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Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin.

Kinner A, Wu W, Staudt C, Iliakis G - Nucleic Acids Res. (2008)

Bottom Line: In higher eukaryotic cells, DSBs in chromatin promptly initiate the phosphorylation of the histone H2A variant, H2AX, at Serine 139 to generate gamma-H2AX.This has allowed the development of an assay that has proved particularly useful in the molecular analysis of the processing of DSBs.We conclude with a critical analysis of the strengths and weaknesses of the approach and present some interesting applications of the resulting methodology.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, Hufelandstrasse 55, 45122 Essen, Germany.

ABSTRACT
DNA double-strand breaks (DSBs) are extremely dangerous lesions with severe consequences for cell survival and the maintenance of genomic stability. In higher eukaryotic cells, DSBs in chromatin promptly initiate the phosphorylation of the histone H2A variant, H2AX, at Serine 139 to generate gamma-H2AX. This phosphorylation event requires the activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs, ATM, and ATR, and serves as a landing pad for the accumulation and retention of the central components of the signaling cascade initiated by DNA damage. Regions in chromatin with gamma-H2AX are conveniently detected by immunofluorescence microscopy and serve as beacons of DSBs. This has allowed the development of an assay that has proved particularly useful in the molecular analysis of the processing of DSBs. Here, we first review the role of gamma-H2AX in DNA damage response in the context of chromatin and discuss subsequently the use of this modification as a surrogate marker for mechanistic studies of DSB induction and processing. We conclude with a critical analysis of the strengths and weaknesses of the approach and present some interesting applications of the resulting methodology.

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γ-H2AX foci are preferentially formed in regions of euchromatin. Elutriated G2 cells of DSB repair-deficient LIG4−/− MEFs were exposed to 16 Gy X-rays and analyzed for γ-H2AX immunofluorescence 3 h later. The picture on the left shows a DAPI-stained nucleus. Bright areas correspond to nuclear regions with increased DNA presence, thought to reflect densely packaged heterochromatin. The picture at the center shows γ-H2AX immunofluorescence obtained as described in Figure 5. The picture on the right shows an overlay of the two images with DNA displayed in blue and γ-H2AX foci in red. Note the nearly complete absence of γ-H2AX foci from heterochromatic areas, as well as from areas in the nucleus with reduced amounts of DNA.
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Figure 7: γ-H2AX foci are preferentially formed in regions of euchromatin. Elutriated G2 cells of DSB repair-deficient LIG4−/− MEFs were exposed to 16 Gy X-rays and analyzed for γ-H2AX immunofluorescence 3 h later. The picture on the left shows a DAPI-stained nucleus. Bright areas correspond to nuclear regions with increased DNA presence, thought to reflect densely packaged heterochromatin. The picture at the center shows γ-H2AX immunofluorescence obtained as described in Figure 5. The picture on the right shows an overlay of the two images with DNA displayed in blue and γ-H2AX foci in red. Note the nearly complete absence of γ-H2AX foci from heterochromatic areas, as well as from areas in the nucleus with reduced amounts of DNA.

Mentions: An additional confounding factor in the analysis of induction and repair of DSBs via γ-H2AX foci quantification comes from the observation that H2AX phosphorylation is diminished in areas of heterochromatin (128,129). We have also noted similar trends that are particularly striking in LIG4−/−-deficient MEFs exposed to high doses of IR. Figure 7 shows a representative example of a cell exposed to 16 Gy and analyzed 3 h later. Because of the DSB repair defect in these cells, γ-H2AX foci formation is still at a maximum at this time. It is evident that very few γ-H2AX foci are detected in the darkly stained areas of heterochromatin—despite the fact that the increased amount of DNA in these areas will lead to increased presence of DSBs after IR. Differential formation, or detection, of γ-H2AX in regions of chromatin with different organization will bias DSB analysis based on γ-H2AX foci formation. Weak H2AX phosphorylation in heterochromatin is also found in yeast, and in mouse fibroblasts, an increase of γ-H2AX foci size is observed after chromatin becomes more accessible (130). Notably, recent results indicate eviction of heterochromatin protein 1β (HP1 β) bound to lysine-9-methylated histone H3 after DNA damage through phosphorylation on Thr51 possibly by CK2 (131). This modification promotes H2AX phosphorylation and suggests a mechanism for γ-H2AX generation in areas of heterochromatin. Notably, a recent report postulates that ATM signaling temporarily perturbs heterochromatin via KAP-1 to facilitate DSB repair in these rather inaccessible regions of chromatin (132). However, H2AX phosphorylation is significantly slower in mitotic as compared to G1 CHO cells (119), in agreement with reduced γ-H2AX formation under conditions of condensed chromatin.Figure 7.


Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin.

