Abstract
High concentration of NaCl increases DNA breaks both in cell culture and in vivo. The breaks remain elevated as long as NaCl concentration remains high and are rapidly repaired when the concentration is lowered. The exact nature of the breaks, and their location, has not been entirely clear, and it has not been evident how cells survive, replicate, and maintain genome integrity in environments like the renal inner medulla in which cells are constantly exposed to high NaCl concentration. Repair of the breaks after NaCl is reduced is accompanied by formation of foci containing phosphorylated H2AX (γH2AX), which occurs around DNA double-strand breaks and contributes to their repair. Here, we confirm by specific comet assay and pulsed-field electrophoresis that cells adapted to high NaCl have increased levels of double-strand breaks. Importantly, γH2AX foci that occur during repair of the breaks are nonrandomly distributed in the mouse genome. By chromatin immunoprecipitation using anti-γH2AX antibody, followed by massive parallel sequencing (ChIP-Seq), we find that during repair of double-strand breaks induced by high NaCl, γH2AX is predominantly localized to regions of the genome devoid of genes (“gene deserts”), indicating that the high NaCl-induced double-strand breaks are located there. Localization to gene deserts helps explain why the DNA breaks are less harmful than are the random breaks induced by genotoxic agents such as UV radiation, ionizing radiation, and oxidants. We propose that the universal presence of NaCl around animal cells has directly influenced the evolution of the structure of their genomes.
Keywords: hypertonicity, salt, DNA damage, kidney, mIMCD3 cells
High extracellular NaCl increases the number of DNA breaks in mammalian cells in tissue culture (1, 2), mouse renal inner medullary cells in vivo (1), cells of the soil nematode Caenorhabditis elegans (3), and marine invertebrates (4). Acute elevation of NaCl in cell culture increases the number of DNA breaks (2, 5) and transiently arrests cells in all phases of the cell cycle (6, 7). After several hours, the cells begin proliferating again, despite the continued presence of high NaCl (7). However, even after cells adapt to high NaCl and reenter the cell cycle, numerous DNA breaks persist (1). Excessive elevation of NaCl causes apoptosis (7). However, the increased DNA breaks that occur at levels of NaCl that cells survive and to which they adapt differs from the chromatin fragmentation that occurs during apoptotic cell death. Thus, high NaCl increases DNA breaks in viable cells without the activation of caspases, nuclear condensation, or formation of apoptotic bodies characteristic of apoptosis (8, 9).
The increase of DNA breaks caused by high NaCl is not limited to proliferating cells in culture. High NaCl also induces DNA breaks in normal cells in animal tissues in vivo. Thus, numerous DNA breaks are normally present in the mouse renal inner medulla (1), where high interstitial NaCl provides the driving force for concentration of the urine (10). The excess breaks in the inner medulla disappear quickly when the high intercellular NaCl concentration in the renal medulla is lowered by the diuretic furosemide (1). The soil nematode, C. elegans is able to adapt to and live in a high NaCl environment (11), and adaptation of C. elegans to high NaCl is accompanied by increased DNA breaks (3). Finally, according to some estimates, ≈80% of all Earth's life lives in the ocean, which has a high osmolality of ≈1,000 mosmol/kg, the dominant solute being NaCl. Many marine invertebrates are osmoconformers, i.e., the NaCl in their extracellular fluids is as high as in seawater (12). Cells in tissues of osmoconforming marine invertebrates have many DNA breaks that disappear if the seawater in which they are immersed is gradually diluted to 300 mosmol/kg (4). Thus, increased DNA breaks in cells exposed to high NaCl is an evolutionarily conserved phenomenon. However, the nature of the DNA breaks, their location, and the mechanism of their induction has not been entirely clear.
A striking feature of adaptation to high NaCl is that despite increased DNA breaks, the cells do not activate the DNA damage response (1, 5, 8). However, the DNA damage response is activated quickly when NaCl is lowered. Thus, reducing NaCl to total osmolality of 300 mosmol/kg (the level normally maintained in mammalian blood and body fluids by osmoregulatory mechanisms) results in rapid repair of the DNA breaks (1, 5). This repair is accompanied by rapid phosphorylation of histone H2AX (called formation of γH2AX) (1, 5), the histone modification that normally accompanies repair of double-strand breaks (13, 14).
