Nucleoplasmic Lamin A/C controls replication fork restart upon stress by modulating local H3K9me3 and ADP-ribosylation levels

Nucleoplasmic Lamin A/C controls replication fork restart upon stress by modulating local H3K9me3 and ADP-ribosylation levels | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Nucleoplasmic Lamin A/C controls replication fork restart upon stress by modulating local H3K9me3 and ADP-ribosylation levels Veronica Cherdyntseva , Joanna Paulson , Selin Adakli , Jean-Philippe Gagné , Moses Aouami , Patricia Ubieto-Capella , Daniel González-Acosta , Collin Bakker , Guy G. Poirier , Nitika Taneja , View ORCID Profile Massimo Lopes doi: https://doi.org/10.1101/2025.01.18.633737 Veronica Cherdyntseva 1 Institute of Molecular Cancer Research, University of Zurich , Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Joanna Paulson 2 Department of Molecular Genetics, Oncode Institute, Erasmus University Medical Center, Erasmus MC Cancer Institute , Rotterdam, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Selin Adakli 1 Institute of Molecular Cancer Research, University of Zurich , Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jean-Philippe Gagné 3 Department of Molecular Biology, Medical Biochemistry and Pathology , Université Laval, Quebec City, Canada 4 CHU de Quebec Research Center, CHUL Pavilion , Oncology Axis, Quebec City, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Moses Aouami 1 Institute of Molecular Cancer Research, University of Zurich , Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Patricia Ubieto-Capella 1 Institute of Molecular Cancer Research, University of Zurich , Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Daniel González-Acosta 1 Institute of Molecular Cancer Research, University of Zurich , Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Collin Bakker 2 Department of Molecular Genetics, Oncode Institute, Erasmus University Medical Center, Erasmus MC Cancer Institute , Rotterdam, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Guy G. Poirier 3 Department of Molecular Biology, Medical Biochemistry and Pathology , Université Laval, Quebec City, Canada 4 CHU de Quebec Research Center, CHUL Pavilion , Oncology Axis, Quebec City, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nitika Taneja 2 Department of Molecular Genetics, Oncode Institute, Erasmus University Medical Center, Erasmus MC Cancer Institute , Rotterdam, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Massimo Lopes 1 Institute of Molecular Cancer Research, University of Zurich , Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Massimo Lopes For correspondence: lopes{at}imcr.uzh.ch Abstract Full Text Info/History Metrics Preview PDF Abstract Mild replication interference is a consolidated strategy for cancer chemotherapy. Tolerance to mild replication stress (RS) relies on active fork slowing, mediated by transient fork reversal and RECQ1-assisted restart, and modulated by PARP1 and nuclear architectural components via yet-elusive mechanisms. We combined acute protein inactivation with cell biology and single-molecule approaches to investigate the role of Lamin A/C upon mild RS. We found that Lamin A/C dynamically interacts with replication factories throughout the nucleus and, together with its nucleoplasmic partner LAP2α, is required to induce active fork slowing and maintain chromosome stability upon mild genotoxic treatments. Inactivating nucleoplasmic Lamin A/C reduces poly-ADP-ribosylation (PAR) levels at nascent DNA, triggering deregulated RECQ1-mediated restart of reversed forks. Moreover, we found that the heterochromatin mark H3K9me3, previously reported at stalled forks, also accumulates in response to mild RS. H3K9me3 accumulation requires Lamin A/C, which prevents its premature removal by the histone demethylase JMJD1A/KDM3A. H3K9me3 loss per se phenocopies Lamin A/C inactivation, reducing PAR levels and deregulating RECQ1 activity at forks. Hence, nucleoplasmic Lamin A/C, H3K9me3 and PARylation levels are crucial, mechanistically-linked modulators of fork slowing, remodelling and restart upon mild RS, with important implications for chemotherapy response and Lamin A/C deregulation in human disease. Introduction DNA replication is an essential process for the transmission of genetic information during somatic cell division. Completeness and accuracy of this process are essential to maintain proper genome duplication across generations. DNA replication is challenged by multiple endogenous and exogenous sources of genotoxic stress – collectively reported as replication stress (RS) – including various types of DNA lesions, limited nucleotide levels and interference with transcription 1 . The mechanisms by which these obstacles are tackled to ensure genome stability are highly relevant to avoid cancer onset and contribute to known mechanisms of resistance of cancer cells to commonly used chemotherapeutics 1 , 2 . Cells respond to RS by activating signaling pathways and replication fork protection mechanisms, which are best characterized upon conditions that block DNA synthesis and induce prolonged fork stalling. In fact, to limit toxicity in normal cells and arrest the uncontrolled proliferation of cancer cells, commonly used chemotherapeutic regimens are compatible with residual DNA synthesis. They rather trigger specialized mechanisms of RS tolerance that capitalize on replication fork plasticity, allowing to continue DNA synthesis in unfavorable conditions, albeit at a slower pace 3 . Even upon mild RS-inducing treatments, a high fraction of replication forks in human cells undergo transient remodeling into 4-way junctions, in a process known as replication fork reversal 4 . Reversed fork formation is modulated by a growing number of replication accessory factors, some of which have been previously implicated in classical DNA repair 5 . Also restart of reversed forks requires specialized enzymes, such as the RECQ1 helicase, which is controlled by its interaction with poly-ADP-ribose (PAR), synthesized by PARP1 on itself and additional targets 3 , 6 , 7 . An accurate balance of fork reversal and restart is crucial to mediate active fork slowing upon mild RS and to resist drug-induced conditions of RS, impacting on cancer therapy response in multiple tissues 2 . The DNA replication template is packed in nucleosomes, containing specific epigenetic marks and is organized in a higher order chromatin structure, which determines the gene expression profile of each cell. Hence, disassembly and restoration of chromatin marks, as well as chromatin organization during DNA replication are of crucial importance to maintain cell identity 8 , 9 . RS adds complexity to this already challenging task, by affecting simultaneous and complete DNA synthesis on the two template strands and requiring spatiotemporally controlled access of specific accessory factors to replicating chromatin 10 . Deposition of specific histone variants and histone marks on replicated DNA are known to assist replication-coupled repair and maintain fork integrity 11 , 12 . Transient accumulation of heterochromatic marks – typically associated with silent chromatin – on newly replicated DNA was recently shown to mediate the cellular response to replication fork stalling, by limiting the access of DNA synthesis restart factors, such as Primpol 13 . Whether similar mechanisms assist the immediate response to mild and clinically relevant genotoxic treatments – affecting DNA synthesis without stalling replication forks – has remained elusive. Besides the established role of chromatin organization in the RS response, several components of nuclear architecture and nuclear dynamics are also emerging as key players upon replication interference. Nuclear actin filaments were shown to assist repair of DNA breaks 14 , 15 and to relocate forks experiencing prolonged stalling 16 . More recently, the nuclear actin network was shown to protect the stability of stalled forks 17 , 18 and to modulate fork progression upon mild RS, by limiting recruitment of Primpol to transiently challenged forks 19 . Considering that replication fork remodeling appears to extend in the nucleus beyond the forks directly experiencing obstacles 20 , these recent findings suggest that proper coordination of the RS response requires local and global control of nuclear architecture and 3D genome organization 21 . Regulated loading and unloading of cohesin – another key player in genome organization and nuclear dynamics – controls replication timing in the nucleus 21 , 22 , but was also shown to support fork stability, progression and restart in different conditions of RS 23 . Based on this growing evidence, it seems likely that several additional components of nuclear architecture play pivotal roles upon RS, possibly impacting genome stability and cellular resistance to cancer chemotherapy. Lamin A/C is another key component of nuclear architecture, best known for its role within the dense fibrillar network of intermediate filaments supporting structurally the nuclear membrane (lamina) 24 . Mutations impairing this structural function have