Progeny counts are in Table S1. P values were calculated by the Chi-square test.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000435-g007: dcr-2 mutants display increased spontaneous DNA damage in heterochromatin.A) Analysis of the ratios of γH2Av foci to total cell numbers at different cell cycle stages are shown for wild type and dcr-2. γH2Av foci in dcr-2 cells only increased during S phase (p<0.05). P values were calculated by Chi-square test. B) The histograms show cell cycle stage analysis of wild type and dcr-2 cells. The percent of G1 cells in the two groups do not differ significantly (p>0.05). The percentage of S phase cells is not significantly lower in dcr-2 compared to wild type (p>0.05), but the percent of wild-type cells in G2 is significantly lower than in dcr-2 (p<0.05). The mitotic index in dcr-2 cells is 11-fold over wild type (p<0.001), and the percent of apoptotic cells (whole nuclei contain TUNEL signals, instead of foci) in dcr-2 is 18-fold over wild type (p<0.001). P values were calculated by Student's t test, and n>1000 cells for each genotype. C) The chart lists the viability of double mutants of dcr-2 with mutations in the DNA damage checkpoint and mitotic checkpoint pathways. Viability was calculated relative to single homozygous checkpoint mutants, which are less viable than dcr-2 single mutants. Progeny counts are in Table S1. P values were calculated by the Chi-square test.
Mentions: We have investigated whether patterns of DNA damage and repair are affected by loss of Dcr2, as reported here for Su(var)3-9 mutants. Quantitative analyses of gH2Av and Rad51 foci in dcr-2 mutant cells showed significant increases in spontaneous DNA damage and repair (2.1- and 3.5- fold over wild type, respectively; Figures S2A and B). Foci localized to DAPI-bright regions in dcr-2 increased 24-fold over wild type for γH2Av and 33-fold for TUNEL (Figure S2C and D). Foci in DAPI-weak regions do not differ significantly between wild type and dcr-2. Therefore, increased DNA damage in dcr-2 occurs in heterochromatic DNAs of dcr-2 mutant cells. IF analyses showed that γH2Av foci in dcr-2 mutant oocytes increased over wild type by 1.5- (foci number) to 3- fold (volumetric analysis). In both meiotic and mitotic cells, the increases in spontaneous DNA damage were significant, but less severe than in Su(var)3-9 mutants (Figure S3). Su(var)3-9 mutant cells contained elevated frequencies of damage in all interphase cell cycle stages (Figure 5B). However, gH2Av foci enrichment in dcr-2 mutant cells only occurred during S phase (Figure 7A), suggesting that increased DNA breaks are repaired in S phase and do not persist into G2.

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