In the present studies, we find that the high NaCl-induced double-strand DNA breaks are not randomly distributed in the mouse genome, but are predominantly located in gene deserts, which are regions of the genome devoid of genes. Our findings are summarized on Fig. S1.
Results
High NaCl Induces Double-Strand Breaks That Are Rapidly Repaired When the NaCl Is Lowered.
We added 100 mM NaCl (which elevates the osmolality to ≈500 mosmol/kg) for 22 h to the medium bathing mIMCD3 cells. This addition of NaCl causes immediate G2/M arrest that lasts ≈6 h (6). Then, the cells begin proliferating again. By 22 h, the cell cycle distribution, appearance of the cells, and their rate of proliferation return to the condition before salt was elevated (ref. 1 and Fig. S2).
It has remained an open question what kind of DNA breaks are present in cells exposed to high NaCl. The breaks induced immediately by acute elevation of high NaCl for 1 h were originally characterized as double-strand DNA breaks (DSBs) by pulsed field gel electrophoresis (PFGE), which detects DSBs but not single-strand breaks (SSBs) (2). However, the methods used to study DNA breaks after cells adapt to high NaCl and resume proliferation were not specific to DSBs. Those methods included a version of the neutral comet assay originally developed by Ostling and Johanson (15), alkaline comet assay (1), and in vitro labeling of their 3′-OH ends with biotinylated deoxynucleotides in a reaction catalyzed by terminal deoxynucleotidyl transferase (1, 3, 4). All these methods do not distinguish between DSBs and SSBs (16–18). In the present studies, to clarify whether DNA breaks that remain induced by high NaCl in adapted cells include DSBs, we used a modification of the neutral comet assay that is specific for DSBs as opposed to SSBs (18, 19).
To confirm specificity of the neutral comet assay (19) that we used here for DSBs, we exposed cells to hydrogen peroxide (H2O2), known to induce only single-strand breaks (20). DNA breaks after H2O2 are not detected by the modified neutral comet assay but are detected by the alkaline comet assay, consistent with their single-stranded nature (Fig. 1A). We also tested mIMCD3 cells exposed to the topoisomerase inhibitor Etoposide, known to induce both double-strand and single-strand breaks in a proportion similar to ionizing radiation (20). The DNA breaks induced by Etoposide are detected by both the modified neutral and alkaline comet assay, consistent with presence of both double- and single-strand breaks (Fig. 1A). The breaks induced by high NaCl are detected by the modified neutral comet assay (Fig. 1A), indicating that they are double-stranded. The DSBs induced by high NaCl are repaired within several hours after the NaCl concentration is reduced to a total osmolality of 300 mosmol/kg (Fig. 1 B and C). Thus, an increased level of DSBs persists after cells have adapted to high NaCl and appear otherwise normal. In addition, there may also be more SSBs, but the effect of high NaCl on SSBs has not been specifically tested.
ATM/ATR-Dependent DNA Double-Strand Break Repair Response Is Activated During Repair of High NaCl-Induced DNA Breaks.
Common DNA damage repair responses include transient cell cycle arrest, during which the DNA repair occurs. We tested to see whether that happens during the disappearance of the DNA breaks after reduction of high NaCl (Fig. 2). Indeed, phosphorylation of checkpoint kinase 1 (Chk1), which contributes to all defined cell cycle checkpoints (21), occurs rapidly, and the phosphorylation is reduced by caffeine, which is an ATM/ATR inhibitor (22) (Fig. 2A). Similarly, G2/M cell cycle arrest activates rapidly when NaCl decreases, and this arrest is abrogated by caffeine (Fig. 2B). Repair of the breaks after NaCl is reduced is accompanied by formation of foci containing phosphorylated H2AX (γH2AX) (1, 5). This histone modification occurs around DNA double-strand breaks and contributes to their repair (13, 14). γH2AX is induced to a maximal level within 15 min after lowering NaCl, then gradually decreases (Fig. 2C) accompanying repair of the DSBs (Fig. 1C). The γH2AX induction is also sensitive to inhibition of ATM/ATR by caffeine (Fig. 2C). These results indicate that, when elevated NaCl is lowered, a classical ATM/ATR-dependent DNA damage response becomes activated, and they further confirm that DNA DSBs increase upon exposure to high NaCl.
