A novel role for the condensin II complex in cellular senescence

<p>Although cellular senescence is accompanied by global alterations in genome architecture, how the genome is restructured during the senescent processes is not well understood. Here, we show that the hCAP-H2 subunit of the condensin II complex exists as either a full-length protein or an N-terminus truncated variant (ΔN). While the full-length hCAP-H2 associates with mitotic chromosomes, the ΔN variant exists as an insoluble nuclear structure. When overexpressed, both hCAP-H2 isoforms assemble this nuclear architecture and induce senescence-associated heterochromatic foci (SAHF). The hCAP-H2ΔN protein accumulates as cells approach senescence, and hCAP-H2 knockdown inhibits oncogene-induced senescence. This study identifies a novel mechanism whereby condensin drives senescence via nuclear/genomic reorganization.</p

Model of the Swi1-Swi3 FPC-dependent telomere length maintenance.

<p>For details, see text.</p

Swi1 loss causes DNA damage at telomeres.

<p><b>(A)</b> Genome-wide (ChIP-seq) analysis of Rad52 distribution at the subtelomeres (left arm of chromosome 1) in asynchronous cultures of wild-type (green) and <i>swi1Δ</i> (blue) cells. Enrichment of Rad52 is displayed as enrichment scores (y-axis). Chromosome coordinates [x-axis, in megabases, (Mb)] were downloaded from the <i>S</i>. <i>pombe</i> Genome Project (Sanger Center: <a href="http://www.sanger.ac.uk/Projects/S_pombe" target="_blank">www.sanger.ac.uk/Projects/S_pombe</a>). The strains used for our experiments are both heterothallic <i>h</i>^<i>+</i> strains. <b>(B)</b> Box plots for log2 ratios of Rad52 enrichment at subtelomeric regions vs other regions. The boxes were limited by the 25th and 75th percentiles, with the black lines representing the median ratios between <i>swi1</i>Δ and wild-type. The whiskers are extended out to the most extreme data points that are at most 1.5 times the interquartile range from the box. The black circles represent outliers. The <i>p</i>-value was evaluated by Mann-Whitney U test. <b>(C)</b> ChIP assays showing Rad52 enrichment at telomeres in wild-type and <i>swi1</i>Δ cells. Rad52-12Pk was immunoprecipitated from the indicated cells, and associated DNA was subjected to competitive multiplex PCR to amplify DNA fragments from the <i>TAS1</i> region and a gene-free region (GFR2), which was used as an internal amplification control. Chromatin association of Rad52-12Pk at <i>TAS1</i> was presented as relative enrichment over the association at GFR2. Data are expressed as the mean of three independent experiments. Error bars represent the standard deviation. <i>p</i>-value was determined by two-tailed Student's t-test. <b>(D-E-F)</b> Wild-type and <i>swi1Δ</i> cells expressing Rad52-YFP and Taz1-mCherry were grown in minimal medium at 25°C until mid-log phase. Nuclei (n = 102) were analyzed for the percentage of TIF-positive nuclei in (D) as well as for the total number of Rad52-YFP and Taz1-mCherry foci in (E). Accumulation of Rad52-YFP foci (E) and a significant increase in TIF-positive nuclei were observed in the <i>swi1Δ</i> cells (D). Data shown is the mean of three independent experiments. Error bars represent the standard deviation. <i>p</i>-values were determined by two-tailed Student's t-test. (F) Representative microscopic images. Pink arrows indicate TIFs.</p

The Swi1-Swi3 FPC is required for telomere maintenance.

<p><b>(A)</b> Schematic diagram showing the position of the <i>Apa</i>I restriction site at <i>S</i>. <i>pombe</i> telomeres and the <i>TAS1</i> (Telomere-Associated Sequences) region [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005943#pgen.1005943.ref087" target="_blank">87</a>]. Terminal telomeric repeats are shown in red. The position of the telomeric probe used in this study is marked in green. <b>(B)</b> Southern blot analysis of <i>Apa</i>I-digested genomic DNA from independently isolated strains of the indicated genotypes. Each strain was passaged at least 8 times before DNA isolation, in order to allow for telomere length stabilization. <i>Apa</i>I-telomere fragments were detected with a telomere-specific DNA probe. <b>(C)</b> <i>swi1</i>Δ cells were transformed with the pJK148-Swi1 plasmid or with the control vector pJK148. Transformants were isolated and passaged at least 8 times before genomic DNA preparation. Southern blot analysis of the <i>Apa</i>I-telomere fragments was performed as described above. Telomeres of two or three independent isolates from each transformation are shown. <b>(D)</b> Swi1 is recruited to <i>TAS1</i> in a replication-dependent manner. <i>swi1-13Myc cdc25-22</i> cells were synchronized at the G2/M boundary by incubation at 36°C for 3 h and then released into fresh YES medium with or without 15 mM hydroxyurea (HU), an inhibitor of DNA replication. Cells were collected at the indicated times and processed for Swi1-13Myc ChIP, and telomere association of Swi1 was determined by real-time PCR as described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005943#pgen.1005943.ref127" target="_blank">127</a>]. Swi1-13Myc was associated with <i>TAS1</i> as cells replicate DNA. Previously determined peaks of binding for leading (Pol ε) and lagging (Pol α and Pol δ) strand DNA polymerase [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005943#pgen.1005943.ref052" target="_blank">52</a>] are indicated. Addition of HU, which prevented telomere replication, inhibited Swi1-13Myc recruitment at <i>TAS1</i>. <b>(E)</b> Southern blot analysis of <i>Apa</i>I-telomere fragments from the indicated cells was performed as described in <i>B</i>.</p

