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. 2012;7(10):e46807.
doi: 10.1371/journal.pone.0046807. Epub 2012 Oct 3.

Nucleotide excision repair, mismatch repair, and R-loops modulate convergent transcription-induced cell death and repeat instability

Yunfu Lin  1 , John H Wilson
Affiliations

Nucleotide excision repair, mismatch repair, and R-loops modulate convergent transcription-induced cell death and repeat instability

Yunfu Lin et al. PLoS One. 2012.

Abstract

Expansion of CAG•CTG tracts located in specific genes is responsible for 13 human neurodegenerative disorders, the pathogenic mechanisms of which are not yet well defined. These disease genes are ubiquitously expressed in human tissues, and transcription has been identified as one of the major pathways destabilizing the repeats. Transcription-induced repeat instability depends on transcription-coupled nucleotide excision repair (TC-NER), the mismatch repair (MMR) recognition component MSH2/MSH3, and RNA/DNA hybrids (R-loops). Recently, we reported that simultaneous sense and antisense transcription-convergent transcription-through a CAG repeat not only promotes repeat instability, but also induces a cell stress response, which arrests the cell cycle and eventually leads to massive cell death via apoptosis. Here, we use siRNA knockdowns to investigate whether NER, MMR, and R-loops also modulate convergent-transcription-induced cell death and repeat instability. We find that siRNA-mediated depletion of TC-NER components increases convergent transcription-induced cell death, as does the simultaneous depletion of RNase H1 and RNase H2A. In contrast, depletion of MSH2 decreases cell death. These results identify TC-NER, MMR recognition, and R-loops as modulators of convergent transcription-induced cell death and shed light on the molecular mechanism involved. We also find that the TC-NER pathway, MSH2, and R-loops modulate convergent transcription-induced repeat instability. These observations link the mechanisms of convergent transcription-induced repeat instability and convergent transcription-induced cell death, suggesting that a common structure may trigger both outcomes.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Structure of the HPRT minigenes in DIT7 and DIT7-R103 cells.
DIT7 cells carry a CAG95 repeat tract and DIT7-R103 cells, which were derived from DIT7 cells by contraction of the repeat, carry a CAG15 repeat tract. In both cell lines, the CAG tract is centered in the 2.1-kb intron in the single, randomly integrated HPRT minigene. The CAG repeat is about 1.6 kb downstream of the sense promoter and about 2.5 kb upstream of the antisense promoter.
Figure 2
Figure 2. Effects of knockdown of TC-NER components on convergent transcription-induced cell death.
(A) siRNA knockdowns in DIT7 cells. Frequencies of cell death are: vimentin, 47%; XPA-1, 55%; XPA-2, 63%; CSB-1, 52%; CSB-2, 51%; ERCC1-1, 61%; ERCC1-2, 53%; XPG-1, 51%; XPG-2, 57%. (B) siRNA knockdowns in DIT7-R103 cells. Frequencies of cell death are: vimentin, 22%; XPA-1, 33%; XPA-2, 31%; CSB-1, 29%; CSB-2, 26%; ERCC1-1, 39%; ERCC1-2, 31%; XPG-1, 25%; XPG-2, 40%. Frequency of cell death was calculated as the number of nonadherent cells divided by the sum of adherent and nonadherent cells. Data are the average frequencies of cell death from at least 6 independent siRNA knockdown experiments. Error bars indicate standard deviations. Statistical significance relative to the vimentin control is indicated: *P<0.05; **P<0.001; ***P<0.0001.
Figure 3
Figure 3. Binding of XPA to CAG- and CTG-containing DNA duplexes.
Duplexes without hairpins, with a CAG13 hairpin, or a CTG13 hairpin were attached to magnetic beads (see Methods) and incubated with a whole cell extract from human cells. The proteins bound to the DNA were then analyzed by Western blot analysis, using antibodies against actin or XPA. Actin served as a control for nonspecific binding. WCE stands for whole cell extract.
Figure 4
Figure 4. Effects of MSH2 knockdown on convergent transcription-induced cell death.
(A) siRNA knockdowns in DIT7 cells. Frequencies of cell death are: vimentin, 47%; MSH2-1, 41%; MSH2-2, 42%. (B) siRNA knockdowns in DIT7-R103 cells. Frequencies of cell death are: vimentin, 22%; MSH2-1, 4%; MSH2-2, 9%. Frequency of cell death was calculated as the number of nonadherent cells divided by the sum of adherent and nonadherent cells. Data are the average frequency of cell death from at least 6 independent siRNA knockdown experiments. Error bars indicate standard deviations. Statistical significance relative to the vimentin control is indicated: *P<0.05; **P<0.001; ***P<0.0001.
Figure 5
Figure 5. Effects of RNase H knockdown on convergent transcription-induced cell death.
(A) siRNA knockdowns in DIT7 cells. Frequencies of cell death are: vimentin, 47%; RNase H1-1, 49%; RNase H2A-1, 46%; RNase H1-1 plus RNase H2A-1, 54%. (B) siRNA knockdowns in DIT7-R103 cells. Frequencies of cell death are: vimentin, 22%; RNase H1-1, 27%; RNase H2A-1, 28%; RNase H1-1 plus RNase H2A-1, 44%. Frequency of cell death was calculated as the number of nonadherent cells divided by the sum of adherent and nonadherent cells. Data are the average frequency of cell death from at least 6 independent siRNA knockdown experiments. Error bars indicate standard deviations. Statistical significance relative to the vimentin control is indicated: *P<0.05; **P<0.001; ***P<0.0001.
Figure 6
Figure 6. Effects of knockdowns of MSH2, XPA and RNase H on convergent transcription-induced CAG repeat contraction.
Contraction frequencies were calculated as the number of HPRT+ colonies divided by the number of viable cells, averaged over at least 6 independent siRNA knockdown experiments. Error bars indicate standard deviations. Statistical significance relative to the vimentin control is indicated: *P<0.05; **P<0.01.
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