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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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Su(var)3-9 somatic cells exhibit genome instability phenotypes.A) DAPI staining of mitotic chromosomes from wild type and Su(var)3-9 mutant diploid cells. Structural defects in Su(var)3-9 mitotic chromosomes are indicated by white arrows. Each image is an optical section; bar = 2 mm. B) The chart shows quantitation of defective Su(var)3-9 mitotic chromosomes. Some mitotic chromosomes exhibited more than one defect. C) Chromosome painting of mitotic chromosomes from wild type and Su(var)3-9 mutant. Red = 3rd chromosomes, green = 2nd chromosomes, and blue = X chromosomes. Fourth and Y chromosomes are only stained with DAPI. Structural defects, such as deletions and translocations, are indicated by white arrows. Each image is an optical section; bar = 2 mm. D) Quantitation showed that 1.1% of the Su(var)3-9 mitotic chromosomes exhibited structural defects, compared to 0% for wild type (p<0.05 by Chi-square test). E) The diagram illustrates the genetic assay used to quantitate genome instability (loss of heterozygosity) in wild type and Su(var)3-9 animals. Wild type virgin flies were mated with males hemizygous for w− (recessive white1118 mutation) to produce females heterozygous for w− and w+. A clone of w− adult eye cells (pictured) arises during larval development when the w+ allele is lost due to mitotic recombination, deletion, or chromosome loss events. f) Quantitative analysis of w− clone frequencies in wild type and Su(var)3-9 animals. The p values were calculated using the Chi-square test.
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pgen-1000435-g003: Su(var)3-9 somatic cells exhibit genome instability phenotypes.A) DAPI staining of mitotic chromosomes from wild type and Su(var)3-9 mutant diploid cells. Structural defects in Su(var)3-9 mitotic chromosomes are indicated by white arrows. Each image is an optical section; bar = 2 mm. B) The chart shows quantitation of defective Su(var)3-9 mitotic chromosomes. Some mitotic chromosomes exhibited more than one defect. C) Chromosome painting of mitotic chromosomes from wild type and Su(var)3-9 mutant. Red = 3rd chromosomes, green = 2nd chromosomes, and blue = X chromosomes. Fourth and Y chromosomes are only stained with DAPI. Structural defects, such as deletions and translocations, are indicated by white arrows. Each image is an optical section; bar = 2 mm. D) Quantitation showed that 1.1% of the Su(var)3-9 mitotic chromosomes exhibited structural defects, compared to 0% for wild type (p<0.05 by Chi-square test). E) The diagram illustrates the genetic assay used to quantitate genome instability (loss of heterozygosity) in wild type and Su(var)3-9 animals. Wild type virgin flies were mated with males hemizygous for w− (recessive white1118 mutation) to produce females heterozygous for w− and w+. A clone of w− adult eye cells (pictured) arises during larval development when the w+ allele is lost due to mitotic recombination, deletion, or chromosome loss events. f) Quantitative analysis of w− clone frequencies in wild type and Su(var)3-9 animals. The p values were calculated using the Chi-square test.

Mentions: Persistent DNA damage could lead to chromosomal structural defects, rearrangements and aneuploidy. To test this hypothesis, we first examined wild type and Su(var)3-9 mitotic chromosomes by DAPI-staining. All wild-type mitotic chromosomes exhibited banding patterns characteristic of heterochromatin (Figure 3A, first panel). In contrast, Su(var)3-9 mitotic chromosomes exhibited a variety of phenotypes such as hypo-condensation (Figure 3A, second panel) and extra DAPI-bright bands (Figure 3A, third panel; complete list of phenotypic analyses is in Figure 3B).


Heterochromatic genome stability requires regulators of histone H3 K9 methylation.

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

Su(var)3-9 somatic cells exhibit genome instability phenotypes.A) DAPI staining of mitotic chromosomes from wild type and Su(var)3-9 mutant diploid cells. Structural defects in Su(var)3-9 mitotic chromosomes are indicated by white arrows. Each image is an optical section; bar = 2 mm. B) The chart shows quantitation of defective Su(var)3-9 mitotic chromosomes. Some mitotic chromosomes exhibited more than one defect. C) Chromosome painting of mitotic chromosomes from wild type and Su(var)3-9 mutant. Red = 3rd chromosomes, green = 2nd chromosomes, and blue = X chromosomes. Fourth and Y chromosomes are only stained with DAPI. Structural defects, such as deletions and translocations, are indicated by white arrows. Each image is an optical section; bar = 2 mm. D) Quantitation showed that 1.1% of the Su(var)3-9 mitotic chromosomes exhibited structural defects, compared to 0% for wild type (p<0.05 by Chi-square test). E) The diagram illustrates the genetic assay used to quantitate genome instability (loss of heterozygosity) in wild type and Su(var)3-9 animals. Wild type virgin flies were mated with males hemizygous for w− (recessive white1118 mutation) to produce females heterozygous for w− and w+. A clone of w− adult eye cells (pictured) arises during larval development when the w+ allele is lost due to mitotic recombination, deletion, or chromosome loss events. f) Quantitative analysis of w− clone frequencies in wild type and Su(var)3-9 animals. The p values were calculated using the Chi-square test.
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Related In: Results  -  Collection

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pgen-1000435-g003: Su(var)3-9 somatic cells exhibit genome instability phenotypes.A) DAPI staining of mitotic chromosomes from wild type and Su(var)3-9 mutant diploid cells. Structural defects in Su(var)3-9 mitotic chromosomes are indicated by white arrows. Each image is an optical section; bar = 2 mm. B) The chart shows quantitation of defective Su(var)3-9 mitotic chromosomes. Some mitotic chromosomes exhibited more than one defect. C) Chromosome painting of mitotic chromosomes from wild type and Su(var)3-9 mutant. Red = 3rd chromosomes, green = 2nd chromosomes, and blue = X chromosomes. Fourth and Y chromosomes are only stained with DAPI. Structural defects, such as deletions and translocations, are indicated by white arrows. Each image is an optical section; bar = 2 mm. D) Quantitation showed that 1.1% of the Su(var)3-9 mitotic chromosomes exhibited structural defects, compared to 0% for wild type (p<0.05 by Chi-square test). E) The diagram illustrates the genetic assay used to quantitate genome instability (loss of heterozygosity) in wild type and Su(var)3-9 animals. Wild type virgin flies were mated with males hemizygous for w− (recessive white1118 mutation) to produce females heterozygous for w− and w+. A clone of w− adult eye cells (pictured) arises during larval development when the w+ allele is lost due to mitotic recombination, deletion, or chromosome loss events. f) Quantitative analysis of w− clone frequencies in wild type and Su(var)3-9 animals. The p values were calculated using the Chi-square test.
Mentions: Persistent DNA damage could lead to chromosomal structural defects, rearrangements and aneuploidy. To test this hypothesis, we first examined wild type and Su(var)3-9 mitotic chromosomes by DAPI-staining. All wild-type mitotic chromosomes exhibited banding patterns characteristic of heterochromatin (Figure 3A, first panel). In contrast, Su(var)3-9 mitotic chromosomes exhibited a variety of phenotypes such as hypo-condensation (Figure 3A, second panel) and extra DAPI-bright bands (Figure 3A, third panel; complete list of phenotypic analyses is in Figure 3B).

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