profound consequences at cellular and organismic level, impacting the mechanical properties of the cells in specific tissues and leading to a heterogeneous set of diseases, collectively called laminopathies 25 . Although most cellular Lamin A/C is assembled in the nuclear lamina, a significant fraction of the protein resides in the nucleoplasm in a more soluble and less detectable form, bound to its specific nucleoplasmic partner LAP2α 26 , 27 . Nucleoplasmic Lamin A/C and LAP2α appear to modulate chromatin mobility in the nuclear interior 27 , 28 . Moreover, Lamin A/C was shown to interact directly with the histone lysine methyltransferase SUV39H1 29 ; however, whether and how Lamin A/C-dependent modulation of chromatin organization affects gene expression and other nuclear functions is still elusive. Lamin A/C was previously investigated in the context of replication fork stalling and shown to promote fork restart, possibly by mediating efficient recruitment of ssDNA-binding proteins RPA and RAD51 30 , 31 . A role in RPA binding and recruitment to damaged chromatin was also recently proposed for LAP2α and reported to depend on PARP1 32 . Intriguingly, both Lamin A/C and LAP2α were recently found by proximity proteomics as interactors of PARP1 33 , which – besides the established role at DNA breaks – is also recruited and activated at persistent discontinuities on nascent DNA 34 , 35 . A general limitation in previous investigations of Lamin A/C roles in DNA replication is the use of prolonged or permanent inactivation of the protein; considering the crucial structural functions of Lamin A/C, cells may need to adapt to its absence, promoting alternative mechanisms of nuclear organization and masking potentially interesting phenotypes. Overall, it is still unclear whether the role of Lamin A/C in DNA replication is limited to prolonged fork stalling or extends to mild RS conditions, whether this function entails the lamina or its nucleoplasmic pool, and whether it relates to the emerging links of Lamin A/C with chromatin organization. Here we show that Lamin A/C interacts with replication factories throughout the nucleus and that acute inactivation of Lamin A/C abolishes active fork slowing and increases genomic instability upon mild RS. These defects are phenocopied by genetic ablation of LAP2α and are linked to an impaired accumulation of PAR at replication forks, triggering the deregulated restart of reversed forks by the RECQ1 helicase. Moreover, we report that the accumulation of heterochromatic marks (H3K9me3) at replication forks is also detected upon mild genotoxic treatments and their maintenance strictly requires Lamin A/C. Strikingly, impairing heterochromatic mark maintenance upon mild genotoxic treatments recapitulates all molecular defects observed upon Lamin A/C inactivation, suggesting that the control of chromatin compaction by nucleoplasmic Lamin A/C plays a key role in modulating PAR levels and RECQ1-mediated restart upon mild genotoxic stress. Results Lamin A/C interacts dynamically with replication factories throughout the nucleus To investigate whether and where Lamin A/C establishes contacts with replication factories within the nucleus, we performed proximity ligation assays (PLA) between Lamin A/C and EdU, a thymidine analogue that was briefly incorporated during DNA synthesis before cell preparation. We selected HCT116 colon cancer cells for these imaging analyses, especially due to the consistent round shape of their nuclei. Expectedly, Lamin A/C was mostly detectable by immunofluorescence (IF) at the nuclear periphery, but confocal imaging and 3D nuclei reconstruction showed numerous Lamin A/C-EdU PLA foci throughout the nucleus, suggesting that – besides the nuclear lamina - also low-abundant Lamin A/C within the nucleoplasm is in close contact with replication centers ( Fig. 1a-b , Extended Data Fig. 1a; Extended Data Video 1). As expected, LMNA downregulation decreased the number of Lamin A/C-EdU PLA foci, supporting the specificity of the antibody to target Lamin A/C (Extended Data Fig. 1b). To assess how Lamin A/C interaction with replication factories is affected upon mild conditions of RS, we exposed U2OS human osteosarcoma cells to mild treatments with the topoisomerase I inhibitor camptothecin (CPT) or the topoisomerase II inhibitor etoposide (ETP), which were previously shown in these cells to significantly impact DNA replication without inducing detectable double-strand breaks 6 . Similarly to HCT116 cells, widespread PLA signals were detectable by widefield IF imaging also in U2OS cells, but were significantly decreased upon both mild genotoxic treatments ( Fig. 1c-e ), reflecting Lamin A/C release from replication centers, or – as recently shown for other replisome components 36 – a switch to a more distant or dynamic interaction with nascent DNA during the RS response. LAP2α downregulation in U2OS cells decreased Lamin A/C-EdU PLA foci (Extended Data Fig. 1c-e), in line with its established role modulating other nucleoplasmic functions of Lamin A/C 27 , 28 . Overall, these data show a widespread interaction of Lamin A/C with DNA replication centers throughout the nucleus, which is promoted by its nucleoplasmic interactor LAP2α and modulated upon mild RS. Download figure Open in new tab Figure 1. Lamin A/C dynamically interacts with replication factories throughout the nucleus. a. Representative confocal microscopy image of HCT116 cells showing Lamin A/C in proximity to nascent DNA (EdU), detected by Lamin A/C:EdU PLA (magenta), Lamin A/C IF staining (yellow) . While Lamin A/C is mainly detected at the nuclear periphery, its interaction with nascent DNA (EdU) is detected throughout the nucleus and in different axial perspectives (top left: XY view, bottom left: YZ view, top right: XZ view). Scale bar, 5 μm. b. Schematic representation of the Proximity Ligation assay used. EdU incorporation is followed by ClickIT chemistry with biotin-azide. Antibodies against the target protein and biotin are recognized by secondary antibodies carrying probes. When the target protein and EdU are in close proximity (< 40 nm), probes are ligated and amplified giving rise to a fluorescent PLA signal c. Experimental design for the IF/PLA experiment in c. The duration of the EdU pulse is adapted to allow comparable incorporation of EdU despite the genotoxic treatments. d. Representative U2OS nuclei (DAPI) – untreated or treated for 1h with 100 nM CPT or 20 nM ETP – and stained for DNA synthesis (EdU), Lamin A/C and its physical proximity to nascent DNA (Lamin A/C:EdU PLA). Scale bar, 10 μm. e. Quantification of Lamin A/C PLA signals from c. Signal was quantified in at least 100 EdU+ nuclei, in 4 independent experiments. EdU-cells are used as negative control. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values. Statistical analysis was applied on the median values, using one-way ANOVA test with Bonferroni’s post hoc correction. Acute depletion of Lamin A/C or its nucleoplasmic partner LAP2 α abolishes active fork slowing upon stress The roles of Lamin A/C in DNA replication or RS response have been explored so far upon prolonged or permanent (chronic) inactivation of the protein and in response to a complete replication block induced by nucleotide depletion 30 , 31 . To investigate functional roles of Lamin A/C upon mild RS, we took advantage of the AID2 technology 37 and developed an auxin-inducible degron HCT116 cell line targeting the LMNA protein by fusing the mAID2-mClover construct on the endogenous loci ( mAID2-LMNA ; Extended Data Fig. 2a). Hence, this cell line expresses a fluorescently-detectable protein (due to mClover) that can be efficiently degraded upon the addition of 5-Ph-IAA (auxin) to the culture media. We isolated two clones (13 and 22) for further experiments in which we determined that full LMNA depletion is achieved 24h after auxin addition (Extended Data Fig. 2b). We confirmed that 24h after auxin addition mAID2-LMNA HCT116 cells do not experience any marked alteration of their cell cycle progression (Extended Data Fig. 2c-d). Similarly, we found that marked LMNA downregulation can be achieved in U2OS cells 48h after transfection with a specific siRNA ( Fig. 2a-b ) and that at this time point the cells do not yet display delayed cell cycle progression, impaired S phase entry or activation of the DNA damage response (Extended Data Fig. 2e-f). We used these controlled conditions of acute Lamin A/C depletion ( Fig. 2a-b ) to investigate replication fork progression at single-molecule level by spread DNA fiber assays 38 , providing cells with halogenated nucleotide analogues and with mild doses of ETP or CPT. These treatments were previously shown to induce marked fork slowing and reversal, with no major impact on cell cycle progression and cell viability 6 . As expected, ETP and CPT markedly affected replication fork progression in both HCT116 and U2OS cells ( Fig. 2c-e and Fig. 2f-h ). However, the active fork slowing observed in these conditions is significantly rescued by auxin-inducible Lamin A/C degradation in both LMNA-mAID2-mClover HCT116 clones ( Fig. 2c-e ) or