Chromatin Compaction Protects Genomic DNA from Radiation Damage

1Genomic DNA is wrapped around histones to form a nucleosome structure. Although the higher-order chromatin structure in eukaryotic cells is not fully understood, several reports, including our recent cryo-microscopy and synchrotron X-ray scattering analyses, have demonstrated that chromatin consists of irregularly folded nucleosome fibers (10-nm fibers) in cells; for review see,. Based on these studies, we suggested that interphase chromatin forms numerous compact chromatin domains, which resemble “chromatin liquid drops”, in the interphase cells. This view is in line with the predictions of the chromosome territory-interchromatin compartment (CT-IC) model. In the CT-IC model, each CT is built from a series of interconnected, megabase-sized chromatin domains, which were originally identified using pulse labeling as DNA replication foci that were shown to persist stably during subsequent cell generations. Recent high-throughput 3C studies such as Hi-C and 5C have also proposed the physical packaging of genomic DNA which has been termed “topologically associating domains”, “topological domains”, or “physical domains”.

2Although chromatin compaction is essential for mitosis to maintain the integrity of genomic information, whether compact chromatin domains confer other advantages, particularly in interphase cells, has not been elucidated. In previous in vitro studies, DNA compaction was shown to play a key role in protection against double-strand breaks (DSBs) generated by γ-rays. Therefore, we explored the significance of higher-order chromatin structures in the DSB generation process. Left unrepaired, DSBs caused by radiation can lead to chromosome fragmentation, chromosome loss, and the rearrangement of genetic information, events that are frequently associated with tumor formation and progression; also, see.

3Much is already known regarding the mechanism(s) of DSB repair; however, little is known about how chromatin structure influences DSB induction processes, especially the quantitative and mechanistic aspects, although the involvement of reactive hydroxyl radicals in the induction of DSBs has been suggested. Whether sensitivity to DSB induction differs for “open” chromatin configurations and inactive “condensed” regions has not yet been resolved, because of the following reasons: In vivo, difficulty in the manipulation of chromatin structure, the lack of an efficient damage detection system, and regional differences in DNA repair efficiency have precluded drawing a decisive conclusion. In vitro, since long genomic DNA can be damaged during experimental manipulations, no efficient in vitro system has been developed for the manipulation of long chromatin and the quantitative detection of generated DSBs.

4In the present study, we developed a novel system for chromatin manipulation and sensitive DNA damage detection, and succeeded in quantifying the DNA damage caused by ionizing irradiation. Importantly, the frequency of radiation-induced DSBs in fully decondensed chromatin was 5–50-fold higher than that in the condensed chromatin, indicating the existence of a DNA damage protection mechanism that is mediated by higher-order chromatin.

5To examine whether the higher-order chromatin structure is directly involved in the induction of DNA damage (e.g., following exposure to γ-rays), we developed a novel system for chromatin manipulation and DNA damage detection (Figure 1A). To analyze DNA on the genome scale without causing physical damage, we used permeabilized nuclei that were attached to glass surfaces. The nuclei were isolated from HeLa cells and attached to poly-l-lysine-coated coverslips by gentle centrifugation (Figure 1A). Since chromatin is negatively charged, the compaction states of the nuclei and chromatin were controlled by altering the Mg2+ concentration in the environment; at 5 mM Mg2+, chromatin becomes highly condensed, whereas it decondenses in the absence of Mg2+ (Figure 1A). Based on the nuclear volume and known size of the genomic DNA, we estimated the DNA or chromatin concentration in the environment. This “solid-phase system” allowed us to perform very gentle and quantitative handling of the genome-sized DNA (Figure 1A). For the direct detection of DNA damage in the chromatin on the glass surface, we fluorescently labeled the DSB sites using terminal deoxynucleotidyl transferase (TdT) (i.e., a TUNEL assay ) (Figure S1). Thus, we detected DSBs in the chromatin without inducing additional breakages, which distinguishes our method from traditional gel electrophoresis-based assays (Figure 1A).

6As shown in Figure 1B, the nuclei and associated chromatin on the glass surface were condensed in the presence of 5 mM Mg2+ (∼400 µm3). In the absence of Mg2+, the nuclei and chromatin swelled or decondensed by approximately 15-fold (∼6000 µm3) (Figure 1B and C). To check whether decondensation could induce DNA damage, we recondensed the nuclei and chromatin (Figure 1B) to a size comparable to that of the condensed nuclei (∼450 µm3) by increasing the Mg2+ concentration (Figure 1C).

