Disease-associated CAG·CTG triplet repeats expand rapidly in non-dividing mouse cells, but cell cycle arrest is insufficient to drive expansion

Abstract

Genetically unstable expanded CAG·CTG trinucleotide repeats are causal in a number of human disorders, including Huntington disease and myotonic dystrophy type 1. It is still widely assumed that DNA polymerase slippage during replication plays an important role in the accumulation of expansions. Nevertheless, somatic mosaicism correlates poorly with the proliferative capacity of the tissue and rates of cell turnover, suggesting that expansions can occur in the absence of replication. We monitored CAG·CTG repeat instability in transgenic mouse cells arrested by chemical or genetic manipulation of the cell cycle and generated unequivocal evidence for the continuous accumulation of repeat expansions in non-dividing cells. Importantly, the rates of expansion in non-dividing cells were at least as high as those of proliferating cells. These data are consistent with a major role for cell division-independent expansion in generating somatic mosaicism in vivo. Although expansions can accrue in non-dividing cells, we also show that cell cycle arrest is not sufficient to drive instability, implicating other factors as the key regulators of tissue-specific instability. Our data reveal that de novo expansion events are not limited to S-phase and further support a cell division-independent mutational pathway.

Figure 1.

Analysis of cell cycle arrest. (A) Cell culture dynamics. The graphs show mean cell survival (±SD) of six replicate cultures over time. Cell survival of chemically treated cultures, cells expressing p16^INK4a and p21^WAF1 following mifepristone A induction, and serum-starved MEFs, was calculated relative to the day zero culture. Statistically significant differences between two consecutive time points are illustrated with asterisks (*, P < 0.05; **, P < 0.01; Mann–Whitney U test). (B) Proportion of BrdU positive cells. The histograms show the percentage of BrdU immunopositive cells and the error bars indicate the 95% confidence intervals for control and treated cultures. Statistically significant reductions in BrdU staining relative to controls are indicated (***, P < 0.001; Fisher's exact test). (C) Relative PCNA expression levels. The graph on the left shows the quantitative analysis of PCNA protein expression levels in arrested cells relative to the corresponding proliferating controls. The graph on the right shows the quantitative analysis of PCNA protein expression (±SD) in mifepristone A-induced cells, co-transfected with pSwitch regulatory plasmid and pGENE/V5-His A/p16^INK4a (p16) or pGENE/V5-His A/p21^WAF1 (p21), relative to un-induced transfected controls. Statistically significant reductions in PCNA protein levels relative to proliferating controls are indicated (*, P < 0.05; Mann–Whitney U test). AU, arbitrary units.

Figure 2.

BrdU incorporation patterns. To measure levels of DNA synthesis we performed a BrdU incorporation assay. Representative low magnification images (left) reveal the relative proportion of BrdU immunostaining (green) nuclei counter-stained with DAPI (blue). Representative high-magnification images (right) illustrate BrdU immunostaining patterns within individual cells and reveal differences between chemical treatments.

Figure 3.

The expanded CAG·CTG repeat continues to expand in chemically arrested cells. (A) Representative SP-PCRs of the expanded CAG·CTG repeat in replicate D2763Kc2 cell cultures (1-6)

Figure 4.

The CAG·CTG repeat expansion continues to expand in genetically arrested cells. (A) Representative SP-PCRs of the expanded CAG·CTG repeat in replicate D2763Kc2 cell cultures (1-6)

Figure 5.

Cell cycle arrest is not sufficient to mediate CAG·CTG repeat expansion. Representative SP-PCRs of the expanded CAG·CTG repeat in chemically arrested D2763L lung cell cultures (A) and MEF cell cultures arrested by serum starvation (B) are shown. Also included are the relevant dividing cell controls, the embryo from which the MEF line was established, the progenitor culture from which all cultures were derived at day zero and the vehicle DMSO control. For clarity, only one representative replicate from each set of six replicate cultures, and data from only one of the three MEF cell lines analyzed, is shown. D2763L lung cells were treated for 90 days with 500 nM apicidin or 100 nM trichostatin A, or for 45 days with 30 μM MMC. The scale on the right indicates the DNA molecular weight markers converted into number of CAG·CTG repeats.