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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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G2, mitotic, and apoptotic cell percentages are increased in Su(var)3-9.A) The histograms show cell cycle stage analysis of wild type and Su(var)3-9 cells. The percent of G1 cells do not differ significantly (p>0.05). The percentage of S phase cells in Su(var)3-9 is significantly lower than wild type (p<0.05). The percent of wild-type cells in G2 is significantly lower than in Su(var)3-9. Mitotic indicex in Su(var)3-9 is 4-fold over wild type (p<0.001). The percent of apoptotic cells (whole nuclei contain TUNEL signals, instead of foci) in Su(var)3-9 is 9-fold over wild type (p<0.001). P values were calculated by Student's t test, and n>1000 cells for each genotype. B) The chart shows γH2Av foci numbers in G1, S, and G2 cells of wild type and Su(var)3-9. Analysis of the ratios of γH2Av foci to total cell numbers during the cell cycle in wild type and Su(var)3-9. γH2Av foci in Su(var)3-9 cells increased over wild type in G1, S, and G2 (p<0.01). P values were calculated by Chi-square test, and n>40 cells for each genotype.
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pgen-1000435-g005: G2, mitotic, and apoptotic cell percentages are increased in Su(var)3-9.A) The histograms show cell cycle stage analysis of wild type and Su(var)3-9 cells. The percent of G1 cells do not differ significantly (p>0.05). The percentage of S phase cells in Su(var)3-9 is significantly lower than wild type (p<0.05). The percent of wild-type cells in G2 is significantly lower than in Su(var)3-9. Mitotic indicex in Su(var)3-9 is 4-fold over wild type (p<0.001). The percent of apoptotic cells (whole nuclei contain TUNEL signals, instead of foci) in Su(var)3-9 is 9-fold over wild type (p<0.001). P values were calculated by Student's t test, and n>1000 cells for each genotype. B) The chart shows γH2Av foci numbers in G1, S, and G2 cells of wild type and Su(var)3-9. Analysis of the ratios of γH2Av foci to total cell numbers during the cell cycle in wild type and Su(var)3-9. γH2Av foci in Su(var)3-9 cells increased over wild type in G1, S, and G2 (p<0.01). P values were calculated by Chi-square test, and n>40 cells for each genotype.

Mentions: Cells in G2 were identified by staining for CycA, but not PCNA or PH3, replicating cells were stained by PCNA, and G1 cells were not stained with CycA, PCNA, or PH3. Comparative analysis showed that the percentage of Su(var)3-9 cells in S phase was lower than in wild type, and that the percentages of Su(var)3-9 cells in G2, mitosis, and apoptosis increased relative to wild type (Figure 5A). These data suggest that G2 and mitotic checkpoints are activated in these mutant cells. Increased apoptosis in the mutant animals is likely caused by unrepaired DNA damage, which is mediated by the p53 pathway [28]. It is important to note that although the fold increases for mitotic and apoptotic cells in Su(var)3-9 are large, the actual percent of cells are low (1 to 3%), in comparison to the 24% of cells in G2 (4.5 fold over wild type).


Heterochromatic genome stability requires regulators of histone H3 K9 methylation.

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

G2, mitotic, and apoptotic cell percentages are increased in Su(var)3-9.A) The histograms show cell cycle stage analysis of wild type and Su(var)3-9 cells. The percent of G1 cells do not differ significantly (p>0.05). The percentage of S phase cells in Su(var)3-9 is significantly lower than wild type (p<0.05). The percent of wild-type cells in G2 is significantly lower than in Su(var)3-9. Mitotic indicex in Su(var)3-9 is 4-fold over wild type (p<0.001). The percent of apoptotic cells (whole nuclei contain TUNEL signals, instead of foci) in Su(var)3-9 is 9-fold over wild type (p<0.001). P values were calculated by Student's t test, and n>1000 cells for each genotype. B) The chart shows γH2Av foci numbers in G1, S, and G2 cells of wild type and Su(var)3-9. Analysis of the ratios of γH2Av foci to total cell numbers during the cell cycle in wild type and Su(var)3-9. γH2Av foci in Su(var)3-9 cells increased over wild type in G1, S, and G2 (p<0.01). P values were calculated by Chi-square test, and n>40 cells for each genotype.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654965&req=5

pgen-1000435-g005: G2, mitotic, and apoptotic cell percentages are increased in Su(var)3-9.A) The histograms show cell cycle stage analysis of wild type and Su(var)3-9 cells. The percent of G1 cells do not differ significantly (p>0.05). The percentage of S phase cells in Su(var)3-9 is significantly lower than wild type (p<0.05). The percent of wild-type cells in G2 is significantly lower than in Su(var)3-9. Mitotic indicex in Su(var)3-9 is 4-fold over wild type (p<0.001). The percent of apoptotic cells (whole nuclei contain TUNEL signals, instead of foci) in Su(var)3-9 is 9-fold over wild type (p<0.001). P values were calculated by Student's t test, and n>1000 cells for each genotype. B) The chart shows γH2Av foci numbers in G1, S, and G2 cells of wild type and Su(var)3-9. Analysis of the ratios of γH2Av foci to total cell numbers during the cell cycle in wild type and Su(var)3-9. γH2Av foci in Su(var)3-9 cells increased over wild type in G1, S, and G2 (p<0.01). P values were calculated by Chi-square test, and n>40 cells for each genotype.
Mentions: Cells in G2 were identified by staining for CycA, but not PCNA or PH3, replicating cells were stained by PCNA, and G1 cells were not stained with CycA, PCNA, or PH3. Comparative analysis showed that the percentage of Su(var)3-9 cells in S phase was lower than in wild type, and that the percentages of Su(var)3-9 cells in G2, mitosis, and apoptosis increased relative to wild type (Figure 5A). These data suggest that G2 and mitotic checkpoints are activated in these mutant cells. Increased apoptosis in the mutant animals is likely caused by unrepaired DNA damage, which is mediated by the p53 pathway [28]. It is important to note that although the fold increases for mitotic and apoptotic cells in Su(var)3-9 are large, the actual percent of cells are low (1 to 3%), in comparison to the 24% of cells in G2 (4.5 fold over wild type).

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