7The three types (condensed, decondensed, and recondensed) of nuclei were irradiated with various doses of cobalt-60 γ-rays (Figure 1A). To promote the formation of a uniform compact state, which ensured equal chromatin accessibility and handling among the three types, the irradiated nuclei were treated with 1 mM Mg2+ then fixed with formaldehyde (Figure 1A). As a result, the nuclei became similar in size. DSBs in the chromatin were then detected using a TUNEL assay (Figure S1).

8As shown in Figure 2A, while the DSB signal intensity in the decondensed chromatin increased in a radiation dosage-dependent manner, the condensed and recondensed chromatin showed only faint signals. The suppressive effect observed for the recondensed chromatin excluded the possibility that the DNA damage was caused by the decondensation process (Figure 2A). In addition, we obtained similar results using a comet assay, which is a widely used damage detection method based on agarose gel electrophoresis (Figure S2), although stronger DSB signals were observed even in the condensed chromatin, probably due to the non-specific breakages during manipulation steps (e.g., nuclear embedding in hot agarose). Taken together, these results indicate that condensed chromatin is much more resistant to γ-ray irradiation than decondensed chromatin.

9We next quantified the DSB signals (Figure 2B). The signal intensities fit well to a quadratic curve in this irradiation dose range: I = kD2 (I, DNA breakage; k, a constant; and D, the irradiation dose). By comparing the k values, we found that the condensed chromatin had 16-fold greater damage suppression effects than decondensed chromatin (Figure 2C). The quadratic curve suggests that DSB induction is caused by two independent single-strand DNA breaks (SSBs). Consistently, under the assumption that DSBs occurred when two random SSBs were generated within ten bases in the double strands (Figure S3A), a simulation also showed that the number of DSBs created increased quadratically with the number of SSBs generated (Figure S3B).

10We next focused on a lower dose range of irradiation (<100 Gy). To detect DSBs, we used an electron-multiplying charge-coupled device (EMCCD) camera, which can detect single photons. Using the EMCCD, DSB signals within the low dose range were readily detectable (Figure 3A). We found that the condensed chromatin had ∼5-fold more damage suppression effects, even at 5 Gy of irradiation (Figure 3B). Moreover, in contrast with high-dose irradiation, the damage frequency increased linearly, suggesting that the DSBs were formed in a single step, which is consistent with the notion that DSBs are induced linearly with irradiation dose (see also the Discussion).

11Previous studies have suggested that chromatin-associated proteins (non-histone proteins) are involved in the induction of DNA damage: the more proteins carried by the chromatin, the more protected the chromatin is against ionizing irradiation. However, we found that the total level and composition of associated proteins were similar between decondensed and recondensed chromatin, indicating that the protective effect on condensed chromatin is not due to the level of associated proteins (Figure S4). Moreover, no significant irradiation-induced degradation of proteins was observed (Figure S5). These results demonstrate that chromatin compaction is important for the protection of genomic DNA against ionizing irradiation.

12To confirm these observations, we performed similar experiments using isolated human mitotic chromosomes. In the absence of Mg2+, the mitotic chromosomes swelled approximately 50-fold, as compared with those in a compact state (Figure 4A and B). The results obtained using chromosomes were more striking than those obtained using nuclei, in that the compact chromatin had approximately 50-fold greater resistance to γ-ray irradiation than the decondensed chromatin (Figure 4C and D). As the chromosomes contained fewer non-histone proteins than the nuclei (Figure S6), they were under fewer physical constraints and were more decondensed in the absence of Mg2+. These results provide further evidence that the protective effect is due to the chromatin concentration (volume) rather than the number of chromatin-associated proteins.

13We next used polyethylene glycol (PEG) instead of Mg2+ to induce chromatin condensation, since a high concentration of macromolecules (100–200 mg/ml) in cells might condense chromatin through a molecular crowding effect. The addition of 12.5% PEG increased the compaction of decondensed chromatin, with a 5-fold nuclear volume reduction (Figure S7). We found a notable inhibitory effect (Figure 5A and B). We also considered that PEG might be contributing to the damage inhibition effect not only as a molecular crowding agent, but also as a scavenger of radicals (see below).

14We demonstrated that the chromatin concentration plays a critical role in the suppression of DNA damage by ionizing irradiation. Since reactive radicals arising from the radiolysis of water molecules might be a major contributor to DNA damage, a lower concentration of chromatin, which entails more water molecules and reactive radicals, could increase the risk of attack by radicals. Thus, we examined the effects of the radical scavenger dithiothreitol (DTT) on DNA damage. When the decondensed chromatin was irradiated in the presence of DTT, DNA damage was suppressed in a dose-dependent manner (Figure 5A and C). Since the sizes of the decondensed nuclei in the presence of 100 mM and 0 mM DTT were similar (Figure S7), the inhibitory effect observed in the presence of DTT was due to radical scavenging rather than chromatin compaction.

