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UHRF1 is a genome caretaker that facilitates the DNA damage response to gamma-irradiation.

Mistry H, Tamblyn L, Butt H, Sisgoreo D, Gracias A, Larin M, Gopalakrishnan K, Hande MP, McPherson JP - Genome Integr (2010)

Bottom Line: UHRF1-depleted cells exhibit increased sensitivity to gamma-irradiation, suggesting a compromised cellular response to DSBs.UHRF1-depleted cells show impaired cell cycle arrest and an impaired accumulation of histone H2AX phosphorylation (gammaH2AX) in response to gamma-irradiation compared to control cells.We also demonstrate that UHRF1 is required for genome integrity, in that UHRF1-depleted cells displayed an increased frequency of chromosomal aberrations compared to control cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, M5 S 1A8, Canada. peter.mcpherson@utoronto.ca.

ABSTRACT

Background: DNA double-strand breaks (DSBs) caused by ionizing radiation or by the stalling of DNA replication forks are among the most deleterious forms of DNA damage. The ability of cells to recognize and repair DSBs requires post-translational modifications to histones and other proteins that facilitate access to lesions in compacted chromatin, however our understanding of these processes remains incomplete. UHRF1 is an E3 ubiquitin ligase that has previously been linked to events that regulate chromatin remodeling and epigenetic maintenance. Previous studies have demonstrated that loss of UHRF1 increases the sensitivity of cells to DNA damage however the role of UHRF1 in this response is unclear.

Results: We demonstrate that UHRF1 plays a critical role for facilitating the response to DSB damage caused by gamma-irradiation. UHRF1-depleted cells exhibit increased sensitivity to gamma-irradiation, suggesting a compromised cellular response to DSBs. UHRF1-depleted cells show impaired cell cycle arrest and an impaired accumulation of histone H2AX phosphorylation (gammaH2AX) in response to gamma-irradiation compared to control cells. We also demonstrate that UHRF1 is required for genome integrity, in that UHRF1-depleted cells displayed an increased frequency of chromosomal aberrations compared to control cells.

Conclusions: Our findings indicate a critical role for UHRF1 in maintenance of chromosome integrity and an optimal response to DSB damage.

No MeSH data available.


Related in: MedlinePlus

Decreased irradiation-induced γH2AX in UHRF1-depleted cells. UHRF1 and control shRNA-expressing cells were exposed to either 1 Gy (A) or 5 Gy (B) γ-irradiation and harvested following 0, 1, 3, 6, 12 or 24 h. Representative histograms plot γH2AX expression as measured by γH2AX-FITC intensity/cell (y-axis) vs. DNA content (x-axis) (left hand side of panel). Numbers indicate the percentage of cells showing elevated γH2AX levels. The percentage of cells with elevated γH2AX expression plotted is the mean ± S.D. of three independent experiments (right hand side of panel). (C) The decreased accumulation of γH2AX in UHRF1-depleted cells was confirmed by indirect immunofluorescence 12 h after 5 Gy γ-irradiation.
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Figure 4: Decreased irradiation-induced γH2AX in UHRF1-depleted cells. UHRF1 and control shRNA-expressing cells were exposed to either 1 Gy (A) or 5 Gy (B) γ-irradiation and harvested following 0, 1, 3, 6, 12 or 24 h. Representative histograms plot γH2AX expression as measured by γH2AX-FITC intensity/cell (y-axis) vs. DNA content (x-axis) (left hand side of panel). Numbers indicate the percentage of cells showing elevated γH2AX levels. The percentage of cells with elevated γH2AX expression plotted is the mean ± S.D. of three independent experiments (right hand side of panel). (C) The decreased accumulation of γH2AX in UHRF1-depleted cells was confirmed by indirect immunofluorescence 12 h after 5 Gy γ-irradiation.

Mentions: Phosphorylation of H2AX on serine 139 (γH2AX) is an early event after introduction of DSBs [26]. To monitor the kinetics and extent of γH2AX following irradiation, we measured γH2AX content 0, 1, 3, 6, 12 and 24 h in controls and UHRF1-depleted cells following 1 Gy or 5 Gy of γ-irradiation (Figure 4). Non-irradiated cells depleted of UHRF1 showed a nominally higher percentage of cells positive for γH2AX compared to control cells. Following 1 Gy of exposure, control cells showed a dramatic increase in the percentage of cells positive for γH2AX as expected, with maximal levels at 6 h that decreased to a level similar to non-irradiated cells by 24 h (Figure 4A). This time course of γH2AX induction corresponded to the degree of cell cycle arrest observed at 1 Gy (Figure 3A). Interestingly, the percentage of cells positive for γH2AX was markedly lower when UHRF1 was depleted compared to control cells. Following exposure to 5 Gy of irradiation, control cells showed a more drastic increase of γH2AX-positive cells with maximal levels 1-6 h following irradiation. Again, UHRF1-depleted cells showed an attenuated increase in the percentage of γH2AX-positive cells (Figure 4B). These findings were confirmed by detection of γH2AX by indirect immunofluorescence (Figure 4C). Taken together, these findings suggest that accumulation of γH2AX is defective in UHRF1-depleted cells following γ-irradiation.


