Copy number variants are produced in response to low-dose ionizing radiation in cultured cells

Abstract

Despite their importance to human genetic variation and disease, little is known about the molecular mechanisms and environmental risk factors that impact copy number variant (CNV) formation. While it is clear that replication stress can lead to de novo CNVs, for example, following treatment of cultured mammalian cells with aphidicolin (APH) and hydroxyurea (HU), the effect of different types of mutagens on CNV induction is unknown. Here we report that ionizing radiation (IR) in the range of 1.5-3.0 Gy effectively induces de novo CNV mutations in cultured normal human fibroblasts. These IR-induced CNVs are found throughout the genome, with the same hotspot regions seen after APH- and HU-induced replication stress. IR produces duplications at a higher frequency relative to deletions than do APH and HU. At most hotspots, these duplications are physically shifted from the regions typically deleted after APH or HU, suggesting different pathways involved in their formation. CNV breakpoint junctions from irradiated samples are characterized by microhomology, blunt ends, and insertions like those seen in spontaneous and APH/HU-induced CNVs and most nonrecurrent CNVs in vivo. The similarity to APH/HU-induced CNVs suggests that low-dose IR induces CNVs through a replication-dependent mechanism, as opposed to replication-independent repair of DSBs. Consistent with this mechanism, a lower yield of CNVs was observed when cells were held for 48 hr before replating after irradiation. These results predict that any environmental DNA damaging agent that impairs replication is capable of creating CNVs.

Keywords: CNV; ionizing radiation; replication stress.

Fig. 1

IR induces de novo CNVs in normal human fibroblasts. Three iterations of the experiment with different cell handling are shown. (A) Incidence of de novo CNVs in normal, hTERT-immortalized human fibroblasts treated with 0-1.5 Gy IR. Fifteen independent clones each of untreated, 0.5 Gy, 1.0 Gy, and 1.5 Gy-treated cells were analyzed. Cells were plated for cloning immediately after irradiation. Error bars indicate SE. (B) Colony-forming ability of IR-treated cells in (A) compared to untreated cells. Error bars indicate SD. (C) Incidence of de novo CNVs in normal, hTERT-immortalized human fibroblasts treated with 0-3.0 Gy IR. Twelve independent clones of untreated cells, 15 clones of 1.5 Gy, and 14 clones of 3.0 Gy-treated cells were analyzed. Cells were plated for cloning immediately after irradiation. Error bars indicate SE. (D) Colony-forming ability of IR-treated cells in (C). (E) Incidence of de novo CNVs in normal, hTERT-immortalized human fibroblasts treated with 0-3.0 Gy IR. Ten independent clones of untreated cells and 11 clones each of 1.5 and 3.0 Gy-treated cells were analyzed. Unlike (A) and (C), cells in (E) were plated for cloning 48 hours after irradiation. Error bars indicate SE. (F) Colony-forming ability of IR-treated cells in (E) compared to untreated cells. Error bars indicate SD.

Fig. 2

Size distribution of CNVs. (A) Fraction of CNVs by size. Untreated (blue circles), IR-treated (red squares). (B) Fraction of CNVs by size for CNVs induced by APH and HU (blue circles) [Arlt, et al., 2011] and IR (red squares).

Fig. 3

Spatial distribution of CNVs. (A) Locations of IR-induced CNVs. Red circles indicate IR-induced CNVs, blue squares indicate spontaneously arising CNVs in untreated cells. Bars are used to indicate large CNVs spanning more than a chromosomal band. Markers above and below chromosomes represent duplications and deletions, respectively. Asterisks (*) mark three large regions of uniparentaldisomy on chromosome 9p. Ideograms were adapted from the University of California, Santa Cruz genome browser (http://genome.ucsc.edu) [Kent, et al., 2002]. Precise coordinates for all de novo CNVs are listed in Dataset S1. (B) Examples of physically shifted hotspot CNVs after IR (red bars) and APH/HU (blue bars). In addition, CNVs induced by IR in these regions are predominantly duplications whereas those induced by APH/HU are mostly deletions

Fig. 4

Non-random association of IR-induced CNVs and APH/HU-induced CNVs. IR CNV regions were randomly distributed around the genome in 10,000 permutations and then compared to the locations of CNV regions induced by APH and HU (AH) to create an expected random distribution of overlap between the two groups. The actual observed number of IR CNV regions that overlapped AH CNV regions is indicated by the vertical green line. The black line represents a single iteration in which the IR CNV regions were offset 10Mb to the right. Blue lines and associated red Gaussian curve-fit show the distribution of values observed for the 10,000 random permutations. Analyses were performed for (A) singleton IR CNVs, (B) regions with more than one IR CNV, and (C) all IR CNV regions combined. Numbers in red above each plot indicate the p-value for the actual value calculated from the fit curve (p), as well as the cumulative frequency of iterations that had as many or more IR CNV regions crossing AH regions as the actual value (f).

Fig. 5

CNV breakpoint junctions. (A) De novo CNV breakpoint junction sequence homology in IR-treated cells (red) compared to the expected distribution if microhomology usage was random (gray). (B) A complex CNV with two junctions at 17q23.2 in 3.0 Gy-treated clone 3D5. Based on aCGH data, this CNV was called as a deletion, but sequencing of the breakpoint junctions revealed that this CNV was complex, containing a 97.7 kb deletion (red), as well as a duplication-insertion of 530 bp (blue) at the deletion boundary.

Fig. 6

Immediate trypsinization and replating of cells following IR reduces CHK1 activation. (A) Western blots showing the induction by IR of CHK1 phosphorylation on residue S317. (B) Quantitation of CHK1 S317 phosphorylation in (A), normalized to tubulin, respectively.

Fig. 7

Model of physically shifted deletion and duplication formation via single- and double-fork failure. When two nearby replication forks collapse (bottom), as might occur during global replication stress, MMBIR can occur between the two forks to restart replication, resulting in the deletion of intervening DNA (red bars). In the event of a single-fork failure (top), the absence of a nearby collapsed fork results in an MMBIR event into a nearby, already-replicated region, resulting in a duplication (blue bars).