15We next considered whether the protective effect of chromatin compaction is limited to γ-ray irradiation. To address this question, we examined the protective effect against heavy ion (carbon ion beam) irradiation. Carbon ion radiotherapy is an emerging modality in cancer therapy, and its clinical applications are increasing worldwide. We found a 7-fold higher damage suppressive effect in compact chromatin compared with decondensed chromatin (Figure 5D and E). This demonstrates that chromatin compaction protects DNA not only from γ-rays, but also heavy ions.

16We also tested the protective effect of chromatin compaction against chemical attack. Since we used ethylenediaminetetraacetic acid (EDTA) for chromatin decondensation, DNA cleavage reagents, which often contain metal ions, were not applicable to our study. Instead, we used cisplatin, which is widely used as an anti-tumor drug. Cisplatin forms covalent adducts with genomic DNA, thereby interfering with DNA replication and/or transcription, and eventually leading to apoptotic cell death. We treated the compact and decondensed chromatin with cisplatin, extracted the DNA, and measured the number of cisplatin adducts by inductively coupled plasma mass spectrometry (ICP-MS) (Figure 5F). We found that the DNA in the decondensed chromatin had 10-times more cisplatin adducts than the condensed chromatin (Figure 5G), suggesting that chromatin compaction protects genomic DNA against chemical-induced damage.

17In the present study, we found that condensed chromatin had 16-times (nuclei) and 50-times (chromosomes) more resistance to γ-rays than decondensed chromatin, demonstrating that damage induction depends on the chromatin concentration (or volume) (Figures 2, 4, and 5). Importantly, this dependency suggests that the contributions of Mg2+ and other components in the system to the suppression of damage are negligible. As suggested previously, the damage-suppressive effect of compaction is likely because the higher-concentration (compacted) chromatin has fewer water molecules per chromatin, thereby generating fewer reactive radicals. Compared with previous studies, which suggested that relaxed nuclear chromatin has 3.1–4.5-fold more DSBs than compacted chromatin, our results are particularly striking because the solid-phase system we used generates fewer background signals (non-specific DNA breakages) and allows for greater chromatin decondensation than agarose-embedded cells. Moreover, we were able to evaluate the suppressive effect in a quantitative fashion, as described below.

18Using low-dose γ-ray irradiation, the DSB frequency increased linearly (Figure 3B), in contrast to high-dose irradiation. This suggests that the creation of DSBs by γ-ray irradiation involves a single step and two independent steps (“DSB efficiency” = a × D+b × D2, where a and b are constants and D is the dose). The single-step creation of DSBs (a × D) appears to be dominant at lower radiation doses (Figure 3B), while the two-step creation of DSBs (b × D2) is more influential as the dose increases (Figure 2B), whereupon the single-step process becomes negligible. Our simple simulation (Figure S3C), which includes rare single-step DSBs (a × D) and frequent two-step DSBs (b × D2), has a profile similar to that shown in Figure 2B. In addition, the linear inhibitory effect at low irradiation doses (Figure 3B) demonstrates that the radiation dose can directly generate DSBs. This is also the case for heavy ion irradiation (Figure 5D and E).

19It is also interesting to consider the in vivo situation. To detect DSBs in eukaryotic cells, immunostaining to detect serine-139 phosphorylation in histone H2Ax (γH2AX) is commonly used, although the percentage of DSBs detected as γH2AX foci and their direct relationship remain unclear. A number of studies have demonstrated that γH2AX foci occur frequently in euchromatin regions, and less frequently in heterochromatic regions. Furthermore, it was demonstrated that hypotonic treatment of cells, in which the chromatin is presumably decondensed, produced more γH2AX foci. Although these in vivo results reflect many indirect effects, making it difficult to draw simple conclusions, they appear to be in good agreement with our in vitro findings. For further in vivo study, the use of an ion microbeam or UV laser microirradiation would be useful, since both allow the targeted irradiation of heterochromatic and euchromatic nuclear areas and may enable a direct comparison of the generation of DSBs in these compartments either by a TUNEL assay or the identification of γH2AX.

20Since we demonstrated that condensed chromatin has fewer DSBs induced by γ-rays and heavy ions, and is less susceptible to attack by chemical agents such as cisplatin, the present study indicates that chromatin compaction is advantageous, even in interphase cells (Figure 6A and B). This is consistent with the previously proposed idea of a chromatin domain model (Figure 6C). In this model, interphase chromatin forms compact chromatin domains. This compact state is the “default”. The only transcribed region, which is minimally decondensed and could be sensitive to radiation or other types of damage, might be looped out from the domain or at the surface of the domain (Figure 6C). This type of genomic organization could be beneficial for cells, especially in the long run.

