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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 somatic cells display increased DNA damage in heterochromatin.A) γH2Av (red) and Rad51 (green) IF in whole-mount diploid tissues from wild type and Su(var)3-9 mutants are shown. Each image is an optical section. γH2Av- and Rad51-positive cells in Su(var)3-9 are 96- and 11- fold increase over wild type. The p values were <0.01 by the Student's t test, and n>800 cells for each group. The scale bars = 25 mm in γH2Av IF images and 8 um in Rad51 images. B) An optical section shows a wild type diploid cell stained with DAPI; dashed lines encircle the DAPI-bright regions. Bar = 0.8 mm. Right panel are optical sections of Su(var)3-9 mutant diploid cells stained by the TUNEL assay (red foci). The foci are double-stranded breaks recognized by TdT. Enlarged images showed examples of TUNEL foci in DAPI-weak and DAPI-bright regions. Bar = 4 mm. C) shows quantitative analysis of γH2Av and TUNEL signal localizations in wild type and Su(var)3-9 cells. The distribution of γH2Av and TUNEL signals in DAPI-weak regions do not differ significantly between wild type and Su(var)3-9 (p>0.05 by Chi-square test; n>40 for each genotype). Compared to wild type, γH2Av foci localized to DAPI-bright regions in Su(var)3-9 is 96-fold higher and 88-fold for TUNEL (p<0.001 by Chi-square test; n>35 for each genotype).
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pgen-1000435-g001: Su(var)3-9 mutant somatic cells display increased DNA damage in heterochromatin.A) γH2Av (red) and Rad51 (green) IF in whole-mount diploid tissues from wild type and Su(var)3-9 mutants are shown. Each image is an optical section. γH2Av- and Rad51-positive cells in Su(var)3-9 are 96- and 11- fold increase over wild type. The p values were <0.01 by the Student's t test, and n>800 cells for each group. The scale bars = 25 mm in γH2Av IF images and 8 um in Rad51 images. B) An optical section shows a wild type diploid cell stained with DAPI; dashed lines encircle the DAPI-bright regions. Bar = 0.8 mm. Right panel are optical sections of Su(var)3-9 mutant diploid cells stained by the TUNEL assay (red foci). The foci are double-stranded breaks recognized by TdT. Enlarged images showed examples of TUNEL foci in DAPI-weak and DAPI-bright regions. Bar = 4 mm. C) shows quantitative analysis of γH2Av and TUNEL signal localizations in wild type and Su(var)3-9 cells. The distribution of γH2Av and TUNEL signals in DAPI-weak regions do not differ significantly between wild type and Su(var)3-9 (p>0.05 by Chi-square test; n>40 for each genotype). Compared to wild type, γH2Av foci localized to DAPI-bright regions in Su(var)3-9 is 96-fold higher and 88-fold for TUNEL (p<0.001 by Chi-square test; n>35 for each genotype).

Mentions: Spontaneous DNA damage in whole-mount (three dimensional) larval brain and imaginal disc tissues from wild type and Su(var)3-9 mutants (generated from mothers and fathers) was examined by indirect immunofluorescence (IF) with antibodies specific to γH2Av and Rad51 (Figure 1A). γH2Av is the phosphorylated form of the histone variant H2Av (at serine 137) and is associated with DNA repair sites [35]. The Rad51 protein facilitates repair of double-stranded breaks via homologous recombination [36]. We observed that Su(var)3-9 somatic cells contained significantly increased frequencies of γH2Av and Rad51 foci in comparison to wild type (6.9-fold and 11-fold, respectively; p<0.001). Both γH2Av and Rad51 localization indicate sites of double-stranded breaks (DSBs), which we confirmed by the TUNEL (TdT-mediated dUTP nick end labeling) assay (Figure 1B; Materials and Methods).


Heterochromatic genome stability requires regulators of histone H3 K9 methylation.

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

Su(var)3-9 mutant somatic cells display increased DNA damage in heterochromatin.A) γH2Av (red) and Rad51 (green) IF in whole-mount diploid tissues from wild type and Su(var)3-9 mutants are shown. Each image is an optical section. γH2Av- and Rad51-positive cells in Su(var)3-9 are 96- and 11- fold increase over wild type. The p values were <0.01 by the Student's t test, and n>800 cells for each group. The scale bars = 25 mm in γH2Av IF images and 8 um in Rad51 images. B) An optical section shows a wild type diploid cell stained with DAPI; dashed lines encircle the DAPI-bright regions. Bar = 0.8 mm. Right panel are optical sections of Su(var)3-9 mutant diploid cells stained by the TUNEL assay (red foci). The foci are double-stranded breaks recognized by TdT. Enlarged images showed examples of TUNEL foci in DAPI-weak and DAPI-bright regions. Bar = 4 mm. C) shows quantitative analysis of γH2Av and TUNEL signal localizations in wild type and Su(var)3-9 cells. The distribution of γH2Av and TUNEL signals in DAPI-weak regions do not differ significantly between wild type and Su(var)3-9 (p>0.05 by Chi-square test; n>40 for each genotype). Compared to wild type, γH2Av foci localized to DAPI-bright regions in Su(var)3-9 is 96-fold higher and 88-fold for TUNEL (p<0.001 by Chi-square test; n>35 for each genotype).
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Related In: Results  -  Collection

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pgen-1000435-g001: Su(var)3-9 mutant somatic cells display increased DNA damage in heterochromatin.A) γH2Av (red) and Rad51 (green) IF in whole-mount diploid tissues from wild type and Su(var)3-9 mutants are shown. Each image is an optical section. γH2Av- and Rad51-positive cells in Su(var)3-9 are 96- and 11- fold increase over wild type. The p values were <0.01 by the Student's t test, and n>800 cells for each group. The scale bars = 25 mm in γH2Av IF images and 8 um in Rad51 images. B) An optical section shows a wild type diploid cell stained with DAPI; dashed lines encircle the DAPI-bright regions. Bar = 0.8 mm. Right panel are optical sections of Su(var)3-9 mutant diploid cells stained by the TUNEL assay (red foci). The foci are double-stranded breaks recognized by TdT. Enlarged images showed examples of TUNEL foci in DAPI-weak and DAPI-bright regions. Bar = 4 mm. C) shows quantitative analysis of γH2Av and TUNEL signal localizations in wild type and Su(var)3-9 cells. The distribution of γH2Av and TUNEL signals in DAPI-weak regions do not differ significantly between wild type and Su(var)3-9 (p>0.05 by Chi-square test; n>40 for each genotype). Compared to wild type, γH2Av foci localized to DAPI-bright regions in Su(var)3-9 is 96-fold higher and 88-fold for TUNEL (p<0.001 by Chi-square test; n>35 for each genotype).
Mentions: Spontaneous DNA damage in whole-mount (three dimensional) larval brain and imaginal disc tissues from wild type and Su(var)3-9 mutants (generated from mothers and fathers) was examined by indirect immunofluorescence (IF) with antibodies specific to γH2Av and Rad51 (Figure 1A). γH2Av is the phosphorylated form of the histone variant H2Av (at serine 137) and is associated with DNA repair sites [35]. The Rad51 protein facilitates repair of double-stranded breaks via homologous recombination [36]. We observed that Su(var)3-9 somatic cells contained significantly increased frequencies of γH2Av and Rad51 foci in comparison to wild type (6.9-fold and 11-fold, respectively; p<0.001). Both γH2Av and Rad51 localization indicate sites of double-stranded breaks (DSBs), which we confirmed by the TUNEL (TdT-mediated dUTP nick end labeling) assay (Figure 1B; Materials and Methods).

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