During Repair of High NaCl-Induced DNA Breaks, γH2AX Is Mainly Located in Gene Deserts, Whereas Bleomycin and UV Induce γH2AX at Random Locations Throughout Genome.
Given that high NaCl induces DSBs that persist as long as the level of NaCl stays high, it has not been clear how cells survive and function in the continued presence of those breaks, why the cell cycle does not remain arrested, how DNA transcription and replication can proceed despite the breaks, and how mutations and genomic instability are prevented. To begin answering those questions, we have determined the genomic location of the breaks. Our strategy is based on the fact that during repair of DSBs, γH2AX is induced in distinct foci (Fig. 3). γH2AX-containing foci occur at DSBs (13, 14), and the number of γ-H2AX foci approximates the number of DSBs induced (ref. 14 and reviewed in ref. 23), making foci of γH2AX consistent and quantitative markers of DSBs. Therefore, to detect locations of DSBs, we performed chromatin immunoprecipitation (ChIP) by using anti-γH2AX antibody, followed by massive parallel sequencing of isolated DNA fragments (γH2AX ChIP-Seq) (24). Given that the maximal intensity of γH2AX foci occurs 15 min after NaCl is lowered and that the intensity of the foci already decreases by 30 min (Fig. 3B), we determined the genomic locations of the γH2AX foci that are present 15 min after lowering NaCl. In addition, we performed ChIP-Seq to determine the genomic locations of the γH2AX induced by bleomycin and UV radiation. Both bleomycin and UV increase γH2AX (Fig. 2D). However, pattern of γH2AX immunostaining is different (Fig. 3A) because of the different nature of damage induced by bleomycin and UV. Thus, bleomycin induces DSBs directly (25) and produces distinct γH2AX foci (Fig. 3A), whereas, after UV radiation, DSBs arise only indirectly as a result of the action of repair or degradation of arrested replication forks (26). After UV radiation, γH2AX is increased both in foci and in more diffused staining (Fig. 3A) that might not be related to DSBs but to some other types of DNA damage (27). Thus, by γH2AX ChIP-Seq we analyzed genomic locations of γH2AX induced by bleomycin, UV, and during repair of high NaCl-induced DNA breaks. We aligned sequence reads (tags) to the mouse genome and analyzed the tag density in the University of California, Santa Cruz (UCSC) genome browser (Fig. 4 and Fig. S5).
There are clusters of increased density of mapped tags (γH2AX peaks) in cells after reduction of high NaCl (Fig. 4A and Fig. S5). The peaks occur mainly in large intergenic regions (gene deserts). Thus, H2AX becomes phosphorylated at specific locations within gene deserts during repair of the high NaCl-induced DSBs, showing that the high NaCl-induced DSBs are not randomly distributed throughout genome but occur within gene deserts. The peaks do not occur in DNA from cells maintained continuously at 300 or 500 mosmol/kg (Fig. 4A, “Control” and “High NaCl, 22h”). Bleomycin and UV also increase γH2AX (Figs. 2D and 3), but peaks do not occur in DNA from cells in which it has been damaged by Bleomycin or UV irradiation (Fig. 4A, “Bleomycin” and “UV”). Thus, the DNA damage caused by Bleomycin and UV occurs randomly throughout the genome, in marked contrast to the localized DSBs caused by high NaCl.
The localization of peaks to gene deserts is apparent by visual examination of tag density in the genome browser. Representative regions are shown in Fig. 4 and Fig. S5. To apply this observation to the whole genome, we extracted information about peak coordinates from the UCSC genome browser and analyzed gene density over the regions of identified peaks in comparison with artificial peaks of the same width and number that were randomly generated throughout genome (Fig. 4B, Upper). We identified 215 peaks of mean width of 2.4 Mb (95% confidence interval between 2.2 Mb and 2.7 Mb). Gene density over the peak regions is greatly reduced compared with random peaks or average gene density in mouse genome (Fig. 4B, Lower).
