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. 2024 Jan 6;27(2):108814.
doi: 10.1016/j.isci.2024.108814. eCollection 2024 Feb 16.

The fragile X locus is prone to spontaneous DNA damage that is preferentially repaired by nonhomologous end-joining to preserve genome integrity

Daman Kumari  1 , Rachel Adihe Lokanga  2 , Cai McCann  1 , Thomas Ried  2 , Karen Usdin  1
Affiliations

The fragile X locus is prone to spontaneous DNA damage that is preferentially repaired by nonhomologous end-joining to preserve genome integrity

Daman Kumari et al. iScience. .

Abstract

A long CGG-repeat tract in the FMR1 gene induces the epigenetic silencing that causes fragile X syndrome (FXS). Epigenetic changes include H4K20 trimethylation, a heterochromatic modification frequently implicated in transcriptional silencing. Here, we report that treatment with A-196, an inhibitor of SUV420H1/H2, the enzymes responsible for H4K20 di-/trimethylation, does not affect FMR1 transcription, but does result in increased chromosomal duplications. Increased duplications were also seen in FXS cells treated with SCR7, an inhibitor of Lig4, a ligase essential for NHEJ. Our study suggests that the fragile X (FX) locus is prone to spontaneous DNA damage that is normally repaired by NHEJ. We suggest that heterochromatinization of the FX allele may be triggered, at least in part, in response to this DNA damage.

Keywords: Epigenetics; Human Genetics; Molecular Genetics.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
A-196 treatment does not affect FMR1 expression (A) Schematic showing different FMR1 alleles seen in the human population. (B) FXS cells were treated with DMSO, 5 μM A-196 or 10 μM A-196 for 3 days and 7 days and collected for RNA isolation. (C) FXS cells were treated with 10 μM AZA for 3 days and then the medium was changed to either with DMSO or 5 μM A-196. Samples were collected for RNA isolation at indicated time. The levels of FMR1 and GUSB mRNA were measured by RT-qPCR. FMR1 mRNA levels are shown as a percentage of GUSB mRNA. The data shown are an average from two independent treatments and the error bars represent standard deviation.
Figure 2
Figure 2
A-196 treatment decreases H4K20me3 (A‒C) FXS cells were treated with 10 μM AZA for 3 days and then the medium was changed to either with DMSO or 5 μM A-196. The cells were grown for another 4 days and then processed for lysate preparation for western blotting and chromatin preparation for ChIP assay. (A) A representative Western blot is shown. (B) Quantitation for H4K20me3 levels from three independent western blots. The levels of H4K20me3 were normalized to the levels of total H4 and are shown relative to AZA/DMSO. The error bars represent standard deviation. The two-tailed p value was calculated using paired t-test, ∗p = 0.012, ∗∗p = 0.0031. (C) ChIP assay was done with an antibody to H4K20me3. The % input data for FMR1 exon1 region were calculated and normalized to AZA/DMSO. Data shown are an average from 3 independent treatments. Error bars represent standard deviation. Two-tailed p value was calculated using the paired t-test, ∗p = 0.048, ∗∗p = 0.01.
Figure 3
Figure 3
A-196 treatment causes an increase in chromosomal duplications at the FX locus (A) Representative metaphases seen in FXS lymphoblastoid cells after treatment with 0.1 μM FdU and/or 5 μM A-196. (i) FISH image showing X chromosome with breakage of one sister chromatid and loss of the FRAXA region on the other. (ii) X chromosome showing breakage of both sister chromatids. (iii) FISH showing X chromosome in which one chromatid shows what appears to be a partial duplication of part of the FRAXA region. (iv, v) X chromosome in which both sister chromatids show duplication of the FRAXA region. (vi) Example of duplication of the FRAXA region seen in interphase nucleus. (B‒D) Percentage of metaphases showing fragile sites and duplications after treatment of FXS (B), typical (C) and premutation (D) cell lines with 0.1 μM FdU for 18 h and with DMSO or 5 μM A-196 for 3 days. For A-196 + FdU treatment, cells were treated with 5 μM A-196 for 3 days and 0.1 μM FdU was added for the last 18h of the treatment. Two-tailed p value was calculated using Fisher’s exact test using the actual number of metaphases showing duplications and/or fragile sites. No significant difference was seen in the percentage of metaphases showing fragile sites and duplications after treatment with A-196 in typical and premutation cells. See also Table S1.
Figure 4
Figure 4
Inhibition of nonhomologous end-joining is responsible for chromosomal duplications seen at the FX locus (A) FXS cells were treated with DMSO and 5 μM A-196 for 3 days and ChIP was done using an antibody to H4K20me2. The abundance of H4K20me2 at the FMR1 exon 1 was calculated as % input and is shown relative to DMSO. Data shown are an average from 3 independent experiments for GM07294 and 2 independent experiments for GM04025. Error bars represent standard deviation, two-tailed p value was calculated by paired t-test, ∗p = 0.009. ∗∗p = 0.005. (B) FXS cells were treated with DMSO and 5 μM A-196 for 3 days and ChIP was done using an antibody to 53BP1. The abundance of 53BP1 at the FMR1 exon 1 was calculated as % input and is shown relative to DMSO. Data shown are an average from 3 independent ChIP experiments. Error bars represent standard deviation, two-tailed p value was calculated by paired t-test, ∗p = 0.039. ∗∗p = 0.001. (C) Percentage of metaphases showing fragile sites and duplications in FXS cells after treatment with 5 μM A-196 or 10 μM SCR7 for 3 days. Two-tailed p value was calculated using Fisher’s exact test using the actual number of metaphases showing duplications and/or fragile sites. See also Figure S1 and Table S1.
Figure 5
Figure 5
Model for CGG repeat expansion induced FMR1 gene silencing Expansion of CGG repeats leads to increased replication stalling/fork collapse due to the formation of secondary structures that block fork progression., This problem is further exacerbated by replication-transcription conflicts in cells carrying active FM alleles. Stalled replication forks could lead to increased genomic instability including chromosome fragility and repeat contraction. Contraction of the repeats would reduce the likelihood of replication fork stalling to some degree. The stable R-loop associated with transcription of the FM alleles would facilitate recruitment of G9a and PRC2 leading to gene silencing, which in turn would reduce the risk further by reducing replication-transcription conflicts. While silencing would reduce replication-transcription conflicts, residual fork stalling could still threaten genome integrity. Recruitment of H3K9me3 and H4K20me3 may mitigate this danger by facilitating NHEJ in part by maximizing 53BP1 binding.
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