γH2AX foci on apparently intact mitotic chromosomes: not signatures of misrejoining events but signals of unresolved DNA damage

Abstract

The presence of γH2AX foci on apparently intact mitotic chromosomes is controversial because they challenge the assumed relationship between γH2AX foci and DNA double-strand breaks (DSBs). In this work, we show that after irradiation during interphase, a variety of γH2AX foci are scored in mitotic cells. Surprisingly, approximately 80% of the γH2AX foci spread over apparently undamaged chromatin at Terminal or Interstitial positions and they can display variable sizes, thus being classified as Small, Medium and Big foci. Chromosome and chromatid breaks that reach mitosis are spotted with Big (60%) and Medium (30%) Terminal γH2AX foci, but very rarely are they signaled with Small γH2AX foci. To evaluate if Interstitial γH2AX foci might be signatures of misrejoining, an mFISH analysis was performed on the same slides. The results show that Interstitial γH2AX foci lying on apparently intact chromatin do not mark sites of misrejoining, and that misrejoined events were never signaled by a γH2AX foci during mitosis. Finally, when analyzing the presence of other DNA-damage response (DDR) factors we found that all γH2AX foci-regardless their coincidence with a visible break-always colocalized with MRE11, but not with 53BP1. This pattern suggests that these γH2AX foci may be hallmarks of both microscopically visible and invisible DNA damage, in which an active, although incomplete or halted DDR is taking place.

Keywords: AU, arbitrary units; DSB, DNA double-strand break; FI, fluorescence intensity; FISH, fluorescence in situ hybridization; IRIF, ionizing radiation induced foci; M phase; MRE11; MRN complex, MRE11-Rad50-Nbs1 complex; SD, standard deviation; TIF, telomere-dysfunction induced foci; illegitimate joining; mFISH, multicolor fluorescence in situ hybridization; misrepair; residual DSBs; telomeric foci; γH2AX.

Figure 1.

γH2AX foci in mitotic chromosomes present different fluorescence intensities. (A) 665 γH2AX foci from 68 metaphases were scored using FociPicker 3D. The Fluorescence Intensity (FI) of each focus was plotted and these measures were used to classify them. The FI values of all scored foci ranged from 25 to 225 (arbitrary units; AU) and were classified into 3 categories: Small, corresponding to the foci with the lower FI (from 25 to 92.9 AU); Medium, corresponding to the foci with median FI (from 93 to 158.9 AU); and Big, corresponding to the foci with the highest FI (from 159 to 225 AU). The mean FI of Small foci (S) was statistically different from that of Medium (M) and Big foci (B) both before and after irradiation (Si, Mi, Bi). The same differences were found for Medium and Big foci (Dunn's Multiple Comparisons Test; P < 0.05 to P < 0.001). The means and standard deviation (SD) shown in the graph are calculated from 2 different slides in non irradiated cells and from 3 different slides in irradiated cells (Table 1). (B) Examples of γH2AX foci of different FI located either at a Terminal or at an Interstitial position within the chromosome are shown. (C) γH2AX foci were classified regarding their FI (Big, Medium or Small) and their position within the chromosome (Terminal or Interstitial). The percentages of each type of foci are shown. Asterisks show statistically significant differences in the number of foci (Fisher's Exact Test; P < 0.0001).

Figure 2.