PFGE Identifies Increased DNA Fragmentation After Exposure of mIMCD3 Cells to High NaCl, and the Distribution of the Lengths of the Fragments Is Consistent with Spacing of the γH2AX Peaks.
We used PFGE, which separates large DNA fragments according to size (28), to further test whether high NaCl induces double-strand breaks and whether the breaks are not randomly distributed (Fig. 5). Exposure of cells to high NaCl increases the number of DNA fragments that enter the gel and the size of the fragments is not random, consistent with a nonrandom location of the DNA breaks (Fig. 5A). Given the locations of the γH2AX peaks and assuming that there is one DNA break in each peak, we calculated a size distribution of the predicted DNA fragments (Fig. 5B and SI Materials and Methods). The calculated distribution of fragment sizes closely resembles the actual distribution (Fig. 5), which supports the conclusion that high NaCl-induced double-strand breaks occur predominantly in gene deserts.
Discussion
Why there are gene deserts has remained a mystery. They were discovered when whole genome sequencing showed that genes are not evenly distributed. A substantial fraction of mammalian genomes contains gene deserts, defined as long regions (>500 kb) containing no protein-coding sequences. Gene deserts occupy ≈38% of human, 34% of mouse, 23% of rat, and 20% of dog genome (29). It is extremely unlikely that gene deserts reached their observed maximal size of 5.1 Mb with 545 deserts larger than 640 kb by chance (30), which raises the question of what selective pressure might be acting.
Animal cells are universally exposed to NaCl, and the level of NaCl may be high in animals exposed to marine or desiccated terrestrial environments. During evolution of mammals, osmoregulatory mechanisms developed that maintain osmolality of most extracellular fluids close to 300 mosmol/kg. Nevertheless, even in mammals, NaCl concentration is constantly very high in some tissues, particularly the renal medulla. Given our finding that DNA breaks induced by high NaCl are concentrated in gene deserts, we suggest that, as the size of genomes has increased, newly formed regions are susceptible (for unknown reasons) to high NaCl-induced DNA breaks and evolve to contain fewer genes, thus limiting mutations and preventing genomic instability. This suggestion is supported by several observations.
i) The neutral mutation rate (30) and the rate of genome rearrangements associated with appearances of new centromeres (31) are both higher in gene deserts than in regions containing genes.
ii) Before the evolution of vertebrates, the sizes of genomes grew in proportion to the number of genes. However, gene deserts began appearing in fish and increased in size to occupy ≈38% of the genome in humans (29). Over the same period, osmoregulatory mechanisms developed that maintain systemic osmolality close to 300 mosmol/kg. Estimates from molecular clocks of the rates of evolution show that the rates decreased significantly in vertebrates before the origin of Osteichthyes (32). That could have been due to a combination of decreased rate of mutations in protein coding regions owing to more precise osmoregulation and the low abundance of functional genes in gene deserts where they would be susceptible to NaCl-induced breaks.
iii) Recently, a model was proposed relating the rate of molecular evolution and the maximal size of genomes (33). The theory assumes that for an organism to be viable, essential genes must be functional. Further, it predicts that populations become extinct because of lethal mutagenesis when the mutation rate exceeds approximately six mutations per replication in essential parts of the genome in mesophilic organisms and one or two mutations in thermophilic ones. The theory therefore predicts that mutation rate limits essential genome size; in other words, the higher the mutation rate, the smaller the sustainable size of the genome. This theory implies that increasing the size of the genome required that genes not evolve in regions, like the present gene deserts, that are more susceptible to DNA breaks and mutations.
Our finding that high NaCl-induced DSBs are located in gene deserts is an example of nonrandom induction of DNA breaks in higher organisms. Although we are uncertain why high NaCl breaks DNA, the gene deserts apparently have properties that render them more susceptible. Limitation of high NaCl-induced DNA breaks to gene deserts helps explain why they apparently are less harmful than are the random breaks induced by genotoxic agents like UV radiation, ionizing radiation, and oxidants. Further, our finding suggests a possible role of high NaCl in evolution of the structure of the animal genome.
Perspective.