by LMNA downregulation in U2OS ( Fig. 2f-h ). Importantly, complete suppression of ETP/CPT-induced active fork slowing is also observed upon depletion of LAP2α ( Fig. 2f-h ), suggesting that this function specifically requires the nucleoplasmic fraction of Lamin A/C. To assess whether defective fork slowing upon Lamin A/C inactivation is linked to increased genomic instability, we used chromosome spreads from metaphase arrested cells and monitored chromosomal breaks and abnormalities ( Fig. 2i ). Using mild CPT treatments that induce per se mild chromosomal instability in U2OS cells, we observed a significant increase of chromosomal instability upon LMNA downregulation ( Fig. 2j-k ). Overall, these data suggest that Lamin A/C and its nucleoplasmic interaction partner LAP2α are required to induce active fork slowing upon mild RS and to limit the associated genomic instability. Download figure Open in new tab Figure 2. Acute inactivation of Lamin A/C or LAP2α abolishes fork slowing and affects chromosomal stability upon mild RS. a-b. Western Blot analysis of Lamin A/C levels upon siRNA-mediated or 5-Ph-IIA-mediated depletion in the indicated cell lines. H3 is used as loading control. c-e. DNA fiber analysis of HCT116 and HCT116 mAID2-mClover-LMNA cells upon siRNA-mediated or 5-Ph-IIA-mediated depletion of Lamin A/C. c. Schematic CldU/IdU pulse-labeling protocol used to evaluate fork progression upon 20 nM ETP. 5-Ph-IIA was added 24h before the assay. d. Representative DNA fiber images for the experiment in c. e. IdU/CIdU ratio is plotted for a minimum of 100 forks from each of 3-4 independent experiments. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values. Statistical analysis was applied on the median values, using one-way ANOVA test with Bonferroni’s post hoc correction. f-h. DNA fiber analysis of U2OS cells upon siRNA-mediated depletion of Lamin A/C or LAP2α. f. Schematic CldU/IdU pulse-labeling protocol used to evaluate fork progression upon 20 nM ETP or 100 nM CPT. siRNA was transfected 48h before the assay. g. Representative DNA fiber images for the experiment in f. h. IdU/CIdU ratio is plotted for a minimum of 100 forks from each of three independent experiments. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values. Statistical analysis was applied on the median values, using one-way ANOVA test with Bonferroni’s post hoc correction. i. Representative metaphase spread images. Insets show magnified chromosomes, numbered in the overview images. Arrowheads point to chromosomal breaks. Scale bar: 10 μm, in inset 5 μm. j. Schematic design of the metaphase spread experiment in i-k. k. Average number of chromosomal breaks in mock- or Lamin A/C-depleted (siRNA) U2OS cells, optionally treated with 100 nM CPT for 3 hours followed by nocodazole treatment. Bar graph depicts mean +/- SD from three independent experiments (yellow dots). A minimum of 55 metaphases was analyzed per sample and experiment. Statistical analysis: one-way ANOVA test with Bonferroni’s post hoc correction. Unrestrained fork progression in Lamin A/C-LAP2 α depleted cells reflects deregulated fork restart by RECQ1 Accelerated fork progression and/or defective fork slowing upon genotoxic treatments have been frequently reported to depend on uncontrolled Primpol activity, which rapidly re-primes DNA synthesis on extended ssDNA stretches and thereby prevents efficient replication fork reversal 19 , 39 – 41 . Hence, we tested by DNA fiber assays whether defective fork slowing upon Lamin A/C- or LAP2α inactivation may also reflect a similar deregulation. Surprisingly, effective PRIMPOL downregulation by siRNA in ETP-treated U2OS cells did not prevent the unrestrained fork progression induced by Lamin A/C- or LAP2α inactivation (Extended Data Fig. 3a-f), suggesting that defective fork slowing in this context does not reflect an altered equilibrium between fork reversal and repriming. An alternative mechanism reported to drive unrestrained fork progression upon stress depends on deregulated restart of reversed forks by the RECQ1 helicase 6 , 7 . We tested RECQ1 contribution by co-downregulating it with LMNA or LAP2A in U2OS cells and found by DNA fiber assays that RECQ1 depletion could fully rescue the unrestrained fork progression induced by LMNA or LAP2A downregulation, restoring active fork slowing in response to ETP treatment ( Fig. 3a-f ). These data suggest that reversed forks are efficiently formed upon genotoxic treatments but are untimely restarted by the deregulated action of RECQ1, when Lamin A/C or LAP2α are not functional. To test this hypothesis, we exploited an established approach to directly visualize replication intermediates by electron microscopy 42 , 43 , which allows distinguishing standard 3-way replication forks ( Fig. 3g ) from 4-way reversed forks ( Fig. 3h ). In line with previous results 6 , fork reversal is detected at high levels upon CPT treatments (ca. 30% of the forks). Strikingly, reversed fork frequency drops to 10-20% when Lamin A/C or LAP2α are depleted, but is restored to control levels upon co-inactivation of RECQ1 ( Fig. 3i-j ; Extended Data Fig. 3g). Collectively, these data strongly suggest that Lamin A/C and LAP2α are required in response to mild RS to actively slow down replication forks and maintain high levels of reversed forks, by negatively regulating RECQ1 fork restart activity. Download figure Open in new tab Figure 3. Lamin A/C and LAP2α mediate fork slowing and reversal by limiting RECQ1-mediated fork restart. a-c. DNA fiber analysis of U2OS cells upon siRNA-mediated downregulation of LMNA , in combination with RECQ1. a. Western Blot analysis of Lamin A/C and RECQ1 levels levels upon siRNA-mediated depletion for the experiment in a-b. Vinculin is used as loading control. * identifies an unspecific band occasionally recognized by the RECQ1 antibody. b. Schematic CldU/IdU pulse-labeling protocol used to evaluate fork progression upon 20 nM ETP. siRNAs were added 48h before the assay. c. IdU/CIdU ratio is plotted for a minimum of 100 forks from each of 3 independent experiments. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values. Statistical analysis was applied on the median values, using one-way ANOVA test with Bonferroni’s post hoc correction. d-f. DNA fiber analysis of U2OS cells upon siRNA-mediated downregulation of LAP2A , in combination with RECQ1 . d. Western Blot analysis of LAP2α and RECQ1 levels upon siRNA-mediated depletion for the experiment in d-e. H3 and actin are used as loading controls. e. Schematic CldU/IdU pulse-labeling protocol used to evaluate fork progression upon 20 nM ETP. siRNAs were added 48h before the assay. f. IdU/CIdU ratio is plotted for a minimum of 100 forks from each of 3 independent experiments. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values. Statistical analysis was applied on the median values, using one-way ANOVA test with Bonferroni’s post hoc correction. g-h Electron micrographs of a representative normal replication fork (g) and reversed fork (h) from CPT-treated U2OS cells: parental (P) and daughter (D) duplexes, R, regressed arm. The insets show a magnification of the junction. Scale bars: in g 100nm (inset 50 nm), in h 100nm (inset 25 nm). i. Western Blot analysis of Lamin A/C, LAP2α and RECQ1 levels upon siRNA-mediated depletion for the experiment in j. Actin is used as loading control. j. Average frequency of reversed replication forks isolated from U2OS cells upon siRNA-mediated depletion of the indicated factors, and optional treatment with 100 nM CPT for 1 hour. Yellow dots represent the observed percentage of reversed forks in each independent experiment (n=2; see Extended Data Fig. 3g). Total number of molecules analyzed per condition in brackets. Lamin A/C-LAP2 α limit fork progression upon stress promoting PARylation events at replication forks RECQ1 was previously shown to be negatively regulated by its transient interaction with auto-modified (i.e., poly-ADP-ribosylated or PARylated) PARP1, thereby preventing the restart of reversed forks until the resolution of local replication stress 6 , 7 . Both Lamin A/C and LAP2α were recently identified by mass spectrometry as PARP1 proximal interactors and as proteins enriched at forks upon prolonged stalling 33 . We thus considered the hypothesis that Lamin A/C may globally promote PARylation levels, thereby indirectly controlling RECQ1 activity upon replication stress. We confirmed that Lamin A/C and PARP1 physically interact via immunoprecipitation, although this interaction was not detectably altered in response to various treatments interfering with fork progression (Extended Data Fig. 4a). We then used an established procedure to preserve and detect the levels of protein PARylation in U2OS cell extracts, which is expectedly affected by cellular treatments with PARP- and PARG-inhibitors (Extended Data Fig. 4b). Neither mild ETP treatment nor LMNA downregulation in U2OS cells - in the same experimental conditions that showed profound effects on replication fork progression ( Figs. 2 , 3) - significantly altered global protein PAR/MARylation levels in this assay, as detected