Non-random spatial organization of telomeres varies during the cell cycle and requires LAP2 and BAF.

Spatial genome organization within the nucleus influences major biological processes and is impacted by the configuration of linear chromosomes. Here, we applied 3D spatial statistics and modeling on high-resolution telomere and centromere 3D-structured illumination microscopy images in cancer cells. We found a multi-scale organization of telomeres that dynamically evolved from a mixed clustered-and-regular distribution in early G1 to a purely regular distribution as cells progressed through the cell cycle. In parallel, our analysis revealed two pools of peripheral and internal telomeres, the proportions of which were inverted during the cell cycle. We then conducted a targeted screen using MadID to identify the molecular pathways driving or maintaining telomere anchoring to the nuclear envelope observed in early G1. Lamina-associated polypeptide (LAP) proteins were found transiently localized to telomeres in anaphase, a stage where LAP2α initiates the reformation of the nuclear envelope, and impacted telomere redistribution in the next interphase together with their partner barrier-to-autointegration factor (BAF).

Progerin impairs 3D genome organization and induces fragile telomeres by limiting the dNTP pools.

Chromatin organization within the nuclear volume is essential to regulate many aspects of its function and to safeguard its integrity. A key player in this spatial scattering of chromosomes is the nuclear envelope (NE). The NE tethers large chromatin domains through interaction with the nuclear lamina and other associated proteins. This organization is perturbed in cells from Hutchinson-Gilford progeria syndrome (HGPS), a genetic disorder characterized by premature aging features. Here, we show that HGPS-related lamina defects trigger an altered 3D telomere organization with increased contact sites between telomeres and the nuclear lamina, and an altered telomeric chromatin state. The genome-wide replication timing signature of these cells is perturbed, with a shift to earlier replication for regions that normally replicate late. As a consequence, we detected a higher density of replication forks traveling simultaneously on DNA fibers, which relies on limiting cellular dNTP pools to support processive DNA synthesis. Remarkably, increasing dNTP levels in HGPS cells rescued fragile telomeres, and improved the replicative capacity of the cells. Our work highlights a functional connection between NE dysfunction and telomere homeostasis in the context of premature aging.

Methyl Adenine Identification (MadID): High-Resolution Detection of Protein-DNA Interactions.

Mapping the binding sites of DNA- or chromatin-interacting proteins is essential to understand many essential biological processes. Methyl Adenine Identification (MadID) is a proximity methylation-based assay that allows the visualization, quantification, and identification of binding sites from DNA-interacting proteins in eukaryotic cells. Chromatin-binding proteins of interest are fused to the newly described bacterial methyltransferase M.EcoGII. This enzyme catalyzes the methylation of adenine residues with no sequence specificity. Consequently, adenines within and in the vicinity of the protein binding sites will be decorated with a methyl group (m6A), a modification that can be further detected using different methods. M.EcoGII-dependent DNA methylation can be monitored in situ using immunostaining, at the genome-wide level using a combination of m6A-specific immunoprecipitation and whole-genome sequencing, or locally at DNA regions of interest purified by chromatin immunoprecipitation or probe-based capture techniques. MadID is conceptually similar to DNA adenine methyltransferase identification (DamID) that relies on the methylation of GATC motifs. However, MadID provides a higher resolution, deeper coverage, and opens ways for identification of binding sites in genomic regions that were largely inaccessible such as telomeres, centromeres, and repeated elements.

Replication stress induces mitotic death through parallel pathways regulated by WAPL and telomere deprotection.

Mitotic catastrophe is a broad descriptor encompassing unclear mechanisms of cell death. Here we investigate replication stress-driven mitotic catastrophe in human cells and identify that replication stress principally induces mitotic death signalled through two independent pathways. In p53-compromised cells we find that lethal replication stress confers WAPL-dependent centromere cohesion defects that maintain spindle assembly checkpoint-dependent mitotic arrest in the same cell cycle. Mitotic arrest then drives cohesion fatigue and triggers mitotic death through a primary pathway of BAX/BAK-dependent apoptosis. Simultaneously, a secondary mitotic death pathway is engaged through non-canonical telomere deprotection, regulated by TRF2, Aurora B and ATM. Additionally, we find that suppressing mitotic death in replication stressed cells results in distinct cellular outcomes depending upon how cell death is averted. These data demonstrate how replication stress-induced mitotic catastrophe signals cell death with implications for cancer treatment and cancer genome evolution.

