High salt and DNA double-strand breaks.

H igh-salt environments are often toxic to mammalian cells; however, some types, such as murine inner medullary collecting duct cells, are normally exposed to high-salt media. The IMCD3 cell line, derived from this mouse kidney cell type, is able to survive and even proliferate under high-salt conditions after a short period of adaptation (1). Interestingly, this adaptation involves the induction of DNA double-strand breaks (DSBs) (2). The increased level of DSBs remains as long as the cells are exposed to high salt. In an unexpected and striking development, a paper in PNAS presents the finding that these breaks are not randomly distributed throughout the genome but are targeted to specific chromosomal regions with few genes—namely gene deserts (3). This finding strongly suggests that hyperosmotic DSB induction and maintenance are caused by a programmed biological process rather than a random physical phenomenon (Fig. 1) (3). To show this point, the authors (3) grow IMCD3 cell cultures in 500 mosmol/kg NaCl and confirm that they contain DSBs by neutral comet assay and pulse gel electrophoresis but lack signs of DSB repair, such as the presence of γH2AX (histone H2AX phospho-serine 129) foci. When these cells were returned to normosmolality (300 mosmol/kg), DSB repair began including the rapid formation of γH2AX foci (4). Then, ChIP using antiγH2AX antibody followed by massive parallel DNA sequencing was performed. The breaks were mapped predominantly to 10 gene-poor stretches of two regions encompassing ∼40-Mb portions of two chromosomes, 11 and 18. This finding indicates that the high salt-induced DSBs were located in these gene deserts. The specificity of the process seems to be in the cleavage steps. An alternative mechanism might involve random cleavage followed by selective repair in euchromatin, leaving DSBs enriched in the gene deserts. Gene deserts are often in heterochromatic regions, which retard DSB repair (5). However, this type of scenario is unlikely, because pulse-field gel analysis reveals that the pattern of fragments seen after 20 h is already present by 1 h. These gene deserts may already be situated for specific cleavage if they are located in heterochromatin. In a 4.3-Mb region on mouse chromosome 14 that contains four clusters of genes separated by gene deserts, 3D structural studies showed that the gene deserts preferentially aligned with the nuclear periphery (6). These associations may be functionally important, because a lamin mutant that causes the premature aging disorder Hutchinson–Gilford Progeria contributes to a loss of peripheral heterochromatin (7). Thus, the relevant nucleases or topoisomerases may already have binding sites near regions of the hyperosmotically induced DSBs. This finding suggests that these breaks may also be preferentially located to the nuclear periphery, a prediction that should be straightforward to test. Is the failure to repair hyperosmotically induced DSBs because of masking of the breaks or inhibition of repair? DSBs are masked by proteins in several cellular processes. For example, in functional telomeres, the DNA is sufficiently long to bind capping structures, and γH2AX foci do not form. As telomeres erode and the shortened DNA fails to retain the capping structures, the exposed double-strand ends induce γH2AX foci (8). Also, topoisomerase II as well as Spo11 complexes are not seen as DSBs, although the DNA has a single-strand break on each strand (9). It should be noted that these complexes are revealed to harbor DSBs by comet assay and pulse-field gel electrophoresis, because the proteins are digested and removed during these procedures. In contrast, DSBs induced by ionizing radiation are not masked. When IMCD3 cells in high-salt conditions were exposed to ionizing radiation, γH2AX foci also failed to form on the radiation-induced breaks (10). Thus, although hyperosmotically induced DSBs may or may not be complexed with proteins, they fail to be repaired because of an inhibition of DSB recognition rather than masking. IMCD3 cells not only survive in highsalt conditions, they thrive, resuming proliferation after a short adaptation period. Fig. 1. Speculative model for high salt-induced DSBs in IMCD3 cells. (A) The genome includes regions with gene clusters separated by regions lacking coding regions known as gene deserts, some of which are located near the nuclear envelope. (B) Many cell types die in the presence of high-salt media. (C) However, some, including IMCD3 cells, are able to proliferate in high-salt media, accompanied by the induction of DSBs. These breaks are targeted to specific gene deserts and may involve endonucleases and topoisomerases. (D) As long as IMCD3 cells are exposed to high salt, DSB repair is inhibited, even for irradiation-induced DSBs. The DSBs may be in reversible topoisomerase II cleavage complexes, which may explain how IMCD3 cells proliferate in high-salt media. (E) When cells are returned to normosmolality, DSB repair is restored, including the rapid formation of γH2AX foci. More speculative steps are in red.