by two different antibodies (96-10, E6F6A; Extended Data Fig. 4b), suggesting that the control of replication fork progression does not entail major changes in global PARylation events. Stabilization and detection of protein PAR/MARylation is sensitive to specific procedural steps and detection reagents 44 . Hence, we also used a different established procedure to isolate ADP-ribosylated proteins and detect them via an improved ADP-ribose binder 45 . In these experimental conditions, we reproducibly observed a significant reduction in protein PARylation upon LMNA downregulation, while pre-treatment with PARGi restored physiological PAR levels in Lamin A/C depleted cells (Extended Data Fig. 4c-d). These data suggested to us that a specific subset of PARylation events – which may be under/over-represented depending on the experimental procedure – could be modulated by Lamin A/C and possibly responsible for the control of fork progression in response to mild RS. To test this hypothesis, we specifically detected PAR in proximity to replication factories as previously described 34 , i.e. via PLA assays detecting the proximity of PAR (E6F6A antibody) to nascent DNA (EdU). In this assay, mild CPT treatment did not significantly increase PAR levels in proximity to EdU, but LMNA downregulation markedly decreased the PLA signal in both treated and untreated cells, showing a similar effect to the treatment with the PARP inhibitor Olaparib ( Fig. 4a-c ). Treatment with PARGi, as expected, increased the detectable level of PAR in proximity to replication forks, but also fully suppressed its reduction induced by LMNA downregulation ( Fig. 4d-f ). Hence, to assess whether the Lamin A/C-dependent control of PAR levels at replication forks is functionally relevant to modulating replication fork progression upon stress, we used PARGi treatment for DNA fiber assays upon mild ETP treatments. Strikingly, although PARGi treatment per se did not alter fork progression upon stress, it was sufficient to fully restore active fork slowing in ETP-treated Lamin A/C-depleted cells ( Fig. 4g-i ). Moreover, PARGi treatment also fully suppressed the unrestrained fork progression induced by LAP2A downregulation upon ETP treatment (Extended Data Fig. 4e). Collectively, these results strongly suggest that Lamin A/C and LAP2α control a specific subset of PARylation events at replication forks, which is functionally relevant to control RECQ1 activity and mediate active fork slowing upon stress. Download figure Open in new tab Figure 4. Lamin A/C sustains PAR levels at replication forks, thereby allowing fork slowing upon mild RS. a. Experimental design for the IF/PLA experiment in b-c. In a and d, the duration of the EdU pulse is adapted to allow comparable incorporation of EdU despite the genotoxic treatments. The PARP inhibitor olaparib (ola; 10 μM) is used as positive control of reduced PAR accumulation on nascent DNA. b. Representative U2OS nuclei (DAPI) upon optional LMNA downregulation, treated for 1h with 100 nM CPT and stained for DNA synthesis (EdU), PAR and its physical proximity to nascent DNA (PAR:EdU PLA). Scale bar: 10 μm. c. Quantification of PAR:EdU PLA signals from a-b. Signal was quantified in at least 100 EdU+ nuclei, in each of the 3 independent experiments. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values. Statistical analysis was applied on the median values, using one-way ANOVA test with Bonferroni’s post hoc correction. EdU-cells are used as negative control. d. Experimental design for the IF/PLA experiment in e-f. e. Representative U2OS nuclei (DAPI) after LMNA downregulation, treated for 1h with 100 nM CPT and optionally with the PARG inhibitor (PDD0017272, 1 μM), stained as in b. f. Quantification of PAR:EdU PLA signals from d-e. Signal was qunatified in at least 100 EdU+ nuclei, in each of the 3 independent experiments. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values. Statistical analysis was applied on the individual experiments, using Kruskal-Wallis test with Dunn’s post hoc correction. EdU-cells are used as negative control. Scale bar: 10 μm. g-i. DNA fiber analysis of U2OS cells upon siRNA-mediated downregulation of LMNA , and optional treatment with the PARG inhibitor (PDD0017272, 1 μM). g. Schematic CldU/IdU pulse-labeling protocol used to evaluate fork progression upon 20 nM ETP. siRNA was added 48h before the assay, while PARGi was added 2h before. h. Western Blot analysis of Lamin A/C levels upon siRNA-mediated depletion. Actin is used as loading control. PARP1 levels are not affected by PARGi treatment. i. IdU/CIdU ratio is plotted for a minimum of 100 forks from each of 3 independent experiments. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values. Statistical analysis was applied on the median values, using one-way ANOVA test with Bonferroni’s post hoc correction. Lamin A/C promotes heterochromatin maintenance at forks, thereby modulating RECQ1 via ADP-ribosylation Along with histone modifications, Lamin A/C and LAP2α were shown to impact chromatin dynamics and diffusion 28 , 46 , which are emerging as key modulators of DNA repair and DNA replication mechanisms 47 . Transient compaction of replicating chromatin via deposition of heterochromatic marks (e.g., H3K9me3) was recently shown to assist the cellular response to prolonged fork stalling 13 . Moreover, PARP1 efficiently binds heterochromatin at specialized genomic domains and gets activated within condensed chromatin to promote DNA repair 48 , 49 . Based on these findings, we set out to investigate i) whether accumulation of heterochromatin marks on nascent DNA may also occur in RS conditions that are permissive for residual fork progression; ii) whether chromatin modifications could be implicated in this novel role of nucleoplasmic Lamin A/C regulating fork restart upon mild RS. To address the first point experimentally, we tested in HCT116 cells whether G9a/EHMT2 – i.e., the lysine methyl transferase mediating the first steps of H3K9 methylation 50 – is recruited to replicating DNA upon mild RS. EdU-PLA experiments showed that low nM treatments with CPT or ETP do induce significant recruitment of G9a to nascent DNA, comparable to the one reported upon HU-induced fork stalling ( Fig. 5a-b ) 13 . Accordingly, PLA experiments upon the same treatments showed a significant accumulation on nascent DNA of the heterochromatic marker H3K9me3 ( Fig. 5c-d ), which is mediated by the sequential action of G9a and SUV39H1 methyltransferases 13 . These data show that accumulation of heterochromatin marks on newly replicated DNA does not require fork stalling and is also observed upon mild conditions of RS, i.e. those we used here to uncover the role of Lamin A/C in modulating fork remodeling and restart. To test the possible involvement of chromatin compaction in these mechanisms, we performed DNA fiber spreading experiments with UNC0642, a specific and potent catalytic inhibitor of G9a/GLP 51 (G9ai), that was previously used to establish the functional role of chromatin compaction upon fork stalling 13 . Remarkably, we found that – analogously to Lamin A/C and LAP2α inactivation ( Figs. 2 - 4 ) – G9a inhibition leads to unrestrained fork progression upon mild CPT and ETP treatments, and that this defect is suppressed by PARG inhibition ( Fig. 5e-f ). Moreover, similarly to Lamin A/C inactivation, PAR levels at replication factories are reduced by G9a inhibition in CPT-treated U2OS cells and restored by simultaneous inhibition of PARG ( Fig. 5g ). Finally, the unrestrained fork progression observed upon G9a inhibition depends on RECQ1 but not Primpol ( Fig. 5h-i ), consolidating a striking phenocopy of G9a and Lamin A/C-LAP2α inactivation for the control of local ADP ribosylation and replication fork restart (see also Figs. 2 - 4 ). Download figure Open in new tab Figure 5. G9a-dependent H3K9me3 marks accumulate on nascent DNA upon mild RS and limit RECQ1 fork restart activity via ADP ribosylation a. Representative PLA images illustrating G9a enrichment on nascent DNA upon mild RS (G9a-EdU PLA, red). RPE-1 cells were labelled with EdU for 20 min either before optional treatment with 1mM HU (1h) or at the end of 20 nM CPT (1h) and 20nM ETP (1h) treatment. Scale bar: 10 μm. b. Quantification of the total intensity of all G9a-EdU PLA spots per nucleus in a. In b and d, n > 800 S phase cells were analyzed in each condition; Kruskal-Wallis test followed by Dunn’s test were performed to test statistical significance for each of two independent PLA experiments. c. Representative PLA images illustrating H3K9me3 deposition on nascent DNA upon mild RS (H3K9me3-EdU PLA, red). U2OS cells were labelled with EdU for 20 min at the end of optional treatment with 25 nM CPT (1h) and 20nM ETP (1h). Scale bar: 10 μm. d. Distribution of H3K9me3-EdU total PLA spot intensity per nucleus. Statistical analysis as in b. e. Schematic CldU/IdU pulse-labeling protocol used in f to evaluate fork progression upon treatment with 20 nM ETP, 100 nM CPT, G9ai (UNC0642, 1 μM) and/or PARGi (PDD0017272, 1 μM). G9ai and PARGi were added 2h before the assay. f. IdU/CIdU ratio is plotted for a minimum of 100 forks from each of 3 independent experiments. Similar results were observed in all independent experiments. Statistical analysis was applied on an individual experiment, using Kruskal-Wallis test with Dunn’s post hoc correction. g. Quantification of PAR:EdU PLA signals from U2OS cells, treated with 100 nM CPT, G9ai (UNC0642, 1 μM) and PARGi (PDD0017272, 1 μM). Experimental design as in Fig. 4a, d . Signal was qunatified in at least 100 EdU+ nuclei, in each of the 3 independent experiments. PARGi-treated and untreated samples were processed in parallel, but are displayed in different graphs due to different ranges of observed signal. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values. Statistical analysis was applied on an individual experiments, using Kruskal-Wallis test with Dunn’s post hoc correction. Similar results were observed in all independent experiments. h. Schematic CldU/IdU pulse-labeling protocol used in i to evaluate fork progression upon treatment with 20 nM ETP and G9ai (UNC0642, 1 μM), and/or RECQ1 downregulation by siRNA. siRNA was transfected 48h before the assay, while G9ai was added 2h before. i. IdU/CIdU ratio is plotted for a minimum of 100 forks from each of 3 independent experimentsStatistical analysis was applied on the individual experiments, using Kruskal-Wallis test with Dunn’s post hoc correction. Similar results were observed in three independent experiments. Based on these data and on the established role of Lamin A/C and LAP2α in modulating chromatin dynamics within the nucleoplasm 28 , 46 , we hypothesized that nucleoplasmic Lamin A/C may modulate fork restart upon mild RS by mediating chromatin compaction at replication forks. H3K9me3-PLA experiments confirmed that Lamin A/C depletion in our HCT116 mAID2-mClover-LMNA cell line markedly reduces the levels of H3K9me3 detected at forks upon CPT treatment ( Fig. 6a-b ). Moreover, using ChromStretch - a single molecule method to map proteins and epigenetic marks directly on individual replication bubbles 13 – we confirmed in U2OS cells that mild CPT treatment leads to a marked accumulation of H3K9me3 at replication forks, and that Lamin A/C inactivation drastically impairs the levels of H3K9me3 accumulation ( Fig. 6c-d ). Interestingly, we performed similar experiments upon downregulation of Jumonji domain-containing protein 1A (JMJD1A)/Lysine (K)-Specific Demethylase 3A (KDM3A) – i.e. the demethylase shown to remove H3K9me3 from stalled forks during restart 13 – and found that impairing H3K9me3 demethylation was sufficient to fully suppress the defect in H3K9me3 levels at forks induced by Lamin A/C inactivation ( Fig. 6c-d ). These results suggest that accumulation of heterochromatin marks at forks is required to modulate fork restart upon mild RS and is modulated by Lamin A/C likely at the level of H3K9 demethylation, impacting the maintenance of the epigenetic mark on replicated DNA. Download figure Open in new tab Figure 6. Lamin A/C is required to accumulate H3K9me3 at forks upon mild RS, via modulation of the KDM3A demethylase. a. Representative PLA images illustrating H3K9me3 deposition on nascent DNA upon mild RS (H3K9me3-EdU PLA, red) in mAID2-LMNA HCT116 cells. Cells were treated with 5-Ph-IAA for 24 h prior to the experiment, to induce Lamin A/C depletion. Cells were pulsed with EdU for 20 min at the end of the optional treatment with 25 nM CPT (1h). Scale bar: 10 μm. b. Quantification of of H3K9me3-EdU total PLA spot intensity per nucleus. n > 800 S phase cells were analyzed in each condition; Kruskal-Wallis test followed by Dunn’s test were performed to test statistical significance for each of two independent PLA experiments.. c. Representative images of chromatin fibers stained for EdU (red), H3K9me3 (green) and H3 (magenta), from U2OS cells left untreated or treated with 25 nM CPT (1h) treatment, upon optional downregulation of LMNA and/or KDM3A . Cells were pulsed with EdU for 20 min at the end of the optional tretments with 25 nM CPT (1h). Scale bar: 3 μm. d. Quantification of H3K9me3 signal overlapping with EdU spots. n > 70 EdU tracks for each condition were analyzed in two independent experiments. Kruskal-Wallis test followed by Dunn’s test were performed to test statistical significance for PLA and chromatin fiber analysis. e. Model depicting the role of Lamin A/C and LAP2α limiting RECQ1-mediated fork restart upon mild RS, by increasing H3K9me3 and ADP ribosylation levels at replication factories. Lamin A/C, assisted by LAP2α, interacts with replication factories throughout the nucleus and upon mild RS induces heterochromatinization on nascent DNA by limiting access and/or activity of the H3K9me3 demethylase KDM3A. The chromatin compaction induced by H3K9me3 accumulation – mediated by G9ai/SUV39H1 H3K9 methylases – allows efficient PARP1-mediated ADP ribosylation, which limits the engagement of the RECQ1 helicase at replication forks, thereby stabilizing reversed forks and promoting active fork slowing. Both H3K9me3 accumulation and ADP ribosylation are reversible processes, that can be reverted respectively by KDM3A and PARG once the stress is released. Discussion Our data establish a new important role for Lamin A/C in supporting active fork slowing and genome stability upon mild replication interference, by modulating RECQ1 activity at reversed forks. Defective replication fork reversal was not reported in Lamin A/C-depleted cells upon fork stalling by HU treatment 31 . It should be noted, however, that RECQ1 activity is very limited in these conditions, as nucleotide depletion impairs efficient fork restart 6 . Moreover, similarly to most studies on Lamin A/C, its permanent inactivation was achieved in that study by stable shRNA expression, which may prime cellular mechanisms of adaptation, possibly masking specific phenotypes. More in general, acute and transient inactivation of Lamin A/C – induced in our case by transient siRNA transfection or auxin-inducible degradation – may uncover novel functions of the protein, and particularly of its nucleoplasmic fraction, which is predictably less stable than the protein assembled into filaments in the nuclear lamina. Indeed, the phenocopy observed for Lamin A/C and LAP2α inactivation – along with the pan-nuclear interaction with replication factories and the global effects on replication fork progression – strongly suggest that this novel role of Lamin A/C in the RS response entails primarily the nucleoplasmic fraction of the protein. Due to its intrinsic solubility and heterogeneity in structure 52 , nucleoplasmic Lamin A/C is much harder to detect by conventional imaging methods; based on our findings, visualizing at high resolution the organization of Lamin A/C structures at replication factories will represent an important and exciting challenge for future studies. It is in principle surprising that this function of Lamin A/C in response to mild RS correlates with decreased proximity of the protein to short stretches of nascent DNA, as those induced in the experimental conditions of our PLA assays ( Fig. 1 ). We propose that the observed drop in Lamin A/C-EdU PLA signal upon mild RS does not imply release of the protein from replication factories, but instead reflects a different spatial arrangement of the protein in respect to nascent DNA ( Fig. 6e ). This distance may be transiently increased by replication fork remodeling, as elegantly shown for the replicative helicase in similar PLA assays 36 . Although we expect these Lamin A/C-mediated events to take place throughout the nucleoplasm, our data do not exclude that the protein could play a similar role also within the lamina, possibly assisting specific fork restart mechanisms that were shown to entail fork relocation to the nuclear membrane 53 . Our data also highlight the functional role of PARylation events in the control of replication fork progression and restart, suggesting the local PAR accumulation within replication factories is required to control RECQ1 activity and thereby mediate active fork slowing throughout the nucleus. This evidence extends previous studies implicating PAR-mediated mechanisms in replication fork progression, remodelling and restart, even in response to perturbations that do not prevent bulk DNA synthesis, such as defective Okazaki fragment ligation 6 , 7 , 34 , 35 , 54 . How could Lamin A/C control PARylation levels within replication factories? Lamin A/C defects were shown to induce altered levels of NAD+, a crucial co-substrate for PAR synthesis, but this function is tissue-specific and related to mitochondrial defects 55 , 56 . Two lines of evidence in our work rather suggest that Lamin A controls PARylation at replication forks via modulation of local chromatin compaction and accessibility: i) defective H3K9 methylation affects PAR levels at forks even in Lamin A/C-proficient cells and strikingly phenocopies Lamin A/C inactivation, ii) replication defects upon both types of genetic perturbation reflect the deregulated action of RECQ1 and can be suppressed by PARG inhibition, which restores high PAR levels at replication factories. PARP1 