MadID, a Versatile Approach to Map Protein-DNA Interactions, Highlights Telomere-Nuclear Envelope Contact Sites in Human Cells.

Mapping the binding sites of DNA- or chromatin-interacting proteins is essential to understanding biological processes. DNA adenine methyltransferase identification (DamID) has emerged as a comprehensive method to map genome-wide occupancy of proteins of interest. A caveat of DamID is the specificity of Dam methyltransferase for GATC motifs that are not homogenously distributed in the genome. Here, we developed an optimized method named MadID, using proximity labeling of DNA by the methyltransferase M.EcoGII. M.EcoGII mediates N6-adenosine methylation in any DNA sequence context, resulting in deeper and unbiased coverage of the genome. We demonstrate, using m6A-specific immunoprecipitation and deep sequencing, that MadID is a robust method to identify protein-DNA interactions at the whole-genome level. Using MadID, we revealed contact sites between human telomeres, repetitive sequences devoid of GATC sites, and the nuclear envelope. Overall, MadID opens the way to identification of binding sites in genomic regions that were largely inaccessible.

Signaling pathways of replication stress in yeast.

Eukaryotic cells activate the S-phase checkpoint in response to a variety of events affecting the progression of replication forks, collectively referred to as replication stress. This signaling pathway is divided in two branches: the DNA damage checkpoint (DDC) and the DNA replication checkpoint (DRC). Both pathways are activated by the sensor kinase Mec1 and converge on the effector kinase Rad53. However, the DDC operates throughout the cell cycle and depends on the checkpoint mediator Rad9 to activate Rad53, whereas the DRC is specific to S phase and is mediated by Mrc1 and other fork components to signal replication impediments. In this review, we summarize current knowledge on these two pathways, with a focus on the budding yeast Saccharomyces cerevisiae, in which many important aspects of the replication stress response were discovered. We also discuss the differences and similarities between DDC and DRC and speculate on how these pathways cooperate to ensure the complete and faithful duplication of the yeast genome under various replication stress conditions.

The telomere deprotection response is functionally distinct from the genomic DNA damage response.

Loss of chromosome end protection through telomere erosion is a hallmark of aging and senescence. Here we developed an experimental system that mimics physiological telomere deprotection in human cells and discovered that the telomere deprotection response is functionally distinct from the genomic DNA damage response. We found that, unlike genomic breaks, deprotected telomeres that are recognized as DNA damage but remain in the fusion-resistant intermediate state activate differential ataxia telangiectasia mutated (ATM) signaling where CHK2 is not phosphorylated. Also unlike genomic breaks, we found that deprotected telomeres do not contribute to the G2/M checkpoint and are instead passed through cell division to induce p53-dependent G1 arrest in the daughter cells. Telomere deprotection is therefore an epigenetic signal passed between cell generations to ensure that replication-associated telomere-dependent growth arrest occurs in stable diploid G1 phase cells before genome instability can occur.

Human telomeres are tethered to the nuclear envelope during postmitotic nuclear assembly.

Telomeres are essential for nuclear organization in yeast and during meiosis in mice. Exploring telomere dynamics in living human cells by advanced time-lapse confocal microscopy allowed us to evaluate the spatial distribution of telomeres within the nuclear volume. We discovered an unambiguous enrichment of telomeres at the nuclear periphery during postmitotic nuclear assembly, whereas telomeres were localized more internally during the rest of the cell cycle. Telomere enrichment at the nuclear rim was mediated by physical tethering of telomeres to the nuclear envelope, most likely via specific interactions between the shelterin subunit RAP1 and the nuclear envelope protein Sun1. Genetic interference revealed a critical role in cell-cycle progression for Sun1 but no effect on telomere positioning for RAP1. Our results shed light on the dynamic relocalization of human telomeres during the cell cycle and suggest redundant pathways for tethering telomeres to the nuclear envelope.

dNTP pools determine fork progression and origin usage under replication stress.

Intracellular deoxyribonucleoside triphosphate (dNTP) pools must be tightly regulated to preserve genome integrity. Indeed, alterations in dNTP pools are associated with increased mutagenesis, genomic instability and tumourigenesis. However, the mechanisms by which altered or imbalanced dNTP pools affect DNA synthesis remain poorly understood. Here, we show that changes in intracellular dNTP levels affect replication dynamics in budding yeast in different ways. Upregulation of the activity of ribonucleotide reductase (RNR) increases elongation, indicating that dNTP pools are limiting for normal DNA replication. In contrast, inhibition of RNR activity with hydroxyurea (HU) induces a sharp transition to a slow-replication mode within minutes after S-phase entry. Upregulation of RNR activity delays this transition and modulates both fork speed and origin usage under replication stress. Interestingly, we also observed that chromosomal instability (CIN) mutants have increased dNTP pools and show enhanced DNA synthesis in the presence of HU. Since upregulation of RNR promotes fork progression in the presence of DNA lesions, we propose that CIN mutants adapt to chronic replication stress by upregulating dNTP pools.

