Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Sep;30(18):4435-51.
doi: 10.1128/MCB.00332-10. Epub 2010 Jul 20.

Convergent transcription through a long CAG tract destabilizes repeats and induces apoptosis

Yunfu Lin  1 , Mei Leng, Ma Wan, John H Wilson
Affiliations

Convergent transcription through a long CAG tract destabilizes repeats and induces apoptosis

Yunfu Lin et al. Mol Cell Biol. 2010 Sep.

Abstract

Short repetitive sequences are common in the human genome, and many fall within transcription units. We have previously shown that transcription through CAG repeat tracts destabilizes them in a way that depends on transcription-coupled nucleotide excision repair and mismatch repair. Recent observations that antisense transcription accompanies sense transcription in many human genes led us to test the effects of antisense transcription on triplet repeat instability in human cells. Here, we report that simultaneous sense and antisense transcription (convergent transcription) initiated from two inducible promoters flanking a CAG95 tract in a nonessential gene enhances repeat instability synergistically, arrests the cell cycle, and causes massive cell death via apoptosis. Using chemical inhibitors and small interfering RNA (siRNA) knockdowns, we identified the ATR (ataxia-telangiectasia mutated [ATM] and Rad3 related) signaling pathway as a key mediator of this cellular response. RNA polymerase II, replication protein A (RPA), and components of the ATR signaling pathway accumulate at convergently transcribed repeat tracts, accompanied by phosphorylation of ATR, CHK1, and p53. Cell death depends on simultaneous sense and antisense transcription and is proportional to their relative levels, it requires the presence of the repeat tract, and it occurs in both proliferating and nonproliferating cells. Convergent transcription through a CAG repeat represents a novel mechanism for triggering a cellular stress response, one that is initiated by events at a single locus in the genome and resembles the response to DNA damage.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Cell lines for assessing effects of sense and antisense transcription. In all cells, the CAG tract is centered in the 2.1-kb intron in the single, randomly integrated HPRT minigene and the repeat is 1.6 kb downstream of the sense promoter and 2.5 kb upstream of the antisense promoter. (A) DIT cells. Sense and antisense transcription are driven by the inducible promoters pCMVmini, which responds to doxycycline (Dox), and pNEBR-X1, which responds to RSL1. (B) FLAH cells. Sense transcription is driven by pCMVmini; no antisense promoter is present. (C) DIT-NAT cells. Sense transcription is driven by pCMVmini; the antisense promoter pNEBR-X1 is silenced. (D) DH cells. In one HPRT minigene, sense transcription is driven by pCMVmini; no antisense promoter is present. In the other, antisense transcription is constitutively expressed from the pCMV promoter; no sense promoter is present. (E) DIT-NR cells. Sense and antisense transcription are driven by pCMVmini and pNEBR-X1, respectively, but the entire CAG tract plus 120-bp flanking sequence is deleted. (F) DIT7-R cells. These cells were derived by contraction of the repeat in DIT7 cells. (G) DITS-H cells. These HEK293-derived cells have an HPRT minigene in which both sense and antisense transcription are driven by pCMVmini and inducible with doxycycline. (H) Strategy for strand-specific real-time RT-PCR to quantify sense and antisense transcripts from the HPRT minigene. The final PCR product in both cases is 225 bp.
FIG. 2.
FIG. 2.
Convergent transcription and repeat instability. (A) Selection assay for repeat contraction; (B) CAG contraction in DIT7 cells. Sense transcription was induced by doxycycline (200 ng/ml), antisense transcription by RSL1 (500 nM), and convergent transcription by doxycycline plus RSL1. Frequencies of HPRT+ colonies minus the background in the absence of inducers ([1.8 ± 0.9] × 10−6) are sense ([9.2 ± 5.1] × 10−6), antisense ([4.8 ± 2.4] × 10−6), and convergent ([33 ± 12] × 10−6). Frequencies are mean values of eight independent measurements, with error bars indicating standard deviation. (C) CAG contraction in DIT3 cells. Sense transcription was induced by doxycycline (200 ng/ml), antisense transcription by RSL1 (500 nM), and convergent transcription by doxycycline plus RSL1. Contraction frequencies minus the background in the absence of inducers ([2.1 ± 1.6] × 10−6) were sense ([6.9 ± 3.5] × 10−6), antisense ([5.9 ± 2.3] × 10−6), and convergent ([26 ± 9.2] × 10−6). Frequencies were averaged from six independent experiments, with error bars indicating standard deviation. Statistical significance is indicated: n.s., not significant; and ***, P < 0.0001.