is very efficient PARylating itself and this modification is sufficient to inhibit RECQ1 enzymatic activity in vitro 7 , representing a critical regulatory event for fork restart. However, the concomitant accumulation of PAR and histone methylation events at replication forks upon mild RS suggests that additional PARylation events on unknown protein targets or DNA could mediate RECQ1 inhibition within active replication centers, transiently preventing its efficient engagement at replication forks and thereby stabilizing reversed forks to promote fork slowing. Uncovering the molecular mechanisms achieving complete control of RECQ1 recruitment and activity at replication forks will require further investigation. Considering that RECQ1 is the most abundant of the RECQ helicases in human cells and a key factor mediating the balance between fork slowing and restart, it seems likely that its activity entails multiple levels of control. Collectively, our data support a sequential model where upon mild RS Lamin A/C promotes efficient PARylation and RECQ1 control at replication factories by assisting chromatin methylation and compaction ( Figure 6e ). Lamin A/C was previously shown to modulate chromatin organization and gene expression by restricting chromatin mobility and diffusion 46 . Lamin A/C directly interacts with SUV39H1 and stabilizes it, thereby affecting H3K9me3 levels and chromatin compaction 29 . Moreover, Lamin A/C interacts with the histone deacetylases HDAC2 and SIRT6, modulating heterochromatinization and promoting PARylation, as well as DNA repair 57 , 58 . Despite this insight, the detailed mechanisms by which Lamin A/C regulates the deposition or maintenance of heterochromatin marks in different contexts are yet elusive. We here provide evidence that, in the context of replication factories and mild RS, decreased H3K9me3 levels in Lamin A/C-depleted cells can be rescued by inactivation of the KDM3a demethylase, suggesting that Lamin A/C controls chromatin compaction by limiting KDM3a access or activity at replication factories, and thereby stabilizes the heterochromatin mark on genomic regions that experience mild RS ( Fig. 6e ). Upon acute inactivation of Lamin A/C, KDM3a-dependent H3K9 demethylation impairs H3K9me3 accumulation at forks experiencing RS, which in turn affects the equilibirium between PAR synthesis and degradation, reducing PAR levels at replication factories. This ultimately deregulates access and/or activity of RECQ1 at forks that had been reversed upon mild RS, leading to unrestrained fork progression and genomic instability (Extended Data Fig. 5). Beyond the specific role of Lamin A/C in the modulation of replicating chromatin, our data establish a novel, general link between chromatin compaction and PARylation in response to mild RS. How RS-induced heterochromatic marks promote PARylation within replication factories is currently unclear. PARP1 binding was previously reported at various specialized heterochromatic domains, such as telomeres, centromeres and silent rDNA repeats, promoting local and functionally relevant ADP-ribosylation events 48 . Moreover, excessive accumulation of heterochromatin at replication forks was shown to induce local ADP ribosylation by promoting discontinuous DNA synthesis 59 , 60 , which is a potent activator of PARP1 34 . Intriguingly, PARP1 activation was recently shown to follow its initial condensation at damaged sites to promote the subsequent DNA repair steps 49 , suggesting that PARP1 may be recruited and subsequently activated within condensed chromatin. Although our data suggests that chromatin compaction precedes and mediates sufficient levels of PARylation at replication forks, they do not exclude that PARP1 activation may consolidate a protected subnuclear environment within those factories, limiting access to abundant and potentially deleterious proteins, as proposed for Primpol upon fork stalling 13 and here for RECQ1 upon mild RS. Interestingly, PARP1 was shown to promote nucleosome assembly in vitro 61 and to induce local chromatin compaction and silencing in cells 62 . Moreover, several chromatin remodelers promoting a compact chromatin environment are known as PARP1 targets 63 , 64 , suggesting that efficient PARylation may further consolidate chromatin compaction in replicating domains facing RS. Based on our evidence, uncovering the complex mechanistic crosstalk of chromatin compaction, PARylation and replication fork progression will represent a promising and exciting avenue of future research. Recent findings uncovered novel functions for Lamin A/C in the ATR-mediated control of nuclear and micronuclear membrane rupture upon excessive DNA damage 65 , 66 . Our study uncovers another independent function of Lamin A/C, protecting genome stability throughout the nucleus in response to mild genotoxic treatments. The ATR kinase was shown to modulate fork progression and remodeling upon mild RS 20 , but the underlying mechanisms have remained elusive. Importantly, upon HU-induced fork stalling, ATR was also shown to mediate chromatin compaction on replicated DNA 13 , and to induce the relocation of stalled forks towards the nuclear periphery via nuclear F-actin polymerization 16 . Investigating how ATR signaling modulates nuclear architecture and organization throughout the nucleoplasm upon mild replication interference will likely reveal important regulatory mechanisms of the RS response. Gaining molecular insight on the role of nucleoplasmic Lamin A/C upon genotoxic stress may also provide novel mechanistic explanations for the dramatic consequences of Lamin A/C deregulation in laminopathies, so far primarily attributed to defects in the structural support of the nuclear lamina. Methods Key materials Antibodies anti-Histone H3, Abcam Cat # ab1791 anti-Actin, Sigma-Aldrich Cat # A5441 anti-Tubulin, Sigma-Aldrich Cat # T5168 anti-Vinculin, Thermo Fischer Scientific Cat # 700062 anti-RECQ1, Novus Biologicals Cat # NB100-618 Rat anti-Primpol, kindly provided by J. Méndez Rat anti-BrdU (CldU), Abcam Cat # ab6326 anti-SMC1, Thermo Fischer Scientific Cat # PA5-29122 Mouse anti-BrdU (IdU), Becton Dickinson Cat # 347580 Goat anti-mouse-Alexa Fluor™ 488, Thermo Fisher Scientific Cat # A-11001 Goat anti-rabbit-Alexa Fluor™ 488, Thermo Fisher Scientific Cat # A-11001 Goat anti-mouse Alexa Fluor™ 555, Azide Thermo Fisher Scientific Cat # A-21422 Goat anti-rabbit Alexa Fluor™ 555, Azide Thermo Fisher Scientific Cat # A-21428 Donkey anti-rat-Cy3, LubioScience Cat # 712-166.153 Anti-GFP, Abcam # ab290 Mouse anti-gH2AX (Ser139), Millipore Cat # 05-636 Anti-Lamin A, SantaCruz Biotechnology Cat #L12923 Anti-Lamin A/C, Proteintech, Cat # 10298-1-AP Anti-Lamin A/C (E-1), SantaCruz Biotechnology Cat # SC-376248 Anti-Lamin A, Abcam Cat # ab83472 Anti-PARP1, Tulip Biolabs, Cat #2090 Anti-PARP1 (C2-10), produced in-house, kindly provided by J.-P. Gagné and G. G. Poirier anti-LAP2α, Abcam Cat # ab5162 anti-PAR CST (E6F6A), Cell Signaling Technology Cat # 83732 anti-PAR 96-10, produced in-house, kindly provided by J.-P. Gagné and G. G. Poirier anti-MAR/PAR eAf1521-Fc fusion protein, kindly provided by M. O. Hottiger anti-PARP1 (9571), kindly provided by J.-P. Gagné and G. G. Poirier anti-H3K9me2, Active Motif Cat # 39754 anti-H3K9me3, Abcam Cat # ab176916 anti-G9a (EPR18894), Abcam Cat # ab185050 anti-IgG mouse, SantaCruz Biotechnology Cat # sc-2025 Chemicals RNAiMAX, Thermo Fisher Scientific Cat # 13778075 Camptothecin, Sigma-Aldrich Cat # C9911 Etoposide, Sigma-Aldrich Cat # E1383 5-Phenyl-1H-indole-3-acetic acid (5-Ph-IIA), Bioacademia Cat # 30-003 Benzyldimethylalkyl Ammonium Chloride, Sigma-Aldrich Cat # B6295 Formamide, Sigma-Aldrich, Cat # 47680 Glutaraldehyde 25%, EMS Cat # 16200 Uranyl acetate, Fluka Cat # 73943 5-Chloro-2ʹ-deoxyuridine, Sigma-Aldrich Cat # C6891 5-Iodo-2ʹ-deoxyuridine, Sigma-Aldrich Cat # I7125 Nocodazole, Sigma-Aldrich Cat # M1404 ProLong Gold Antifade Mountant Thermo Fisher Scientific Cat # P36930 ibidi mounting medium, ibidi, # Cat 50001 Western Bright ECL-HRP Substrate, Advansta Cat # K-12045 WesternBright Sirius - femtogram HRP Substrate, Advansta, Cat # K-12043 DAPI, Sigma-Aldrich Cat # D9542 Nonidet™ P-40, Merck, Cat # 21-3277 Dynabeads™ Protein G for Immunoprecipitation, Thermo Fischer Scientific Cat #10003D Benzonase, Millipore, Cat # E1014 Biotin-azide, Merck # Cat 762024 Biotin-azide, Jackson ImmunoResearch Cat # AB_2339006 G9ai-UNC0642, MedChemExpress, kindly provided by Taneja lab PARGi PDD0017272, Lucerna-Chem, Cat # HY-133531 PARPi-olaparib, Selleckchem Cat # S1060 Duolink In Situ PLA Probe Anti-Rabbit PLUS, Merck Cat # DUO92002 Duolink In Situ PLA Probe Anti-Mouse MINUS, Merck Cat # DUO92004 Duolink In Situ Detection Reagents Red, Merck Cat # DUO92008 Poly-L-lysine, Sigma-Aldrich Cat # P4832 Cell lines Human osteosarcoma U2OS cells and human colon cancer HCT116 cells were acquired from ATCC. The HCT116 F74G cell line was kindly provided by the Kanemaki laboratory. The mAID2-mClover-LMNA HCT116 F74G cell line was generated in Massimo Lopes’ laboratory. Cell culturing U2OS cells and HCT116 