21We propose that cellular protection by genomic DNA compaction represents a universally conserved function, from viruses to animals, aimed at maintaining genome integrity, because DNA condensation by polyamine was also observed to suppress DSB induction by γ-ray irradiation in previous in vitro studies, including our own. The maintenance of genomic DNA integrity by greater compaction would carry a selective advantage during the evolution of prokaryotes. This selective advantage might have even been more important in early evolutionary times when DNA repair mechanisms were preliminary. In eukaryotic organisms, the maintenance of genetic information would be especially critical in germline cells for the next generation. For example, yeast spores have ∼10-fold more condensed genomic DNA than somatic cells. Human primary oocytes, which take 20–40 years to complete meiosis I, have compact chromosomes, while mouse and rat have highly extended (dictyate-type) chromatin in their primary oocytes. Surprisingly, human primary oocytes are much more resistant to ionizing radiation than mouse or rat primary oocytes, which supports our hypothesis.

22Although we emphasized the importance of chromatin compaction in the maintenance of genomic DNA integrity, chromatin compaction might interfere with DNA repair due to a reduction in chromatin accessibility. This might be partly true; however, we recently observed local nucleosome fluctuations in living mammalian cells and demonstrated that this fluctuation increases chromatin accessibility, especially in compact chromatin regions. This novel mechanism to facilitate chromatin accessibility would play an important role in the DNA repair process in compact chromatin domains. Furthermore, it was reported that chromatin decondenses after DSB induction, increasing chromatin accessibility.

23Finally, it is important to emphasize that our findings provide a theoretical basis for various novel combinations of cancer therapies. Since decondensed chromatin has greater susceptibility to γ-rays, heavy ions, and chemicals, histone deacetyltransferase inhibitors and similar drugs (which decondense chromatin) would greatly enhance the cytotoxic effects of cisplatin and other DNA-damaging drugs in cancer cells, as well as those of radiation therapy involving γ-rays or heavy ions.

24For the isolation of nuclei, HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) containing 5% fetal bovine serum albumin (BSA; Nichirei Biosciences Inc., Tokyo, Japan) at 37°C in a 5% CO2 atmosphere. Collected cells were suspended in nuclei isolation buffer (3.75 mM Tris-HCl [pH 7.5], 20 mM KCl, 0.5 mM EDTA, 0.05 mM spermine, 0.125 mM spermidine, 0.1% Trasylol, 0.1 mM phenylmethylsulphonyl fluoride [PMSF]) and centrifuged at 193 ×g for 7 min at room temperature. The cell pellets were resuspended in nuclei isolation buffer and again centrifuged at 193 ×g for 7 min at room temperature. The cell pellets were then resuspended in nuclei isolation buffer containing 0.05% Empigen (nuclei isolation buffer+) and homogenized immediately with ten downward strokes using a tight Dounce-pestle. The cell lysates were centrifuged at 433 ×g for 5 min. The nuclei pellets were washed in nuclei isolation buffer+ and stored at –20°C in nuclei isolation buffer+ containing 50% glycerol. Chromosome isolation was performed as described previously.

25Isolated nuclei or chromosomes were suspended in HM buffer (10 mM HEPES-KOH [pH 7.4] and 5 mM MgCl2) and attached to poly-l-lysine-coated coverslips by centrifugation at 400 ×g for 5 min. For decondensed chromatin, the nuclei or chromosomes on the coverslips were gently transferred to the buffer that contained 1 mM EDTA (pH 8.0). For recondensation, the decondensed chromatin on the coverslip was placed in HM buffer.

26For high-dose irradiation (all experiments, with the exception of that shown in Figure 3), condensed, decondensed, or recondensed chromatin was irradiated with cobalt-60 γ-rays at 40 Gy/min at the Radiation Research Center, Osaka Prefecture University (Osaka, Japan). For low-dose irradiation (Figure 3), we irradiated the same sets of samples with cesium-137 at 1.67 Gy/min at the irradiation facility of the National Institute of Genetics (NIG; Mishima, Japan). In both irradiation experiments, the coverslips with chromatin were put in a 12-well cell culture plate (Corning Inc. Life Sciences, Tewksbury, MA, USA) with the indicated buffers and irradiated at room temperature. The applied dose was determined using a Fricke dosimeter.