UHRF1 is a genome caretaker that facilitates the DNA damage response to gamma-irradiation.

Mistry H, Tamblyn L, Butt H, Sisgoreo D, Gracias A, Larin M, Gopalakrishnan K, Hande MP, McPherson JP - Genome Integr (2010)

Decreased irradiation-induced γH2AX in UHRF1-depleted cells. UHRF1 and control shRNA-expressing cells were exposed to either 1 Gy (A) or 5 Gy (B) γ-irradiation and harvested following 0, 1, 3, 6, 12 or 24 h. Representative histograms plot γH2AX expression as measured by γH2AX-FITC intensity/cell (y-axis) vs. DNA content (x-axis) (left hand side of panel). Numbers indicate the percentage of cells showing elevated γH2AX levels. The percentage of cells with elevated γH2AX expression plotted is the mean ± S.D. of three independent experiments (right hand side of panel). (C) The decreased accumulation of γH2AX in UHRF1-depleted cells was confirmed by indirect immunofluorescence 12 h after 5 Gy γ-irradiation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2914011&req=5

Figure 4: Decreased irradiation-induced γH2AX in UHRF1-depleted cells. UHRF1 and control shRNA-expressing cells were exposed to either 1 Gy (A) or 5 Gy (B) γ-irradiation and harvested following 0, 1, 3, 6, 12 or 24 h. Representative histograms plot γH2AX expression as measured by γH2AX-FITC intensity/cell (y-axis) vs. DNA content (x-axis) (left hand side of panel). Numbers indicate the percentage of cells showing elevated γH2AX levels. The percentage of cells with elevated γH2AX expression plotted is the mean ± S.D. of three independent experiments (right hand side of panel). (C) The decreased accumulation of γH2AX in UHRF1-depleted cells was confirmed by indirect immunofluorescence 12 h after 5 Gy γ-irradiation.
Mentions: Phosphorylation of H2AX on serine 139 (γH2AX) is an early event after introduction of DSBs [26]. To monitor the kinetics and extent of γH2AX following irradiation, we measured γH2AX content 0, 1, 3, 6, 12 and 24 h in controls and UHRF1-depleted cells following 1 Gy or 5 Gy of γ-irradiation (Figure 4). Non-irradiated cells depleted of UHRF1 showed a nominally higher percentage of cells positive for γH2AX compared to control cells. Following 1 Gy of exposure, control cells showed a dramatic increase in the percentage of cells positive for γH2AX as expected, with maximal levels at 6 h that decreased to a level similar to non-irradiated cells by 24 h (Figure 4A). This time course of γH2AX induction corresponded to the degree of cell cycle arrest observed at 1 Gy (Figure 3A). Interestingly, the percentage of cells positive for γH2AX was markedly lower when UHRF1 was depleted compared to control cells. Following exposure to 5 Gy of irradiation, control cells showed a more drastic increase of γH2AX-positive cells with maximal levels 1-6 h following irradiation. Again, UHRF1-depleted cells showed an attenuated increase in the percentage of γH2AX-positive cells (Figure 4B). These findings were confirmed by detection of γH2AX by indirect immunofluorescence (Figure 4C). Taken together, these findings suggest that accumulation of γH2AX is defective in UHRF1-depleted cells following γ-irradiation.

Bottom Line: UHRF1-depleted cells exhibit increased sensitivity to gamma-irradiation, suggesting a compromised cellular response to DSBs.UHRF1-depleted cells show impaired cell cycle arrest and an impaired accumulation of histone H2AX phosphorylation (gammaH2AX) in response to gamma-irradiation compared to control cells.We also demonstrate that UHRF1 is required for genome integrity, in that UHRF1-depleted cells displayed an increased frequency of chromosomal aberrations compared to control cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, M5 S 1A8, Canada. peter.mcpherson@utoronto.ca.

ABSTRACT

Background: DNA double-strand breaks (DSBs) caused by ionizing radiation or by the stalling of DNA replication forks are among the most deleterious forms of DNA damage. The ability of cells to recognize and repair DSBs requires post-translational modifications to histones and other proteins that facilitate access to lesions in compacted chromatin, however our understanding of these processes remains incomplete. UHRF1 is an E3 ubiquitin ligase that has previously been linked to events that regulate chromatin remodeling and epigenetic maintenance. Previous studies have demonstrated that loss of UHRF1 increases the sensitivity of cells to DNA damage however the role of UHRF1 in this response is unclear.

Results: We demonstrate that UHRF1 plays a critical role for facilitating the response to DSB damage caused by gamma-irradiation. UHRF1-depleted cells exhibit increased sensitivity to gamma-irradiation, suggesting a compromised cellular response to DSBs. UHRF1-depleted cells show impaired cell cycle arrest and an impaired accumulation of histone H2AX phosphorylation (gammaH2AX) in response to gamma-irradiation compared to control cells. We also demonstrate that UHRF1 is required for genome integrity, in that UHRF1-depleted cells displayed an increased frequency of chromosomal aberrations compared to control cells.

Conclusions: Our findings indicate a critical role for UHRF1 in maintenance of chromosome integrity and an optimal response to DSB damage.

No MeSH data available.


Related in: MedlinePlus