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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

Su(var)3-9 mutant oocytes and nurse cells display increased DNA damage in heterochromatin.A) The images show γH2Av (white in top panel and red in bottom panel) and C(3)G (green) IF in whole-mount germaria from wild type, Su(var)3-9 and mei-W68; Su(var)3-9. C(3)G is part of the synaptonemal complex and used to distinguish oocytes from nurse cells, both of which contain DSBs. Each image is an optical section; bar = 7 mm. B) and C) The graphs show the average numbers and volumes (relative to total nuclear volumes) of γH2Av foci in nurse cells and oocytes from wild type, Su(var)3-9 and mei-W68; Su(var)3-9. Both quantitation methods showed that γH2Av foci in Su(var)3-9 nurse cells were significantly increased over wild type (p<0.001). γH2Av foci in Su(var)3-9 oocytes were significantly increased over wild type (p<0.001). The numbers of γH2Av foci in mei-W68; Su(var)3-9 nurse cells and oocytes were lower than Su(var)3-9 alone and not significantly different from wild type (p<0.001). Error bars indicate standard deviations, p values were calculated by Student's t test, and n>15 for each cell type. D) Combined γH2Av IF (red) and satellite FISH (green) in wild-type and Su(var)3-9 germaria; C(3)G (grey) staining identifies the oocytes. Percent of oocyte and nurse cells that displayed overlap between γH2Av and satellite signals are shown. Each image is an optical section, and cells are 5 mm wide.
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pgen-1000435-g002: Su(var)3-9 mutant oocytes and nurse cells display increased DNA damage in heterochromatin.A) The images show γH2Av (white in top panel and red in bottom panel) and C(3)G (green) IF in whole-mount germaria from wild type, Su(var)3-9 and mei-W68; Su(var)3-9. C(3)G is part of the synaptonemal complex and used to distinguish oocytes from nurse cells, both of which contain DSBs. Each image is an optical section; bar = 7 mm. B) and C) The graphs show the average numbers and volumes (relative to total nuclear volumes) of γH2Av foci in nurse cells and oocytes from wild type, Su(var)3-9 and mei-W68; Su(var)3-9. Both quantitation methods showed that γH2Av foci in Su(var)3-9 nurse cells were significantly increased over wild type (p<0.001). γH2Av foci in Su(var)3-9 oocytes were significantly increased over wild type (p<0.001). The numbers of γH2Av foci in mei-W68; Su(var)3-9 nurse cells and oocytes were lower than Su(var)3-9 alone and not significantly different from wild type (p<0.001). Error bars indicate standard deviations, p values were calculated by Student's t test, and n>15 for each cell type. D) Combined γH2Av IF (red) and satellite FISH (green) in wild-type and Su(var)3-9 germaria; C(3)G (grey) staining identifies the oocytes. Percent of oocyte and nurse cells that displayed overlap between γH2Av and satellite signals are shown. Each image is an optical section, and cells are 5 mm wide.

Mentions: IF analysis showed a dramatic increase in γH2Av signals in Su(var)3-9 mutant germaria compared to wild type (Figure 2A). We performed two kinds of quantitative analyses because a high percentage of each nucleus in mutant cells stained for γH2Av. γH2Av foci can fuse with each other, and thus foci counts can under-represent the phenotypic severity in mutant cells. Quantitative volumetric analysis can address this issue, but can also be influenced by varying intensity values in whole-mount IF experiments. The number of γH2Av foci in Su(var)3-9 nurse cells and oocytes were significantly increased over wild type (Figure 2B, p<0.001). Quantitative analysis of γH2Av volumes (relative to nuclear volumes) in wild type and Su(var)3-9 yielded similar results (Figure 2C). Increased DNA damage foci in germaria may be due to higher frequencies of meiotic breaks or defects in repairing meiotic breaks. However, we did not observe any γH2Av signals in the Su(var)3-9 late stage oocytes (data not shown), where meiotic crossover would have completed, suggesting that increased DNA breaks were not due to defective meiotic break repair in mutants.


Heterochromatic genome stability requires regulators of histone H3 K9 methylation.

