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. 2011 Aug;31(15):3105-12.
doi: 10.1128/MCB.05158-11. Epub 2011 May 31.

Topoisomerase 1 and single-strand break repair modulate transcription-induced CAG repeat contraction in human cells

Leroy Hubert Jr  1 , Yunfu Lin, Vincent Dion, John H Wilson
Affiliations

Topoisomerase 1 and single-strand break repair modulate transcription-induced CAG repeat contraction in human cells

Leroy Hubert Jr et al. Mol Cell Biol. 2011 Aug.

Abstract

Expanded trinucleotide repeats are responsible for a number of neurodegenerative diseases, such as Huntington disease and myotonic dystrophy type 1. The mechanisms that underlie repeat instability in the germ line and in the somatic tissues of human patients are undefined. Using a selection assay based on contraction of CAG repeat tracts in human cells, we screened the Prestwick chemical library in a moderately high-throughput assay and identified 18 novel inducers of repeat contraction. A subset of these compounds targeted pathways involved in the management of DNA supercoiling associated with transcription. Further analyses using both small molecule inhibitors and small interfering RNA (siRNA)-mediated knockdowns demonstrated the involvement of topoisomerase 1 (TOP1), tyrosyl-DNA phosphodiesterase 1 (TDP1), and single-strand break repair (SSBR) in modulating transcription-dependent CAG repeat contractions. The TOP1-TDP1-SSBR pathway normally functions to suppress repeat instability, since interfering with it stimulated repeat contractions. We further showed that the increase in repeat contractions when the TOP1-TDP1-SSBR pathway is compromised arises via transcription-coupled nucleotide excision repair, a previously identified contributor to transcription-induced repeat instability. These studies broaden the scope of pathways involved in transcription-induced CAG repeat instability and begin to define their interrelationships.

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Figures

Fig. 1.
Fig. 1.
Screening the Prestwick chemical library. (A) Selection assay for CAG repeat contractions. Large CAG repeat tracts are spliced aberrantly, preventing expression of functional HRPT and making the cells HAT sensitive (HATS). When the repeat tract is 38 units or fewer, sufficient correctly spliced message is produced to permit expression of enough HPRT to become HAT resistant (HATR). (B) Design of chemical screen. FLAH25 cells were plated at near confluence in 96-well plates, treated for 3 days with chemicals in the presence of doxycycline, and then replated for HAT selection in six-well plates.
Fig. 2.
Fig. 2.
Treatments that interfere with TOP1. (A) Chemical treatments. Acacetin (10 μM) and camptothecin (10 μM) were compared to the DMSO control in the presence (■) or absence (▩) of doxycycline. Relative contraction frequencies were normalized to 1 for DMSO treatment in the presence of doxycycline (8.0 × 10−6 ± 0.8 × 10−6). (B) siRNA treatments. Two TOP1 siRNAs were compared to control siRNA against vimentin in the presence (■) or absence (▩) of doxycycline. Relative contraction frequencies were normalized to 1 for vimentin siRNA in the presence of doxycycline (7.4 × 10−6 ± 1.5 × 10−6). In all cases, error bars indicate standard deviations, and the statistical significance is indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
Fig. 3.
Fig. 3.
Treatments that interfere with TDP1. (A) Chemical treatments. Amikacin (100 μM) was compared to the DMSO control in the presence (■) or absence (▩) of doxycycline. Relative contraction frequencies were normalized to 1 for DMSO treatment in the presence of doxycycline (8.0 × 10−6 ± 0.8 × 10−6). (B) siRNA treatments. Two TDP1 siRNAs were compared to control siRNA against vimentin in the presence (■) or absence (▩) of doxycycline. Relative contraction frequencies were normalized to 1 for vimentin siRNA in the presence of doxycycline (7.4 × 10−6 ± 1.5 × 10−6). In all cases, error bars show the standard deviations, and the statistical significance is indicated (*, P < 0.05; ***, P < 0.001).
Fig. 4.
Fig. 4.
Treatments that interfere with SSBR. (A) Chemical treatments. Betulinic acid (10 μM) was compared to the DMSO control in the presence (■) or absence (▩) of doxycycline. Relative contraction frequencies were normalized to 1 for DMSO treatment in the presence of doxycycline (8.0 × 10−6 ± 0.8 × 10−6). (B) siRNA treatments. Two XRCC1 siRNAs and one PARP1 siRNA were compared to control siRNA against vimentin in the presence (■) or absence (▩) of doxycycline. Relative contraction frequencies were normalized to 1 for vimentin siRNA in the presence of doxycycline (7.4 × 10−6 ± 1.5 × 10−6). In all cases, error bars show the standard deviations, and the statistical significance is indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
Fig. 5.
Fig. 5.
Combination treatments with siRNAs. FLAH25 cells were treated with combinations of siRNAs against vimentin (the control), TDP1, and XPA. Relative contraction frequencies were normalized to 1 for vimentin siRNA (9.5 × 10−6 ± 2.9 × 10−6). In all cases, error bars show the standard deviations, and the statistical significance is indicated (n.s., not significant; *, P < 0.05; ***, P < 0.001).
Fig. 6.
Fig. 6.
Proposed relationship between the TOP1-TDP1-SSBR and the TC-NER pathways. The TOP1-DNA irreversible cleavage complexes (TOP1icc) that arise normally in the course of TOP1 action are usually taken care of by TDP1 and SSBR, which prevent the formation of the large CAG contractions our system is able to detect. When the TDP1-SSBR pathway is compromised, however, RNA polymerase II stalls at the damage, eliciting involvement of TC-NER, which promotes CAG repeat contractions, as described previously (28, 32).
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