Swi1^Timeless Prevents Repeat Instability at Fission Yeast Telomeres

<div><p>Genomic instability associated with DNA replication stress is linked to cancer and genetic pathologies in humans. If not properly regulated, replication stress, such as fork stalling and collapse, can be induced at natural replication impediments present throughout the genome. The fork protection complex (FPC) is thought to play a critical role in stabilizing stalled replication forks at several known replication barriers including eukaryotic rDNA genes and the fission yeast mating-type locus. However, little is known about the role of the FPC at other natural impediments including telomeres. Telomeres are considered to be difficult to replicate due to the presence of repetitive GT-rich sequences and telomere-binding proteins. However, the regulatory mechanism that ensures telomere replication is not fully understood. Here, we report the role of the fission yeast Swi1^Timeless, a subunit of the FPC, in telomere replication. Loss of Swi1 causes telomere shortening in a telomerase-independent manner. Our epistasis analyses suggest that heterochromatin and telomere-binding proteins are not major impediments for telomere replication in the absence of Swi1. Instead, repetitive DNA sequences impair telomere integrity in <i>swi1</i>Δ mutant cells, leading to the loss of repeat DNA. In the absence of Swi1, telomere shortening is accompanied with an increased recruitment of Rad52 recombinase and more frequent amplification of telomere/subtelomeres, reminiscent of tumor cells that utilize the alternative lengthening of telomeres pathway (ALT) to maintain telomeres. These results suggest that Swi1 ensures telomere replication by suppressing recombination and repeat instability at telomeres. Our studies may also be relevant in understanding the potential role of Swi1^Timeless in regulation of telomere stability in cancer cells.</p></div

Neither heterochromatin disruption nor shelterin removal can rescue telomere shortening in <i>swi1Δ</i> cells.

<p><b>(A-B)</b> Loss of heterochromatin-related proteins does not rescue telomere-shortening phenotype of <i>swi1</i>Δ cells. Southern blot analysis of telomere fragments from the indicated mutants. At least two independent segregants of the double deletion mutants were examined. Genomic DNA was prepared after 1, 4, or 8 restreaks after the indicated strains were generated. <i>Apa</i>I-telomere fragments were detected using a telomere-specific probe as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005943#pgen.1005943.g002" target="_blank">Fig 2B</a>. <b>(C)</b> Telomere shortening still occurs in the absence of shelterin components when <i>swi1</i> is deleted. Cells with the indicated genotypes were passaged at least 8 times and subjected to Southern blot analysis as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005943#pgen.1005943.g002" target="_blank">Fig 2B</a>.</p

Swi1 is required to prevent hyperrecombination at telomeres and ALT-like telomere phenotypes in the absence of telomerase.

<p><b>(A)</b><i>swi1</i> deletion significantly increases the chances of telomerase negative cells to undergo ALT-like telomere maintenance. Representative image of telomere Southern blot analysis of wild-type, <i>swi1</i>Δ, <i>est1</i>Δ and <i>est1</i>Δ <i>swi1</i>Δ cells. Multiple segregants with the same genotypes were analyzed after at least 11 passages. <i>Apa</i>I-digested DNA was hybridized with the telomere-specific probe as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005943#pgen.1005943.g002" target="_blank">Fig 2B</a>. Representative results are shown. <b>(B)</b> Quantification of the independent segregants analyzed for each genotype and classification depending on their telomere phenotype.</p

Homologous recombination is not involved in maintaining telomeres in <i>swi1</i>Δ cells.

<p>HR proteins are dispensable for maintaining telomeres in <i>swi1Δ</i> cells. Telomere southern analysis of the indicated strains was performed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005943#pgen.1005943.g002" target="_blank">Fig 2B</a>.</p

Telomere shortening in <i>swi1Δ</i> mutants is not caused by defects in telomerase recruitment at telomeres.

<p><b>(A)</b> Strains of the indicated genotypes were engineered to express Trt1-G9-5FLAG [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005943#pgen.1005943.ref050" target="_blank">50</a>]. ChIP results show that telomerase is preferentially recruited at short telomeres in <i>swi1Δ</i> mutants compared to wild-type length telomeres. Telomerase recruitment was monitored by competitive multiplex PCR at two different subtelomeric regions: Tel3s and TAS1. GFR1 (for Tel3s) and GFR2 (for TAS1) were amplified as internal control regions in multiplex PCR reactions. Trt1 enrichment at Tel3s and TAS1 over control regions was determined. Data is shown as relative fold enrichment in comparison to WT. Error bars represent the standard deviation obtained from three independent experiments. <b>(B)</b> <i>trt1Δ taz1Δ</i> and <i>trt1Δ taz1Δ swi1Δ</i> cells were passaged at least 8 times, and Southern blot analysis of <i>Apa</i>I-digested genomic DNA was performed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005943#pgen.1005943.g002" target="_blank">Fig 2B</a>. <b>(C)</b> Genetic interaction between Swi1 and Rad3-Rad26 in telomere maintenance. Genomic DNA was isolated form the indicated cells, and telomere southern analysis was performed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005943#pgen.1005943.g002" target="_blank">Fig 2B</a>.</p

Plasmid instability associated with telomeric repeats.

<p>(A) Schematic diagram of pAS-SK-telo plasmids. (B) Wild-type and <i>swi1Δ</i> cells were transformed with either the empty pAL-SK plasmid or the pAL-SK carrying a 300 bp. telomere tract in normal (+) or reverse (-) orientation relative to the replication origin of the plasmid. Recovered plasmid from 10 yeast colonies was amplified in bacteria, and 10 bacterial colonies per treatment were analyzed by restriction digestion with <i>PvuII</i> in 3% agarose gels stained with EtBr. Each column represents and independent bacterial colony. Size markers are shown and expected restriction fragment size is shown in bold.</p