H2AX chromatin structures and their response to DNA damage revealed by 4Pi microscopy

Abstract

DNA double-strand breaks (DSBs) caused by cellular exposure to genotoxic agents or produced by inherent metabolic processes initiate a rapid and highly coordinated series of molecular events resulting in DNA damage signaling and repair. Phosphorylation of histone H2AX to form gamma-H2AX is one of the earliest of these events and is important for coordination of signaling and repair activities. An intriguing aspect of H2AX phosphorylation is that gamma-H2AX spreads a limited distance up to 1-2 Mbp from the site of a DNA break in mammalian cells. However, neither the distribution of H2AX throughout the genome nor the mechanism that defines the boundary of gamma-H2AX spreading have yet been described. Here, we report the identification of previously undescribed H2AX chromatin structures by successfully applying 4Pi microscopy to visualize endogenous nuclear proteins. Our observations suggest that H2AX is not distributed randomly throughout bulk chromatin, rather it exists in distinct clusters that themselves are uniformly distributed within the nuclear volume. These data support a model in which the size and distribution of H2AX clusters define the boundaries of gamma-H2AX spreading and also may provide a platform for the immediate and robust response observed after DNA damage.

Fig. 1.

4Pi images of H2AX and γ-H2AX clusters during a time course of DNA damage and repair. HeLa cells exposed to 3 Gy IR were fixed 15, 45, and 180 min after irradiation and immunolabeled with γ-H2AX (red) and H2AX (green) antibodies. Images were obtained by using a 4Pi microscope and deconvolved. 3D- rendered 4Pi data for the 15, 45, and 180 min after IR time points are displayed in a, b, and c, respectively. (Insets) γ-H2AX data alone. Movies for all time points after IR (15, 45, 90, 180, 360, and 720 min) are presented in Movies 1–6.

Fig. 2.

Cluster size as a function of time after DNA damage. The characteristic size of both H2AX and γ-H2AX clusters were determined for each time point during DNA repair by using the autocorrelation function of the deconvolved 4Pi data (see Supporting Materials and Methods). Whereas the H2AX clusters remain at subresolution size during the whole time course, the characteristic size of γ-H2AX clusters increases 2-fold reaching a plateau after ≈90 min.

Fig. 3.

3D distribution of H2AX and γ-H2AX clusters in the nuclear volume. Fluorescence intensities of both the γ-H2AX) (red) and H2AX (green) channels were analyzed in 3D (confocal data are used for this analysis because the microscope collects three channels, whereas the 4Pi instrument collects only two). Examples of xy and xz sections of 3D data sets showing the DAPI (a), H2AX (b), γ-H2AX (c), and overlay (d) signals used for the calculations are shown. Nuclear dimensions were determined by creating a series of shells shown in different colors (f) based on eroding smoothed DAPI stain images (e) in 400-nm steps (see Supporting Materials and Methods). Histograms for t = 0 (g), 15 (h), and 720 (i) min after IR averaged over 10–11 cells are displayed (SE ranges from 5% to 20%) and show a time-dependent change in fluorescence intensity for the γ-H2AX signal (red) and little change in the intensity of the H2AX signal. Both intensities show little to no dependence on the distance from the nuclear periphery (δ). (g–i) Because of an inaccuracy in the identification of the nuclear boundary, defined by a smoothed DAPI signal threshold, a continuous rise in the signal for the first two shells instead of a step-like rise is observed. For the ratios shown between γ-H2AX and H2AX, this cancels out.

Fig. 4.

Lack of colocalization of H2AX and γ-H2AX staining throughout the nuclear volume. (a) Sections in all directions through a typical 3D 4Pi data set show no significant overlap between the red (γ-H2AX) and green (H2AX) signals (dashed lines represent positions of different sections). Analysis of all data sets by cytofluorograms (example shown in b) reveals a minimal signal overlap (note the logarithmic color scale). (b and b Inset) Profiles in b Inset represent the cytofluorogram data along the dotted lines in b. The fact that they are the same shape agrees with the expected coincidental overlap of the resolution-limited signals. The profiles were normalized according to the numbers displayed in b Inset. The dotted arrows (upper left in b) indicate the directions for the cross-talk corrected signal (see Supporting Materials and Methods).

Fig. 5.

Comparison of confocal vs. 4Pi microscopy for endogenous human histone H2AX. (a and b) The data shown represent the H2AX staining in a HeLa cell nucleus as seen with confocal and 4Pi microscopy, respectively, in a maximum projection of 3D data sets. (c and d) The same single xz section taken from the 3D data sets of a and b, respectively. The dotted ellipses mark areas where the resolution enhancement can be seen best. (c Inset and d Inset) The PSFs of confocal and 4Pi microscopy at the same scale as a–d for comparison.