Kinner A, Wu W, Staudt C, Iliakis G - Nucleic Acids Res. (2008)

γ-H2AX foci are preferentially formed in regions of euchromatin. Elutriated G2 cells of DSB repair-deficient LIG4−/− MEFs were exposed to 16 Gy X-rays and analyzed for γ-H2AX immunofluorescence 3 h later. The picture on the left shows a DAPI-stained nucleus. Bright areas correspond to nuclear regions with increased DNA presence, thought to reflect densely packaged heterochromatin. The picture at the center shows γ-H2AX immunofluorescence obtained as described in Figure 5. The picture on the right shows an overlay of the two images with DNA displayed in blue and γ-H2AX foci in red. Note the nearly complete absence of γ-H2AX foci from heterochromatic areas, as well as from areas in the nucleus with reduced amounts of DNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2553572&req=5

Figure 7: γ-H2AX foci are preferentially formed in regions of euchromatin. Elutriated G2 cells of DSB repair-deficient LIG4−/− MEFs were exposed to 16 Gy X-rays and analyzed for γ-H2AX immunofluorescence 3 h later. The picture on the left shows a DAPI-stained nucleus. Bright areas correspond to nuclear regions with increased DNA presence, thought to reflect densely packaged heterochromatin. The picture at the center shows γ-H2AX immunofluorescence obtained as described in Figure 5. The picture on the right shows an overlay of the two images with DNA displayed in blue and γ-H2AX foci in red. Note the nearly complete absence of γ-H2AX foci from heterochromatic areas, as well as from areas in the nucleus with reduced amounts of DNA.
Mentions: An additional confounding factor in the analysis of induction and repair of DSBs via γ-H2AX foci quantification comes from the observation that H2AX phosphorylation is diminished in areas of heterochromatin (128,129). We have also noted similar trends that are particularly striking in LIG4−/−-deficient MEFs exposed to high doses of IR. Figure 7 shows a representative example of a cell exposed to 16 Gy and analyzed 3 h later. Because of the DSB repair defect in these cells, γ-H2AX foci formation is still at a maximum at this time. It is evident that very few γ-H2AX foci are detected in the darkly stained areas of heterochromatin—despite the fact that the increased amount of DNA in these areas will lead to increased presence of DSBs after IR. Differential formation, or detection, of γ-H2AX in regions of chromatin with different organization will bias DSB analysis based on γ-H2AX foci formation. Weak H2AX phosphorylation in heterochromatin is also found in yeast, and in mouse fibroblasts, an increase of γ-H2AX foci size is observed after chromatin becomes more accessible (130). Notably, recent results indicate eviction of heterochromatin protein 1β (HP1 β) bound to lysine-9-methylated histone H3 after DNA damage through phosphorylation on Thr51 possibly by CK2 (131). This modification promotes H2AX phosphorylation and suggests a mechanism for γ-H2AX generation in areas of heterochromatin. Notably, a recent report postulates that ATM signaling temporarily perturbs heterochromatin via KAP-1 to facilitate DSB repair in these rather inaccessible regions of chromatin (132). However, H2AX phosphorylation is significantly slower in mitotic as compared to G1 CHO cells (119), in agreement with reduced γ-H2AX formation under conditions of condensed chromatin.Figure 7.

Bottom Line: In higher eukaryotic cells, DSBs in chromatin promptly initiate the phosphorylation of the histone H2A variant, H2AX, at Serine 139 to generate gamma-H2AX.This has allowed the development of an assay that has proved particularly useful in the molecular analysis of the processing of DSBs.We conclude with a critical analysis of the strengths and weaknesses of the approach and present some interesting applications of the resulting methodology.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, Hufelandstrasse 55, 45122 Essen, Germany.

ABSTRACT
DNA double-strand breaks (DSBs) are extremely dangerous lesions with severe consequences for cell survival and the maintenance of genomic stability. In higher eukaryotic cells, DSBs in chromatin promptly initiate the phosphorylation of the histone H2A variant, H2AX, at Serine 139 to generate gamma-H2AX. This phosphorylation event requires the activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs, ATM, and ATR, and serves as a landing pad for the accumulation and retention of the central components of the signaling cascade initiated by DNA damage. Regions in chromatin with gamma-H2AX are conveniently detected by immunofluorescence microscopy and serve as beacons of DSBs. This has allowed the development of an assay that has proved particularly useful in the molecular analysis of the processing of DSBs. Here, we first review the role of gamma-H2AX in DNA damage response in the context of chromatin and discuss subsequently the use of this modification as a surrogate marker for mechanistic studies of DSB induction and processing. We conclude with a critical analysis of the strengths and weaknesses of the approach and present some interesting applications of the resulting methodology.

Show MeSH
Related in: MedlinePlus