Analysis of replication profiles reveals key role of RFC-Ctf18 in yeast replication stress response.

Maintenance of genome integrity relies on surveillance mechanisms that detect and signal arrested replication forks. Although evidence from budding yeast indicates that the DNA replication checkpoint (DRC) is primarily activated by single-stranded DNA (ssDNA), studies in higher eukaryotes have implicated primer ends in this process. To identify factors that signal primed ssDNA in Saccharomyces cerevisiae, we have screened a collection of checkpoint mutants for their ability to activate the DRC, using the repression of late origins as readout for checkpoint activity. This quantitative analysis reveals that neither RFC(Rad24) and the 9-1-1 clamp nor the alternative clamp loader RFC(Elg1) is required to signal paused forks. In contrast, we found that RFC(Ctf18) is essential for the Mrc1-dependent activation of Rad53 and for the maintenance of paused forks. These data identify RFC(Ctf18) as a key DRC mediator, potentially bridging Mrc1 and primed ssDNA to signal paused forks.

Does interference between replication and transcription contribute to genomic instability in cancer cells?

We have recently reported that topoisomerase 1 (Top1) cooperates with ASF/SF2, a splicing factor of the SR family, to prevent unscheduled replication fork arrest and genomic instability in human cells. Our results suggest that Top1 execute this function by suppressing the formation of DNA-RNA hybrids during transcription, these so-called R-loops interfering with the progression of replication forks. Using ChIP-chip, we have shown that γ-H2AX, a marker of DNA damage, accumulates at gene-rich regions of the genome in Top1-deficient cells. This is best illustrated at histone genes, which are highly expressed during S phase and display discrete γ-H2AX peaks on ChIP-chip profiles. Here, we show that these γ-H2AX domains are different from those induced by camptothecin, a Top1 inhibitor inducing double-strand DNA breaks throughout the genome. These data support the view that R-loops promote genomic instability at specific sites by blocking fork progression and inducing chromosome breaks. Whether this type of transcription-dependent fork arrest contributes to the replication stress observed in precancerous lesions is an important question that deserves further attention.

Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription.

Topoisomerase I (Top1) is a key enzyme in functioning at the interface between DNA replication, transcription and mRNA maturation. Here, we show that Top1 suppresses genomic instability in mammalian cells by preventing a conflict between transcription and DNA replication. Using DNA combing and ChIP (chromatin immunoprecipitation)-on-chip, we found that Top1-deficient cells accumulate stalled replication forks and chromosome breaks in S phase, and that breaks occur preferentially at gene-rich regions of the genome. Notably, these phenotypes were suppressed by preventing the formation of RNA-DNA hybrids (R-loops) during transcription. Moreover, these defects could be mimicked by depletion of the splicing factor ASF/SF2 (alternative splicing factor/splicing factor 2), which interacts functionally with Top1. Taken together, these data indicate that Top1 prevents replication fork collapse by suppressing the formation of R-loops in an ASF/SF2-dependent manner. We propose that interference between replication and transcription represents a major source of spontaneous replication stress, which could drive genomic instability during the early stages of tumorigenesis.

Telomere dysfunction as a cause of genomic instability in Werner syndrome.

Werner syndrome (WS) is a rare human premature aging disease caused by mutations in the gene encoding the RecQ helicase WRN. In addition to the aging features, this disorder is marked by genomic instability, associated with an elevated incidence of cancer. Several lines of evidence suggest that telomere dysfunction is associated with the aging phenotype of the syndrome; however, the origin of the genomic instability observed in WS cells and the reason for the high incidence of cancer in WS have not been established. We previously proposed that WRN helicase activity was necessary to prevent dramatic telomere loss during DNA replication. Here we demonstrate that replication-associated telomere loss is responsible for the chromosome fusions found in WS fibroblasts. Moreover, using metaphase analysis we show that telomere elongation by telomerase can significantly reduce the appearance of new chromosomal aberrations in cells lacking WRN, similar to complementation of WS cells with WRN. Our results suggest that the genome instability in WS cells depends directly on telomere dysfunction, linking chromosome end maintenance to chromosomal aberrations in this disease.

