Topological Analysis of γH2AX and MRE11 Clusters Detected by Localization Microscopy during X-ray-Induced DNA Double-Strand Break Repair

Abstract

DNA double-strand breaks (DSBs), known as the most severe damage in chromatin, were induced in breast cancer cells and normal skin fibroblasts by 2 Gy ionizing photon radiation. In response to DSB induction, phosphorylation of the histone variant H2AX to γH2AX was observed in the form of foci visualized by specific antibodies. By means of super-resolution single-molecule localization microscopy (SMLM), it has been recently shown in a first article about these data that these foci can be separated into clusters of about the same size (diameter ~400 nm). The number of clusters increased with the dose applied and decreased with the repair time. It has also been shown that during the repair period, antibody-labeled MRE11 clusters of about half of the γH2AX cluster diameter were formed inside several γH2AX clusters. MRE11 is part of the MRE11-RAD50-NBS1 (MRN) complex, which is known as a DNA strand resection and broken-end bridging component in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). This article is a follow-up of the former ones applying novel procedures of mathematics (topology) and similarity measurements on the data set: to obtain a measure for cluster shape and shape similarities, topological quantifications employing persistent homology were calculated and compared. In addition, based on our findings that γH2AX clusters associated with heterochromatin show a high degree of similarity independently of dose and repair time, these earlier published topological analyses and similarity calculations comparing repair foci within individual cells were extended by topological data averaging (2nd-generation heatmaps) over all cells analyzed at a given repair time point; thereby, the two dimensions (0 and 1) expressed by components and holes were studied separately. Finally, these mean value heatmaps were averaged, in addition. For γH2AX clusters, in both normal fibroblast and MCF-7 cancer cell lines, an increased similarity was found at early time points (up to 60 min) after irradiation for both components and holes of clusters. In contrast, for MRE11, the peak in similarity was found at later time points (2 h up to 48 h) after irradiation. In general, the normal fibroblasts showed quicker phosphorylation of H2AX and recruitment of MRE11 to γH2AX clusters compared to breast cancer cells and a shorter time interval of increased similarity for γH2AX clusters. γH2AX foci and randomly distributed MRE11 molecules naturally occurring in non-irradiated control cells did not show any significant topological similarity.

Keywords: DNA double-strand breaks; ionizing photon irradiation; persistent homology; single-molecule localization microscopy; topology of MRE11 clusters; topology of γH2AX clusters.

Figure 1

Example of images of γH2AX clusters after 180 min after 2 Gy irradiation of human skin fibroblasts. (a) Clusters detected by the original program used in [21]. (b) Cluster image obtained with the DBSCAN algorithm (cluster parameter values: N_min = 110; ε = 200 nm). (c) The same image as (b) but with the closing function applied. The high co-incidence of (a) and (c) can be observed. Scale bar: 2 µm.

Figure 2

Confocal images of γH2AX (green) and MRE11 (red) focus formation in breast cancer cells exposed to 2 Gy of γ-radiation and left repair DSBs for 30 min (A) or 4 h (B). The top line of both panels represents the maximum immunofluorescence confocal microscopy images composed of 40 optical confocal slices (0.2 µm thick) and shown in all three x-y, x-z, and y-z planes. The bottom lines show a single confocal slice through the nuclei displayed in the top lines. For the post-irradiation period of 4 h, the inserts show a selected γH2AX+MRE11 focus in a magnified view. The white arrows indicate the position of x-y and y-z confocal planes. Chromatin counterstaining was performed with DAPI (artificially blue).

Figure 3

Examples of cluster images of a human skin fibroblast nucleus 60 min after irradiation with 2 Gy. (a) γH2AX labeling can be found in clusters highlighted by red points in closed areas. (b) MRE11 labeling is dispersed over the cell nucleus and clustered, too. (c) The merged image of (a) and (b) indicating the embedding of MRE11 in γH2AX clusters. Scale bar: 2 µm.

Figure 4

Example of the three 1st-generation heatmaps of γH2AX clusters compared with each other for the control group of the MCF-7 cell line (10 min values). (a) The cluster similarities of the components (dim 0), (b) those of the holes (dim 1), and (c) the average values for each of the pixels from (a) and (b) are presented. Note: This example is shown because the differences in the similarity of components and holes is large. In the dim 0 heatmap, many clusters have a similarity of above 0.98 and the lowest values are around 0.96. In contrast, in the dim 1 heatmap, the highest value is at about 0.6 and practically all values are in the range of 0.2 to 0.6, excluding the diagonal ones (identity). Therefore, the average is dominated by the dim 1 values. Moreover, the problematic effect of the diagonal can be seen quite effectively as the range of values can only be observed up to a certain degree of accuracy. The differences between the colors for a relatively large spectrum of values are hardly recognizable.

Figure 5

First-generation heatmaps of γH2AX clusters in MCF-7 cells of (a,b) the irradiated samples (2 Gy) and (c,d) the non-irradiated control at the time point of 30 min after irradiation. (a,c) Heatmaps of the components; (b,d) heatmaps of the holes. Note the different color bars and the significantly different cluster numbers (more than 278 for the irradiated specimen vs. 30 for the non-irradiated specimen).

Figure 6

Averaged 1st-generation heatmaps of γH2AX clusters of the skin fibroblast cell line CCD-1059SK. Pairwise comparison of clusters (a) 30 min, (b) 120 min, (d) 180 min, and (g) 24 h after irradiation. (c) Comparison of 30 min with 120 min clusters, (e) 120 min with 180 min clusters, (f) 30 min with 180 min clusters, and (h) 30 min with 24 h clusters. Note the differences in the color bars of the heatmaps comparing the same time point and comparing different time points.

Figure 7

Second-generation heatmaps of γH2AX clusters of components (a) and holes (b) and average values of both (c) for the skin fibroblast cell line CCD-1059SK (A,B) and the breast cancer cell line MCF-7 (C,D). Each value of a pixel of these heatmaps represents the mean value of one 1st-generation heatmap. Note the differences in the color bars between (a–c), while the visual pattern may be similar.

Figure 8

Second-generation heatmaps of MRE11 clusters of components (a) and holes (b) and average values of both (c) for the skin fibroblast cell line CCD-1059SK (A,B) and the breast cancer cell line MCF-7 (C,D). Each value of a pixel of these heatmaps represents the mean value of one 1st-generation heatmap. Note the differences in the color bars between (a_–c), while the visual pattern may be similar.