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. 2018 Aug;25(8):669-676.
doi: 10.1038/s41594-018-0094-9. Epub 2018 Jul 30.

Mechanisms of genetic instability caused by (CGG)n repeats in an experimental mammalian system

Artem V Kononenko  1 , Thomas Ebersole  1 , Karen M Vasquez  2 , Sergei M Mirkin  3
Affiliations

Mechanisms of genetic instability caused by (CGG)n repeats in an experimental mammalian system

Artem V Kononenko et al. Nat Struct Mol Biol. 2018 Aug.

Abstract

We developed an experimental system for studying genome instability caused by fragile X (CGG)n repeats in mammalian cells. Our method uses a selectable cassette carrying the HyTK gene under the control of the FMR1 promoter with (CGG)n repeats in its 5' UTR, which is integrated into the unique RL5 site in murine erythroid leukemia cells. Carrier-size (CGG)n repeats markedly elevated the frequency of reporter inactivation, making cells ganciclovir resistant. These resistant clones had a unique mutational signature: a change in repeat length concurrent with mutagenesis in the reporter gene. Inactivation of genes implicated in break-induced replication, including Pold3, Pold4, Rad52, Rad51, and Smarcal1, reduced the frequency of ganciclovir-resistant clones to the baseline level that was observed in the absence of (CGG)n repeats. We propose that replication fork collapse at carrier-size (CGG)n repeats can trigger break-induced replication, which results in simultaneous repeat length changes and mutagenesis at a distance.

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Figures

Figure 1
Figure 1. Experimental system to study genome instability caused by (CGG)n repeats
a. FMR1 regulatory domain included in the selectable cassettes is shown by the red bracket; blue line represents human endogenous FMR 1 locus. b. FMR1-CGGn-HyTK cassettes were integrated into the RL5 locus in MEL cells via Cre-loxP recombination replacing the eGFP gene. A cassette can be integrated into the RL5 locus in two different orientations. c. Frequencies of Gc-r clones recovered. For clones with each experimental cassette, 96-well plates containing 106 cells were grown in the presence of ganciclovir, and Gc-r clones were counted. The number of analyzed plates were 17, 8 and 20 for CGG153-HyTK, CGG53-HyTK and CGG0-HyTK cassette, respectively. d. Sample PCR across the (CGG)153 run showing expanded (lanes 3 and 5), contracted (lanes 2 and 6) and unchanged repeats (lanes 1 and 4). e. Distribution of repeat expansions and contractions. f. PCR analysis of rare Gc-r clones containing large-scale repeats expansions (lanes 2–3); lane 1- starting repeat, lane 4 – small-scale expansion. g. qPCR analysis of the HyTK gene transcription in the clones shown in f. Means and standard deviations were calculated from three independent experiments. h. ChIP analysis of the H3K4me3 chromatin mark in the clones shown in f. The murine beta-major globin and the murine amylase genes were used as controls for open and condensed chromatin, respectively. Means and standard deviations were calculated from three independent experiments. Source data for d and f are available online.
Figure 2
Figure 2. Mechanisms of ganciclovir-resistance
a. Loss of function mutations in the HyTK gene leading to Gc-r. DNA bases from the wild-type HyTK gene that underwent mutagenesis are shown inside boxed areas of different cassettes. Distances from the 3′ end of the (CGG)n run are shown at the top. Dashed vertical lines align identical positions. Black arrows show transcription start sites (TSS), red rectangles represent (CGG)n repeats, M stands for translation initiation codon, blue rectangles demarcate the Hy domain. Point substitutions are written above the wild-type DNA bases; orange squares above the wild-type DNA bases designate indels or complex mutations. b. Frequencies of Gc-r clones in the c3-1 cell line carrying FMR1-CGG153-HyTK cassette in the A orientation (Table 1) upon treatment with siRNA targeted against genes implicated in BIR. Means and standard deviations from 4 independent experiments are shown. c. Inactivation of mouse POLD3, POLD4, RAD51, RAD52 and SMARCAL1 genes by Accell siRNAs. Western blot analyses with specific antibodies show dramatic reductions in the expression levels of the corresponding proreins. Protein levels for GAPDH gene are shown as internal control. Source data for c are available online.
Fig. 3
Fig. 3
Proposed mechanism for mutational events triggered by carrier-size (CGG)n repeats. Fork stalling and reversal can lead to the formation of the one-ended DSB. Repair of this DSB via BIR can result in repeat expansion or contractions concordant with mutagenesis at a distance (see text for details). Strands of the repeat are shown in brown and blue; flanking DNA is in black. Proteins involved at various steps of the process are indicated.
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