Functional human telomeres are recognized as DNA damage in G2 of the cell cycle.

Telomeres have to be distinguished from DNA breaks that initiate a DNA damage response. Proteins involved in the DNA damage response have previously been found at telomeres in transformed cells; however, the importance of these factors for telomere function has not been understood. Here, we show that telomeres of telomerase-negative primary cells recruit Mre11, phosphorylated NBS1, and ATM in every G2 phase of the cell cycle. This recruitment correlates with a partial release of telomeric POT1; moreover, telomeres were found to be accessible to modifying enzymes at this time in the cell cycle, suggesting that they are unprotected. Degradation of the MRN complex, as well as inhibition of ATM, led to telomere dysfunction. Consequentially, we propose that a localized DNA damage response at telomeres after replication is essential for recruiting the processing machinery that promotes formation of a chromosome end protection complex.

In the end, it's all structure.

Chromosome end protection is essential for all organisms with linear genomes. Specialized structures, called telomeres, accomplish this protection by forming DNA-protein complexes that hide the natural chromosome ends from the DNA damage machinery. In mammalian cells protection takes place on several levels. Telomeric DNA forms large duplex loops with the help of telomeric proteins, consequently hiding the very tip of the telomere. Telomeric proteins play additional roles in protecting the end from degradation, regulating telomere length, and suppressing the DNA damage response machinery. Here we summarize the current knowledge about telomere structure, and discuss the future directions of the field.

Defective telomere lagging strand synthesis in cells lacking WRN helicase activity.

Cells from Werner syndrome patients are characterized by slow growth rates, premature senescence, accelerated telomere shortening rates, and genome instability. The syndrome is caused by the loss of the RecQ helicase WRN, but the underlying molecular mechanism is unclear. Here we report that cells lacking WRN exhibit deletion of telomeres from single sister chromatids. Only telomeres replicated by lagging strand synthesis were affected, and prevention of loss of individual telomeres was dependent on the helicase activity of WRN. Telomere loss could be counteracted by telomerase activity. We propose that WRN is necessary for efficient replication of G-rich telomeric DNA, preventing telomere dysfunction and consequent genomic instability.

Oxaliplatin-induced mitochondrial apoptotic response of colon carcinoma cells does not require nuclear DNA.

We previously established a model of acquired oxaliplatin resistance derived from the HCT116 oxaliplatin-sensitive cell line (HCT116S) and consisting in two resistant clones (HCT116R1, HCT116R2) and their total or partial revertants (HCT116Rev1 and HCT116Rev2, respectively). Using this cellular model, we explored the contribution of mitochondrial apoptosis and nuclear DNA to oxaliplatin-mediated apoptosis induction and oxaliplatin resistance. We showed that the activity of oxaliplatin is mediated by the induction of Bax/Bak-dependent mitochondrial apoptosis and that oxaliplatin resistance is mediated by a defect in Bax/Bak activation correlating with a reduced loss of the mitochondrial transmembrane potential (DeltaPsim). In addition, we observed that p53 only contributed marginally to oxaliplatin-induced cytotoxicity and was not involved in oxaliplatin resistance. Moreover and surprisingly, depletion of the nucleus in HCT116S cells did not abolish the oxaliplatin-induced DeltaPsim loss indicative of imminent apoptosis. Enucleation abolished the oxaliplatin resistance of HCT116R1 cells, while HCT116R2 cytoplasts conserved their resistant phenotype. Altogether, these data demonstrate that oxaliplatin exerts its cytotoxic effects by inducing mitochondrial apoptosis and that these effects can be initiated by interacting on other cellular structures than nuclear DNA. Resistance to oxaliplatin may imply both nuclear and cytoplasmic compartments.

Drug specific resistance to oxaliplatin is associated with apoptosis defect in a cellular model of colon carcinoma.

To investigate acquired resistance to oxaliplatin, we selected two resistant clones from the HCT116 cell line. We found that the resistant phenotype was associated with resistance to oxaliplatin-induced apoptosis as demonstrated by FACS analysis and by Western blotting of caspase 3 activation. In addition, the resistant phenotype showed a concomitant resistance to lonidamine and arsenic trioxide which are inducers of mitochondrial apoptosis. Furthermore, a complete loss of Bax expression due to a frameshift mutation was observed in the most resistant clone. Taken together, these findings suggest that altered mitochondrial-mediated apoptosis could play a role in oxaliplatin resistance.