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

Su(var)3-9 mutant oocytes and nurse cells display increased DNA damage in heterochromatin.A) The images show γH2Av (white in top panel and red in bottom panel) and C(3)G (green) IF in whole-mount germaria from wild type, Su(var)3-9 and mei-W68; Su(var)3-9. C(3)G is part of the synaptonemal complex and used to distinguish oocytes from nurse cells, both of which contain DSBs. Each image is an optical section; bar = 7 mm. B) and C) The graphs show the average numbers and volumes (relative to total nuclear volumes) of γH2Av foci in nurse cells and oocytes from wild type, Su(var)3-9 and mei-W68; Su(var)3-9. Both quantitation methods showed that γH2Av foci in Su(var)3-9 nurse cells were significantly increased over wild type (p<0.001). γH2Av foci in Su(var)3-9 oocytes were significantly increased over wild type (p<0.001). The numbers of γH2Av foci in mei-W68; Su(var)3-9 nurse cells and oocytes were lower than Su(var)3-9 alone and not significantly different from wild type (p<0.001). Error bars indicate standard deviations, p values were calculated by Student's t test, and n>15 for each cell type. D) Combined γH2Av IF (red) and satellite FISH (green) in wild-type and Su(var)3-9 germaria; C(3)G (grey) staining identifies the oocytes. Percent of oocyte and nurse cells that displayed overlap between γH2Av and satellite signals are shown. Each image is an optical section, and cells are 5 mm wide.
© Copyright Policy
Related In: Results  -  Collection

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pgen-1000435-g002: Su(var)3-9 mutant oocytes and nurse cells display increased DNA damage in heterochromatin.A) The images show γH2Av (white in top panel and red in bottom panel) and C(3)G (green) IF in whole-mount germaria from wild type, Su(var)3-9 and mei-W68; Su(var)3-9. C(3)G is part of the synaptonemal complex and used to distinguish oocytes from nurse cells, both of which contain DSBs. Each image is an optical section; bar = 7 mm. B) and C) The graphs show the average numbers and volumes (relative to total nuclear volumes) of γH2Av foci in nurse cells and oocytes from wild type, Su(var)3-9 and mei-W68; Su(var)3-9. Both quantitation methods showed that γH2Av foci in Su(var)3-9 nurse cells were significantly increased over wild type (p<0.001). γH2Av foci in Su(var)3-9 oocytes were significantly increased over wild type (p<0.001). The numbers of γH2Av foci in mei-W68; Su(var)3-9 nurse cells and oocytes were lower than Su(var)3-9 alone and not significantly different from wild type (p<0.001). Error bars indicate standard deviations, p values were calculated by Student's t test, and n>15 for each cell type. D) Combined γH2Av IF (red) and satellite FISH (green) in wild-type and Su(var)3-9 germaria; C(3)G (grey) staining identifies the oocytes. Percent of oocyte and nurse cells that displayed overlap between γH2Av and satellite signals are shown. Each image is an optical section, and cells are 5 mm wide.
Mentions: IF analysis showed a dramatic increase in γH2Av signals in Su(var)3-9 mutant germaria compared to wild type (Figure 2A). We performed two kinds of quantitative analyses because a high percentage of each nucleus in mutant cells stained for γH2Av. γH2Av foci can fuse with each other, and thus foci counts can under-represent the phenotypic severity in mutant cells. Quantitative volumetric analysis can address this issue, but can also be influenced by varying intensity values in whole-mount IF experiments. The number of γH2Av foci in Su(var)3-9 nurse cells and oocytes were significantly increased over wild type (Figure 2B, p<0.001). Quantitative analysis of γH2Av volumes (relative to nuclear volumes) in wild type and Su(var)3-9 yielded similar results (Figure 2C). Increased DNA damage foci in germaria may be due to higher frequencies of meiotic breaks or defects in repairing meiotic breaks. However, we did not observe any γH2Av signals in the Su(var)3-9 late stage oocytes (data not shown), where meiotic crossover would have completed, suggesting that increased DNA breaks were not due to defective meiotic break repair in mutants.

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