FIG. 3.
FIG. 3.
Convergent transcription and cell death. (A) Convergent transcription in proliferating DIT7 cells. Cells were plated at the same initial density and photographed on different days, as shown. (B) Cell killing with and without convergent transcription in DIT7 cells; (C) cell killing with and without convergent transcription in DIT3 cells; (D) transcription-induced cell death in DIT7 cells. Viable cells were counted after 4 days with no inducer (None), Dox (200 ng/ml), RSL1 (500 nM), or Dox plus RSL1. (E) Cell death as a function of increasing sense transcription with fully induced antisense transcription. (F) Cell death as a function of increasing antisense transcription with highly induced sense transcription. (G) Comparison of cell killing under full or modest induction of convergent transcription in proliferating and serum-starved DIT7 cells. For full induction, 200 ng/ml doxycycline and 500 nM RSL1 were added; for modest induction, 20 ng/ml doxycycline and 50 nM RSL1 were added. For all experiments, percentages of viable cells were calculated as the number of adherent cells divided by the sum of adherent and nonadherent cells and are averaged from at least six independent measurements. Error bars show standard deviations. Statistical significance is indicated: n.s., not significant; *, P < 0.05; **, P < 0.001; and ***, P < 0.0001.
FIG. 4.
FIG. 4.
Effects of convergent transcription and length of CAG repeats on cell death. (A) Convergent transcription and cell death in various cell lines. Cell lines were treated for 4 days with or without Dox plus RSL1. (B) Correlation between CAG tract length and cell death induced by convergent transcription; (C) convergent transcription and apoptosis in DITS-H cells. DITS-H5 and DITS-H8 cells were treated with DMSO, doxycycline plus DMSO, or doxycycline plus zVAD for 7 days. Percentages of viable cells were calculated as described in the legend to Fig. 3 and are averaged from at least six independent measurements. Error bars show standard deviations. Statistical significance is indicated: ***, P < 0.0001.
FIG. 5.
FIG. 5.
Convergent transcription-induced apoptosis. (A) Effect of the caspase inhibitor zVAD (20 μM) on death of proliferating DIT7 cells. Cells were plated at the same initial density, treated with DMSO alone, Dox plus RSL1, or Dox plus RSL1 plus zVAD, and photographed 4 days later. (B) Increased level of active caspase 3 after induction of convergent transcription. The micrograph shows an example of an immunostaining, with a cell negative for active caspase 3 above and a cell positive for active caspase 3 below. The graph shows the mean number ± standard deviation (SD) of cells that were positive for active caspase 3 from 0 to 48 h after induction of convergent transcription. (C) Suppression of convergent-transcription-induced cell killing by zVAD (20 μM) in proliferating DIT7 cells; (D) suppression of convergent-transcription-induced cell killing by zVAD (20 μM) in proliferating DIT3 cells. Percentages of viable cells were calculated as described in the legend to Fig. 3 and are averaged from at least six independent measurements. Error bars show standard deviations. (E) Frequencies of apoptotic cells measured by the PI-Hoechst 33342 staining method. Cells were plated at the same density in six-well plates. Doxycycline (200 ng/ml) and RSL1 (500 nM) were added to different wells at 5 days, 4 days, 3 days, 2 days, and 1 day before staining. For each time point in each experiment, random fields of adherent cells were examined by fluorescence microscopy and a total of at least 1,000 cells were counted. Most dead cells were washed away prior to staining; however, dead cells among the adherent cells increased from 0.3% at day 0 to 3.2% at day 5. Frequencies of apoptotic cells, which are the means of three measurements for each time point, are expressed as a percentage of total adherent cells. Error bars indicate standard deviations. (F) Percentages of live, apoptotic, and dead cells determined by flow cytometry after staining with FTTC, annexin, and PI. The means of three assays are shown. For each time point in each experiment, 10,000 cells were counted by flow cytometry. (G) Frequencies of apoptotic cells measured by flow cytometry after staining with FITC, annexin V, and PI. Frequencies of apoptotic cells increased from 0.3% at day 0 to 8.9% at day 5. Frequencies of apoptotic cells are expressed here as a percentage of live plus apoptotic cells for ease of comparison with the data in panel E. Frequencies are the means of three experiments for each time point, with error bars indicating the standard deviation. For each time point in each experiment, 10,000 cells were counted by flow cytometry. Statistical significance is indicated: n.s., not significant; *, P < 0.05; **, P < 0.001; and ***, P < 0.0001.