Big and small γH2AX foci decorate mitotic chromosomes before and after irradiation. (A) Left: metaphase from a non-irradiated cell where very few γH2AX foci can be scored. The circles highlight a chromosome with a Big Terminal γH2AX foci and a chromosome with a Medium Interstitial γH2AX foci. The square highlights a chromosome with a Small Interstital γH2AX foci. Middle: metaphase spread obtained 48 hours after 1 Gy irradiation, in which many γH2AX foci can be seen located both interstitially and at the chromosome ends. A Big Interstitial γH2AX focus spotting a chromatid break (>) and a Small Terminal γH2AX focus (*) are highlighted. Right: after γH2AX foci scoring, telomeric FISH was applied to the same slides. (B) Telomeric FISH allows identification of terminal chromosome breaks. Terminal γH2AX foci are highlighted with white arrows. In the top row, both γH2AX foci correspond to chromosome breaks, evidenced by the absence of telomeric signals in the affected chromosomes. In the bottom row, the Terminal γH2AX focus does not correspond with a break, as telomeric signals are present. (C) Left: the Terminal γH2AX foci from 14 metaphases were scored after irradiation and classified regarding their FI. Small, Medium and Big foci were statistically different (*) (ANOVA test; P < 0.001). The number of foci scored (N), as well as the mean FI and SD values for each category are shown. In each group, those γH2AX foci that signal chromosome breaks are highlighted using filled circles and their percentage is given below. Right: the chromosome breaks scored are classified according to the size of the γH2AX foci that signals them. (D) Left column: a chromatid break (*) scored after DAPI reverse analysis marked with a Big Interstitial γH2AX foci that spans over both sides of the broken chromatid. Right column: the white box (*) signals a chromosome gap, also marked with a Big Interstitial γH2AX foci.

Figure 3.

Many γH2AX foci locate on apparently normal telomeres. (A) Many of the γH2AX foci scored after irradiation are located at the terminal ends of chromosomes and seem to lie on apparently intact chromatin (>). Most of the telomeres lying beneath these foci show a normal size and structure (>) except for a few shorter telomeric signals (*). (B) Examples of eroded (* top row, right image) or split (* low row, right image) telomeres decorated with γH2AX foci that co-exist with apparently normal telomeres. In turn, some of these apparently normal telomeres can also be signaled with a terminal γH2AX focus (white circle). (C) Western blot showing the presence of wild-type p53 in the cells used. The first lane is a protein molecular weight marker. As expected, the phospho-p53 signal clearly increased after irradiation (IR: irradiation).

Figure 4.

γH2AX foci do not flag misrejoining events. (A) From left to right. After γH2AX foci immunofluorescence, a telomeric-FISH analysis is performed on the same slides. In this metaphase, an apparently intact chromosome with a Medium Interstitial γH2AX focus (upper white box) and a broken chromosome and an acentric fragment with a Big and a Small Terminal γH2AX foci (both inside the lower white box) are highlighted. Top row: the mFISH analysis shows that the chromosome with the internal γH2AX focus is an intact chromosome 8 that has not suffered any misrejoining event. Low row: the 2 broken chromosomes with Terminal γH2AX foci are the 2 pieces resulting from the breakage of chromosome 16. (B) On the top row, a translocation between chromosomes 9 and 10 can be identified. This translocated chromosome has an Interstitial γH2AX focus that, clearly, does not lie on the joining point (denoted by white arrows). In the lower row, 3 chromosomes bearing Interstitial γH2AX foci have been highlighted. Telomeric FISH and mFISH images show that all of them are complete chromosomes that have not suffered any illegitimate rejoining event. (C) In a reverse approach, mFISH was used to identify misrejoining events such as translocations. Once identified, the telomeric and γH2AX foci labeling was analyzed. Top row: a (4;8) translocation is highlighted (white box) in which no γH2AX focus can be observed at the joining point. Low row: a reciprocal translocation between chromosomes 1 and 10 can be identified (white contour) and, again, no γH2AX foci can be observed at either joining site.

Figure 5.

Co-localization pattern of γH2AX foci with MRE11 and 53BP1. (A) A metaphase spread showing γH2AX and MRE11 co-localization. These 2 proteins co-localize at sites of chromatin damage such as a chromosome break (upper row) and at sites of apparently intact chromatin (middle row). In the lower row, an apparently intact chromosome from a different metaphase bears an internal γH2AX focus that also co-localizes with MRE11. (B) Analysis of 53BP1 and γH2AX shows that, after irradiation, 53BP1 co-localizes with γH2AX foci at interphase nuclei (>) but not with γH2AX foci located on mitotic chromosomes, even when these foci are signaling chromatin breaks (*).