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, 41966-029, Life Technologies) supplemented with 10% Fetal Bovine Serum (FBS, GIBCO), 100 U/mL penicillin and 100 mg/mL streptomycin at 37 °C in a humidified atmosphere containing 6% CO2. Cell line generation The mAID2-mClover-LMNA HCT116 F74G cell line was generated as described previously (Natsume et al., 2016, Yesbolatova et al, 2019, Yesbolatova et al, 2020). The gRNA was cloned into the CRISPR-Cas9 containing plasmid pX330 (Addgene Cat # 42230-DNA.cg) according to Ann et al. 2016, using the following oligos for the N-terminal region: 5’-CGCTGCCAACCTGCCGGCCA-3’ (CRISPR gRNA) and 5’-TGGCCGGCAGGTTGGCAGCG-3’ (reverse complement). For the donor plasmid, we used HCT116 genomic DNA to amplify by PCR and clone into the pJET plasmid (CloneJET PCR Cloning Kit, Thermo Fisher Scientific Cat # K1232) a 1kb fragment as homology arms (HAs) at the N-terminus of the LMNA gene containing the first ATG codon, using the following oligos: 5’-CACCCACTCTCCCTCCTTGG-3’ (forward primer) and 5’-GCCCCAACTTGTCCCTGATAC-3’ (reverse primer). The HA containing plasmid was amplified with oligos containing BamHI and SalI restriction sites from the ATG by inverse PCR, followed by digestion with BamHI and SalI. On the other hand, pMK345 (Addgene Cat # 121179) and pMK348 (Addgene Cat # 121182) plasmids were digested with BamHI and SalI, and the fragment containing the antibiotic (Hygro and BSD respectively) was ligated to the HA containing plasmid. After confirmation of the sequence by sequencing, OsTIR1(F74G)-expressing HCT116 cells were transfected with both plasmids, followed by double antibiotic selection. Clones were then expanded and selected for PCR genotyping. Promising clones were checked microscopically (fluorescence), and were further subjected to FACS analysis and Western Blot. RNA interference RNAi transfection was carried out using RNAiMAX following the manufacturer’s instructions. U2OS cells were transfected using the following siRNAs for 48 or 72 hours: si LUC (5ʹ-CGU ACG CGG AAU ACU UCG ATT-3ʹ), si LMNA (5ʹ-CAG UCU GCU GAG AGG AAC ATT-3’), siLAP2A (5ʹ-GAG AAU UGA UCA GUC UAA GTT-3), si RECQ1 (5’-UUA CCA GUU ACC AGC AUU ATT-3’), si PRIMPOL (5’-GAG GAA ACC GUU GUC CUC AGU GUA U-3’) (all purchased from Microsynth), and si KDM3 (ON-TARGETplus SMARTPool Cat # L-017301-00-0005). Protein extraction and Western Blotting To determine the levels of depletion of the proteins of interest, protein extracts from all cell lines were prepared in Laemmli buffer (4% SDS, 20% glycerol, and 120 mM Tris-HCl, pH 6.8) and sonicated with a Bandelin Sonoplus Mini 20-System sonicator (3 pulses of 1.5 second, 70% amplitude). Equal amounts of protein (20 μg) were loaded onto 4%-15% gradient Mini-PROTEAN TGX Precast Protein Gels (BioRad). Proteins were separated by electrophoresis at 16 mA followed by transferring the proteins to Amersham nitrocellulose membranes (Merck) for 1-1.5 hours at 350 mA at 4°C in transfer buffer (25 mM Tris, 192 mM glycine) containing 20% methanol. Upon transferring, membranes were stained with Ponceau and imaged, followed by blocking in 5% milk in 0.1% TBST (TBS 1x supplemented with 0.1% Tween-20) for 1 hour. Next, membranes were incubated in primary antibodies diluted in 5% miklk/TBST overnight at 4°C. eAf1521 in particular was diluted in 2% milk/TBST. Upon washing the membranes three times with 0.1% TBST, secondary antibodies were added for 45 min at room temperature. Membranes were then washed again three times with 0.1% TBST, followed by imaging in Fusion Solo (Vilber Lourmat) using ECL detection reagent or ECL Sirius. Proteins were quantified where necessary using ImageJ 64 software. Determination of PAR levels by CHAPS extracts and Western blot (for 96-10 and E6F6A antibodies specifically) Cell pellets were lysed in 1 mL of lysis buffer (40 mM HEPES pH 7.5, 120 mM NaCl, 0.3% CHAPS) supplemented with cOmplete™ EDTA-free protease inhibitor cocktail (Sigma-Aldrich), 10 μM of PARP-1/2 inhibitor Talazoparib/BMN-673 (Selleckchem) and PARG inhibitor PDD00017272 (Tocris Bioscience) to block PAR turnover. Lysates were briefly sonicated for 30 sec on ice and mixed for 30 min on a rotating mixer in a cold room. Insoluble material and cellular debris were removed by centrifugation for 5 min at 3400 rpm. The supernatant was mixed with an equal volume of 4x Laemmli sample buffer (Bio-Rad) containing 5% β-mercaptoethanol. Lysates were resolved by 4–12% linear gradient SDS-PAGE (Bio-Rad) and transferred onto a 0.2 μm nitrocellulose membrane. PAR was revealed by Western blot using the anti-MAR/PAR antibody E6F6A or the anti-PAR antibody 96-10. The mouse monoclonal antibody clone C2-10 was used to detect PARP1. Lamin A was targeted with the anti-lamin A antibody L1213. Nonspecific antibody binding was blocked using 5% nonfat dried milk in PBS solution containing 0.1% Tween-20 (PBST). Primary antibodies were diluted in 5% milk in PBST and incubated overnight at room temperature on a rocking shaker. Prior to adding the secondary antibodies, the membranes were washed five times using PBST containing 5% milk. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies were allowed to bind at room temperature for 30 min. Membranes were washed in PBST and revealed using Western Lightning Plus-ECL enhanced chemiluminescence substrate (Revvity Health Sciences) according to the manufacturer’s instructions and added to the membrane for 1 min. Excess substrate was removed prior to imaging on autoradiography films. Immunoprecipitation For co-immunoprecipitation (IP), cells were scraped off the plate in IP buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EGTA, 2 mM MgCl 2 , 0.5% NP-40, 1 mM DTT, 1x Complete EDTA-free Protease Inhibitor cocktail, Phosphatase inhibitor cocktail 2 and 3, PARGi 1 μM, and PARPi 10 μM) and incubated 10 min on ice. The samples were then sonicated with a Bandelin Sonoplus Mini 20-System sonicator (3 pulses of 1.5 second, 70% amplitude), followed by centrifugation for 10 min at 4000 rpm. The soluble fractions were optionally treated with 50 U/μl of benzonase and used as input for the IP (1 mg/IP), followed by incubation with the Lamin A/C antibody (E1) (5 μg/IP) overnight at 4°C, and blocking with BSA-blocked protein G dynabeads for 4 hr at 4°C. Beads were washed three times in IP buffer without benzonase, followed by elution of complexes from the beads using SDS PAGE sample buffer. Samples were analyzed by SDS PAGE and immunoblotting. DNA fiber analysis U2OS or HCT116 cells were cultivated asynchronously and subsequently labeled with two different thymidine analogs: 30 μM of chlorodeoxyuridine (CldU) for 25 minutes, followed by three washes with warm PBS 1x, and 250 μM of 5-iodo-2ʹ-deoxyuridine (IdU) for 25 minutes alone or in combination with mild doses of genotoxic treatments (100 nM CPT or 20 nM ETP). To evaluate the impact of ADP-ribosylation on replication fork progression, U2OS cells were pre-incubated in DMEM containing 1 μM PARG inhibitors for two hours before the initiation of the CldU labeling, which were retained during both CldU and IdU labeling. To evaluate the contribution of chromatin compaction on replication fork slowing, U2OS cells were pre-incubated in DMEM containing 1 μM G9a inhibitors which were maintained during both CldU and IdU labeling. In the HCT116 mAID2-LMNA cells, in order to induce degradation of Lamin A/C, 1 μM 5-Ph-IIA was added 24 hours before the CldU/IdU pulse labeling and was maintained during the labeling. Upon IdU labeling, the cells were washed three times with cold PBS 1x, collected by standard trypsinization and resuspended in cold PBS 1x at 3×10 5 cells/mL. 3 μL of this cell suspension were then mixed with 7 μL of lysis buffer (200 mM Tris-HCl, pH 7.5, 50 mM EDTA, and 0.5% (w/v) SDS) on a glass slide placed horizontally. After an incubation of 6 minutes at RT, the slides were tilted at a 45° angle to stretch the DNA fibers onto the slide. The resulting DNA spreads were air-dried, fixed in ice-cold 3:1 methanol/acetic acid for 10 minutes, air-dried once more, and stored at 4°C overnight. The following day, the DNA fibers were denatured by incubation in 2.5 M HCl for 1 hour at RT, washed five times with PBS 1x and blocked with 2% BSA in PBST (PBS 1x supplemented with 0.05% Tween-20) for 40 minutes at RT. The newly replicated CldU and IdU tracks were then stained for 2.5 hours at RT in a humidified chamber, using two different anti-BrdU antibodies recognizing CldU (1:300) and IdU (1:80), respectively. After washing five times with PBST, the slides were stained with anti-mouse AlexaFluor 488 (1:300) and anti-rat Cy3 (1:300) or anti-rat AlexaFluor 555 (1:300) secondary antibodies for 1 hour at RT in the dark in a humidified chamber. After washing another five times with PBST, the slides were air-dried and then mounted in 13 μL Prolong Gold antifade reagent. Microscopy imaging was performed using a Leica DM6 B microscope (HCX PL APO 63x objective). To assess fork progression, the CldU and IdU track lengths of at least 100 fibers per sample were measured using the line tool in ImageJ software and analyzed into IdU/CldU ratio in Microsoft Excel. Graphical and