FIG. 6.
FIG. 6.
Apoptosis in nonproliferating cells. (A) Convergent transcription and cell death in serum-starved DIT7 cells in the presence and absence of zVAD. Serum-starved DIT7 cells were treated with DMSO, doxycycline plus RSL1 (Dox + RSL1), or doxycycline plus RSL1 and zVAD (Dox + RSL1 + zVAD) and photographed 3 days later. (B) Quantification of cell killing in serum-starved DIT7 cells that were treated with doxycycline plus RSL1 or with DMSO alone. (C) Quantification of cell killing in confluent DIT7 cells that were untreated or treated with doxycycline plus RSL1. (D) Quantification of the effect of zVAD on cell killing in serum-starved DIT7 and DIT3 cells. Serum-starved DIT cells were treated for 3 days with DMSO, zVAD, doxycycline plus RSL1, or doxycycline plus RSL1 and zVAD. Percentages of viable cells were calculated as described in the legend to Fig. 3 and are averaged from at least six independent measurements with error bars indicating standard deviations. Statistical significance is indicated: **, P < 0.001; and ***, P < 0.0001.
FIG. 7.
FIG. 7.
ATR response after induction of convergent transcription in DIT7 cells. (A) ATR-S428P; (B) CHK1-S345P; (C) p53-S15P; (D) ATM-S1981P. Individual proteins were assessed at 0, 6, 24, and 48 h by immunostaining. Micrographs show examples of cells that are negative (above) and positive (below). Results were averaged from three determinations, and error bars represent standard deviations. Statistical significance is indicated: n.s., not significant; *, P < 0.05; **, P < 0.001; and ***, P < 0.0001. (E) p53-S15P fluorescence in ATR-S428P-positive cells; (F) ATM-S1981P fluorescence in ATR-S428P-positive cells; (G) active caspase 3 fluorescence in p53-S15P-positive cells.
FIG. 8.
FIG. 8.
Roles of ATR and ATM in downstream phosphoryation. DIT7 cells were treated with ATR or ATM siRNAs (or with vimentin siRNA as a control) prior to and during induction of convergent transcription. Cells positive for phosphorylated proteins were counted 2 days after convergent transcription was induced. Results were averaged from three determinations, and error bars represent standard deviations. Statistical significance is indicated: n.s., not significant; *, P < 0.05; and **, P < 0.001.
FIG. 9.
FIG. 9.
Chromatin immunoprecipitation assay. Enrichment of proteins at two sites, ChIP-1 and ChIP-2, was assayed 48 h after induction of convergent transcription. Relative values are the values determined for ChIP-1 and ChIP-2 (as calculated in Materials and Methods) normalized to values at ChIP-2. Results are averaged from three determinations, and error bars show standard deviations. Statistical significance is indicated: *, P < 0.05; and **, P < 0.001.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Arima, Y., M. Nitta, S. Kuninaka, D. Zhang, T. Fujiwara, Y. Taya, M. Nakao, and H. Saya. 2005. Transcriptional blockade induces p53-dependent apoptosis associated with translocation of p53 to mitochondria. J. Biol. Chem. 280:19166-19176. - PubMed
    1. Bacolla, A., J. E. Larson, J. R. Collins, J. Li, A. Milosavljevic, P. D. Stenson, D. N. Cooper, and R. D. Wells. 2008. Abundance and length of simple repeats in vertebrate genomes are determined by their structural properties. Genome Res. 18:1545-1553. - PMC - PubMed
    1. Belotserkovskii, B. P., E. De Silva, S. Tornaletti, G. Wang, K. M. Vasquez, and P. C. Hanawalt. 2007. A triplex-forming sequence from the human c-MYC promoter interferes with DNA transcription. J. Biol. Chem. 282:32433-32441. - PubMed
    1. Carthew, R. W., and E. J. Sontheimer. 2009. Origins and mechanisms of miRNAs and siRNAs. Cell 136:642-655. - PMC - PubMed
    1. Chen, W. L., J. W. Lin, H. J. Huang, S. M. Wang, M. T. Su, G. J. Lee-Chen, C. M. Chen, and H. M. Hsieh-Li. 2008. SCA8 mRNA expression suggests an antisense regulation of KLHL1 and correlates to SCA8 pathology. Brain Res. 1233:176-184. - PubMed

Publication types

MeSH terms