H2AX phosphorylation within the G1 phase after UV irradiation depends on nucleotide excision repair and not DNA double-strand breaks

Abstract

The variant histone H2AX is phosphorylated in response to UV irradiation of primary human fibroblasts in a complex fashion that is radically different from that commonly reported after DNA double-strand breaks. H2AX phosphorylation after exposure to ionizing radiation produces foci, which are detectable by immunofluorescence microscopy and have been adopted as clear and consistent quantitative markers for DNA double-strand breaks. Here we show that in contrast to ionizing radiation, UV irradiation mainly induces H2AX phosphorylation as a diffuse, even, pan-nuclear staining. UV induced pan-nuclear phosphorylation of H2AX is present in all phases of the cell cycle and is highest in S phase. H2AX phosphorylation in G(1) cells depends on nucleotide excision repair factors that may expose the S-139 site to kinase activity, is not due to DNA double-strand breaks, and plays a larger role in UV-induced signal transduction than previously realized.

Fig. 1.

UV irradiation induces H2AX phosphorylation in all phases of the cell cycle in cycling and contact-inhibited cells. (a) Overlay of unirradiated (filled histogram) and UV-irradiated (open histogram) cells represents flow cytometry data that demonstrate increases in H2AX phosphorylation after exposure to UV irradiation. For the sample used in the H2AX phosphorylation assay, a cell cycle profile also was created by measuring total DNA content with PI. The cell cycle profiles are presented below the corresponding H2AX phosphorylation assay. Data are presented for either cycling cells representing all cells or cells in G₁. (b) Analysis of different levels of γH2AX phosphorylation 4 h after UV irradiation (20 J/m² UV-C).

Fig. 2.

Time course of γH2AX fluorescence after UV irradiation. (a) Histograms represent γH2AX fluorescence intensity at different time points after UV irradiation overlayed on the control sample. Determination of G₁ and S phase population based on analysis of concurrent PI staining. (b) Three-dimensional plots representing cell number (y axis), PI fluorescence (x axis), and gH2Ax fluorescence (z axis).

Fig. 3.

UV irradiation induces predominantly pan-nuclear H2AX phosphorylation in all stages of the cell cycle. (a) Immunofluorescence of γH2AX and CPDs in normal human fibroblasts after exposure to UV, ionizing radiation or mock treatment. Nuclear DNA was counterstained with DAPI. (b) Quantitation of γH2AX staining pattern in human fibroblasts 4 h after exposure to 20 J/m² UV. Roman numerals within the image of UV-irradiated cells correspond to the different types of γH2AX staining that quantitated on the pie chart. (c) Immunofluorescence of γH2AX and BrdU in normal human fibroblasts after UV irradiation. Nuclear DNA was counterstained with DAPI.

Fig. 4.

UV irradiation restricted to subnuclear regions triggers phosphorylation of H2AX exclusively at sites of UV exposure and not throughout the nucleus. Normal human fibroblasts were irradiated through 3.0-μm isopore filters with 100 J/m² UV. Overlay of γH2AX and CPDs images shows that H2AX phosphorylation occurred exclusively at nuclear sites of UV irradiation.

Fig. 5.

H2AX phosphorylation after UV irradiation occurs in the absence of DNA DSBs. (a) Filled histogram represents flow cytometry of γH2AX fluorescence in untreated control cells, whereas open solid line and dashed line histograms represent cells treated with 20 J/m² UV or 5 Gy ionizing radiation, respectively. Cells were gated for G₁ based on the genomic DNA content as determined by PI fluorescence. Immunofluorescence images illustrating the difference between γH2AX foci formation after ionizing radiation and pan-nuclear H2Ax phosphorylation after UV irradiation both were captured at a ×600 total magnification and were captured by using the same exposure time. (b) Immunofluorescence of γH2AX and p53-binding protein 1 (53BP1) after exposure to UV, ionizing irradiation, or mock treatment. Nuclear DNA was counterstained with DAPI. (c) Immunofluorescence of γH2AX and phospho NBS1 (ser343) after exposure to UV, ionizing radiation, or mock treatment. Nuclear DNA was counterstained with DAPI. (d) The kinase inhibitor wortmannin does not prevent H2AX phosphorylation in G₁ cells after UV irradiation. Filled histogram represents untreated control cells, whereas open histograms represent exposure to UV or ionizing radiation either with or without wortmannin (solid red line and solid black line, respectively). For wortmannin kinase inhibition, cells were incubated with 100 μM wortmannin for 30 min before irradiation and for the entirety of the incubation after irradiation. Cells were gated for G₁ based on the genomic DNA content as determined by PI fluorescence.

Fig. 6.

Reduced levels of γH2AX after UV irradiation in NER-deficient cell lines. (a) Flow cytometry of γH2AX levels in normal, XP-A, and XP-C cell lines. Overlay of γH2AX levels of unirradiated cells (filled histogram) compared with cells 4 h after exposure to 20 J/m² UV (open histogram) in either all cells or G₁ cells. (b) γH2AX immunofluorescence in WT, XP-A, and XP-C cells 4 h after exposure to 20 J/m² UV. Images were acquired with the same exposure time and processed equally. All images are representative of at least three replicates.