27After γ-ray irradiation, the condensed, decondensed, or recondensed chromatin on the coverslips was placed in HM buffer, followed by HMK buffer (10 mM HEPES-KOH [pH 7.4], 1 mM MgCl2, and 100 mM KCl), to ensure that the chromatin condensation state was uniform. The chromatin samples were fixed with 1% formaldehyde in HMK buffer at room temperature for 15 min. After washing with 50 mM glycine in HMK buffer and then HMK buffer, the samples were stored in HEN buffer (10 mM HEPES-KOH [pH 7.5], 1 mM EDTA, and 100 mM NaCl) at 4°C until use.

28DNA damage was detected using a Click-iT TUNEL Alexa Fluor Imaging Assay Kit (Invitrogen) according to the manufacturer’s instructions. The fluorescently labeled samples were co-stained with DAPI to visualize DNA then mounted in PPDI (10 mM HEPES-KOH [pH 7.5], 1 mM MgCl2, 100 mM KCl, 80% glycerol, and 1 mg/ml paraphenylene diamine). For the high-dose irradiation samples (Figures 2, 4 and 5), microscopic images to quantify the TUNEL assay signals were acquired under the same imaging conditions using an ECLIPSE E800 fluorescence microscope with a 60× objective lens (Nikon, Tokyo, Japan). For the lower dose irradiation samples (Figure. 3), we used Nikon microscope system Ti-E with a 60× objective lens (Nikon, Tokyo, Japan) using Evolve512 EM-CCD camera (Roper Scientific, USA). The TUNEL assay signals (Alexa 488) were analyzed using NIS-elements BR 3.10 software (Nikon) as follows. The nuclear or chromosomal regions were extracted based on a threshold value of DAPI signal intensity. The mean intensity values of Alexa 488 signals in the extracted nuclear or chromosomal regions were then examined. The mean intensity value in the regions at 0 Gy was used as background. After subtraction of the background signal, the obtained signal intensity values were normalized against that of decondensed chromatin irradiated at 500 Gy. The comet assay was performed according to the manufacturer’s instructions (Trevigen, Gaithersburg, MD, USA).

29After fixation with 1% formaldehyde, the samples were washed with 50 mM glycine and stained with 2 nM TO-PRO-3 solution (Invitrogen) at 37°C for 30 min. After washing, Z-stack images were acquired using a LSM510 META laser scanning confocal microscope (Carl Zeiss, Wetzlar, Germany) with a 100× objective at 0.48-µm intervals. The obtained images were processed using the LSM Image Browser (Carl Zeiss) and ImageJ software (NIH, Bethesda, MD, USA).

30Condensed, decondensed, and recondensed nuclei or chromosomes were collected by centrifugation (3000 rpm, 10 min). The pellets, which contained ∼1.26 mg of DNA, were completely dissolved in Laemmli sodium dodecyl sulfate (SDS) sample buffer by sonication. After boiling at 95°C for 5 min, the lysates were subjected to 10–20% gradient SDS-polyacrylamide gel electrophoresis (PAGE). To quantify the protein levels, BSA was used as a standard. The gel was stained with Coomassie brilliant blue R-250 (CBB) and the image was acquired using an LAS-1000 imaging system (Fujifilm, Tokyo, Japan). A quantitative analysis was performed using ImageJ software.

31The simulation was conducted as follows using Microsoft Visual C++. Two random numbers (SSBs) from 1 to 100,000 (100 kb) were generated independently for strands a and b (Figure S3A). If the difference between the numbers on the two strands was less than 10, we counted the pair as a DSB (Figure S3A). We repeated this process at the indicated times and plotted the number of DSBs against the number of SSBs (Figure S3B). The number of created DSBs was quadratically increased with the number of generated SSBs. For Figure S3C, two random numbers (SSBs) from 1 to 100000 (100 kb) were again generated independently for strands a and b. Every 50 SSBs, a DSB was also created at random.

32Heavy ion treatment was performed using the Heavy Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Sciences (Chiba, Japan). The accelerated ions used in this study were carbon ions (290 MeV/n). Details concerning the characteristics of the carbon ion beams, biological irradiation procedures, and dosimetry may be found elsewhere.

33Isolated nuclei (∼1×107) were suspended in HM buffer or 1 mM EDTA containing buffer and treated with cisplatin at 2 mM overnight at room temperature. After five washes with HMK buffer, half of the samples were subjected to ICP-MS (chromatin fractions). DNA was extracted from the remaining samples and subjected to ICP-MS (DNA fractions). We performed ICP-MS using ELAN DRC II (PerkinElmer, Waltham, MA, USA).