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

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.

Figure 1

Characterization of UHRF1-depleted HeLa cell lines. (A) Stable depletion of UHRF1 by shRNA in HeLa cells as shown by Western analysis (UHRF1 shRNA clones 1-3) compared to clones expressing a nonspecific shRNA (control shRNA clones 1-3). Tubulin levels are shown as loading controls. (B) Representative indirect immunofluorescence of H3K9me3 and H4K20me3 in control and UHRF1 shRNA cells at 40 × magnification. (C-D) Representative indirect immunofluorescence deconvolution of H3K9me3 and H4K20me3 in two UHRF1 shRNA and two control shRNA cell lines. Line traces are shown on the right (blue = DAPI, red = H3K9me3 or H3K20me3) for representative photomicrographs labeled with Greek characters.

Figure 2

Impact of UHRF1 loss on proliferation and sensitivity to γ-irradiation. (A) Cell growth curves of UHRF1-depleted and control cell lines. (B) Susceptibility of UHRF1-depleted cell lines to γ-irradiation. Cell lines were exposed to ionizing radiation and sensitivity was measured by colony formation.

Figure 3

Cell cycle analysis in UHRF1-depleted cells following γ-irradiation. UHRF1 or control shRNA-expressing cells were pulsed with BrdU and then treated with 1 Gy (A) or 5 Gy (B) of γ-irradiation. Cells were harvested at the indicated times and stained with PI and for BrdU. For each time point, representative DNA content histograms (PI stain) are shown on the left with corresponding BrdU-PI bivariate plots on the right. Numerical values indicate percentage of G1, S and G2/M cells for each time point. The percentage change of cells in either G1 phase (C), S phase (D) or G2/M phases (E) compared to non-irradiated cells are plotted as mean ± S.D. of three independent experiments (shown for 1 Gy).

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.

Figure 5

Chromosomal instability in UHRF1-depleted cells. (A-D) Representative photomicrographs showing presence of micronuclei in UHRF1-depleted cells. (E) Percentage of cells with micronuclei in control cells vs. UHRF1-depleted cells. Determinations from each cell line were performed in triplicate (n ≥ 200 cells/determination) and error bars represent standard deviations from the mean. (F) Percentage of control or UHRF1-depleted cells with 1, 2, 3, or 4 centrosomes per cell. (G-I) Representative images of DAPI-stained metaphases from UHRF1-depleted cells showing chromosomal aberrations (image shown in H is magnification of image in G). br = chromosome break, cf = centrosomal fragment, af = acentric fragments. Telomeres are visualized in red, centromeres in green.