More studies are required to decipher why double-strand breaks occur predominantly in gene deserts during exposure to high NaCl. Possibilities that we are considering include decreased DNA repair in gene deserts similar to that in heterochromatin (34), presence of specific target sequences for nucleases activated by high NaCl, and high NaCl-induced alterations of chromatin in gene deserts that makes the DNA there more susceptible to damaging agents.
Materials and Methods
Methods were published for Western blot (1), exposure of cells to UV radiation (5), treatment with H2O2 (20), analysis of cells in mitosis (6), immunostaining, and analysis of brightness of γH2AX foci by laser-scanning cytometry (LSC) (5). More details are included in SI Materials and Methods, as are details of doses and timing of drug application, analysis of gene density at genomic locations enriched with γH2AX-immunoprecipitated sequence tags, and analysis of expected distribution of DNA fragment sizes, based on genomic locations of γH2AX ChIP-Seq peaks.
Cell Culture.
mIMCD3 cells (35) were grown in medium containing 45% DME Low Glucose (Invitrogen), 45% F12 Coon's Modification (No. F6636; Sigma), and 10% FBS (HyClone). Osmolality of control medium was 300–320 mosmol/kg. High NaCl medium was prepared by adding NaCl to the total osmolality of 500 mosmol/kg. All of the experiments were performed on logarithmically growing cells at ≈80% confluence. To elevate NaCl, control medium was replaced by the high NaCl media.
Analysis of Double-Strand and Single-Strand DNA Breaks by Comet Assay.
Two different assays were used: neutral comet assay modified for detection of double-strand breaks and alkaline comet assay, which detects DNA SSBs, double-strand breaks, and alkali-labile sites. Those assays were performed as described (19) with minor modifications. See SI Materials and Methods and Fig. S4 for details.
ChIP and Illumina Library Construction for Sequencing.
ChIP was performed by using Enzymatic Chromatin IP kit (No. 9003; Cell Signaling Technology). Conversion of the ChIP-enriched DNA into libraries suitable for sequencing using the Illumina Genome Analyzer was performed by using the published protocol (36). See SI Materials and Methods and Fig. S4 for the detailed ChIP-Seq protocol.
Solexa Pipeline Analysis.
Sequence tags were obtained and mapped to the mouse genome by using the Solexa Analysis Pipeline as described (37). The unique reads were retained and converted to browser extensible data (BED) files for viewing the data in the UCSC genome browser. The read number and genomic coordinates were summarized in 300-bp windows.
Analysis of DNA Fragmentation by PFGE.
Agarose embedded DNA was prepared by using the CHEF Mammalian Genomic DNA Plug Kit (No. 170–3591; Bio-Rad). Briefly, cells were rinsed with PBS, scraped off the dish, resuspended in 1% CleanCut Agarose from the kit at a final concentration of 12 million cells/mL. The agarose/cell suspension was solidified at 4 °C for 10 min in a casting mold, followed by incubation of the agarose plugs in Proteinase K solution at 50 °C for 3 d to digest proteins. PFGE was performed as described (28) by using the CHEF-DR II system (Bio-Rad) and the following parameters: 1% Megabase Agarose (No. 161–3108; Bio-Rad), 0.5× TBE running buffer, 120° reorientation angle, 6 V⋅cm−1, and 14 °C. Gels were run for 16 h with switch time of 16 s, followed by 30-h run with switch time of 80 s DNA Size Markers were as follows: Schizosaccharomyes pombe, Saccharomyces cerevisiae, and Hansenula wingei chromosomes (Bio-Rad). Gels were stained with SYBR Gold (Invitrogen).
Supplementary Material
Acknowledgments
We thank Drs. Chris Combs and Daniela Malide at the National Heart, Lung, and Blood Institute (NHLBI) Light Microscopy Core Facility for help with microscopy and images processing and Dr. Iouri Chepelev at the NHLBI Laboratory of Molecular Immunology for assistance with sequencing data analysis. This research was supported by the Intramural Research Programs of the National Institutes of Health, NHLBI.
Footnotes
The authors declare no conflict of interest.
Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE32882).
See Commentary on page 20281.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1114677108/-/DCSupplemental.
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