statistical analysis was carried out using GraphPad Prism 10. Analysis of chromosome spreads U2OS cells were transfected with siLUC, siLMNA or siLAP2A and treated with 100 nM CPT for 3 hours. The genotoxic agent was removed by washing three times with PBS 1x and the cells were then released into fresh DMEM medium containing 200 ng/mL nocodazole for 16 hours. Cells were collected at 48 hours upon transfection, washed and resuspended in hypotonic solution (0.075 M KCl) for 20 min at 37 °C. Cells were then fixed with ice-cold fixation buffer (methanol:acetic acid, 3:1). The fixation step was repeated another two times. Cells were then dropped onto pre-hydrated glass slides and air-dried. The following day, slides were mounted with Vectashield medium containing DAPI. Microscopy imaging was performed using a Leica DM6 B microscope (HCX PL APO 63x objective) at 63x magnification equipped with a camera (model DFC360 FX; Leica) and visible chromosome abnormalities per metaphase spread were counted. Graphical and statistical analysis was carried out using GraphPad Prism 10. Proximity Ligation Assays (PLA) Lamin A/C:EdU PLA. U2OS or HCT116 cells were asynchronously grown on sterile ibidi slides (ibidi, Cat # 80827). Cells were then treated with 100 nM CPT or 20 nM ETP for one hour in total, followed by 25 μM EdU (10 minutes for the untreated cells, 12 minutes for the ETP-treated cells, 13 minutes for the CPT-treated cells) before the end of the one hour. The cells were washed with PBS 1x and pre-extracted for 5 minutes using CSK-buffer (10 mM HEPES, 50 mM NaCl, 0.3 M Sucrose, 3 mM MgCl 2 , 1 mM EDTA or 1 mM EGTA, and 0.5% Triton X-100) on ice, followed by fixation in 4% formaldehyde at RT. Upon fixation, cells were washed three times with PBS 1x and permeabilized using 50 mM NH 4 Cl in 0.5% Triton X-100/PBS 1x for 3 minutes, followed by another 3 minutes in 0.5% Triton X-100/PBS 1x. Upon washing three times in PBS 1x, EdU detection was performed using a homemade ClickIT reaction (0.1 M Tris pH 8.5, 0.1 M sodium ascorbate, 2 mM Cu 2 SO 4 and 0.1 mM biotin-azide) for 1.5 hour at 37 °C in a humidified chamber. After another three washes with PBS 1x, cells were incubated in blocking buffer at 37 °C for 1 hour and incubated overnight at 4°C with anti-Lamin A/C (Proteintech). After washing the primary antibody, cells were incubated with PLA probes for 1 h at 37 °C, ligation for 30 min 37 °C, and polymerase reaction for 100 minutes at 37 °C according to the manufacturer’s instructions. After washing, cells were incubated at 37 °C for 30 minutes with secondary antibodies in blocking buffer containing DAPI (0.5 mg/mL). Following three washes in PBST and PBS 1x, the ibidi slides were kept in PBS 1x until being mounted with ibidi mounting medium only right before imaging acquisition. Confocal imaging was performed using Leica SP8 inverse STED 3X and HC PL APO STED WHITE-motCORR 93x magnification. For confocal analysis, deconvolution was performed using Huygens Professional software. Image analysis and 3D-reconstruction was done using Imaris Software. PAR:EdU PLA U2OS or HCT116 cells were asynchronously grown on sterile 12-mm diameter glass coverslips coated with poly-L-lysine and were optionally pre-treated with 1 μM PARG inhibitors or 1 uM G9a inhibitors for two hours. One hour before fixation, cells were treated with 100 nM CPT (optionally in combination with PARGi or G9ai). The protocol was the same as the one for Lamin A/C: EdU with only the following modifications. The CSK buffer was supplemented with 10 µM PARPi and 1 µM PARGi and was used for 5 min on ice. The fixation was performed using 4% formaldehyde at RT, followed by MeOH for 5 mins in -20°C. The permeabilization was performed for 6 minutes in 0.5% Triton X-100/PBS 1x. Upon secondary antibody and DAPI incubation, coverslips were washed twice in PBST and once in PBS 1x, and briefly immersed in distilled water, dried on 3 mm paper and mounted with Prolong Gold antifade reagent. Microscopy imaging was performed using a Leica DM6 B microscope (HCX PL APO 63x objective). PLA quantification was performed using an automated pipeline in Cell Profiler, whereas graphical and statistical analysis using GraphPad Prism 10. H3K9me3:EdU PLA HCT116-mAID2-mClover-LMNA cells were grown on sterile poly-lysine coated coverslips to be 60-70% confluent on the day of experiment. Cells were treated with 1 μM 5-Ph-IAA for 24 hours prior to the experiment to induce Lamin A/C depletion. For camptothecin (25nM) treated samples, cells were pulsed with EdU (10µM) for 20 minutes at the end of 1 hour treatment. After two washes with cold 1x PBS, cells were pre-extracted with 0.5% Triton in ice-cold cytoskeletal (CSK) buffer for 5 min at 4oC and fixed with 4% Formaldehyde in PBS for 15 minutes at room temperature. After thorough washes with PBS 1x, cells were permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature. Samples were washed thoroughly with 1x PBS and then blocked with 5% BSA in PBS for 1 hour at room temperature. EdU was conjugated with biotin azide (Jackson ImmunoResearch) using Click chemistry. Samples were then incubated with rabbit anti-H3K9me3 and mouse anti-biotin primary antibodies (1:1000 dilution in PBS, 5% BSA) overnight at 4°C. PLA experiment was carried out using Duolink PLA probes and Duolink In situ Detection Reagent following manufacturer’s protocol. Images were taken using Metafer 5 and PLA spot intensity (a.u.), the product of no. of spots and the mean intensity of spots per nucleus, was quantified using Metasystem. Electron Microscopy U2OS cells were asynchronously grown and transfected with si LUC , si LMNA or si LAP2A . After 48 hours of transfection and at 70-80% of confluency, cells were treated with 100 nM CPT for 1 hour, followed by collection, resuspension in ice-cold PBS and crosslinking with 4,5ʹ, 8-trimethylpsoralen (10 μg/mL final concentration). Crosslinked cells were irradiated with pulses of UV 365 nm monochromatic light (UV Stratalinker 1800; Agilent Technologies). DNA was extracted according to Muzi-Falconi and Brown, 2018. Briefly, cells were lysed (1.28 M sucrose, 40 mM Tris-HCl [pH 7.5], 20 mM MgCl2, and 4% Triton X-100; Qiagen) and digested (800 mM guanidine-HCl, 30 mM Tris-HCl pH 8.0, 30 mM EDTA pH 8.0, 5% Tween-20, and 0.5% Triton X-100) at 50 °C for 2 h in presence of 1 mg/mL proteinase K. The DNA was purified using chloroform/isoamylalcohol (24:1) and precipitated in one volume of isopropanol. Finally, the DNA was washed with 70% EtOH and resuspended in 200 μL TE (Tris-EDTA) buffer. Restriction enzyme digestion followed (120 U of PvuII HF, New England Biolabs) in order to digest 6 μg of the purified genomic DNA for 5 h at 37oC. RNase A (Sigma–Aldrich, R5503) to a final concentration of 250 ug/ml was added for the last 2 h of this incubation. The digested DNA was then purified using a Silica Bead Gel Extraction kit (Thermo Fisher Scientific) according to manufacturer’s instructions. The Benzyl-dimethyl-alkyl-ammonium chloride (BAC) method was used to spread the DNA on carbon-coated 400-mesh nickel grids (G2400N, Plano Gmbh). Subsequently, DNA was coated with platinum using a High Vacuum Evaporator (EM BAF060, Leica) as described in Zellweger and Lopes (2018). The grids were imaged automatically at 28’000x using a Talos 120 transmission electron microscope (FEI; LaB6 filament, high tension ≤120 kV) with a bottom-mounted CMOS camera BM-Ceta (4096x4096 pixels) and the MAPS 3 software (Thermo Fisher Scientific). For the EM analysis, samples were annotated for replication intermediates using the MAPS offline viewer (V3.28, Thermo Fisher Scientific) and corresponding images were extracted. The replication intermediates were scored blind to the experimental condition using Fiji (Schindelin et al, 2012). For each experimental condition at least 65 replication fork molecules were analyzed in two distinct biological replicates. Chromatin Fiber analysis (ChromStretch) Chromatin fibers were prepared as described in Gaggioli et al., NCB, 2023. Following treatments, cells were harvested and washed with cold PBS 1x. Cells were lysed with 10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM DTT and protease inhibitor (cOmplete, mini, EDTA-free Protease Inhibitor Cocktail, Roche) for 5 min on ice. Samples were centrifuged (1,500g for 5 min) at 4 °C to collect the released nuclei. Nuclei were resuspended in hypotonic buffer (3 mM EDTA, 0.2 mM egtazic acid, 1 mM DTT and protease inhibitor), spotted on Superfrost microscope slides and incubated in a humid chamber. Excess buffer was removed and the slides were allowed to dry for a maximum of 5 minutes. Then, slides were transferred to a chamber containing lysis buffer pH 7 and incubated for 10 minutes. Chromatin fibers were isolated by letting the lysis buffer flow out of the chamber at a constant flow rate to facilitate stretching with the help of an equipment developed in the lab. Stretched chromatin fibers were fixed with 1% Formaldehyde in PBS 1x for 15 minutes at room temperature. Slides were washed thoroughly in PBS 1x and EdU was fluorescently labeled with Alexa Fluor 594 azide using Click chemistry. Slides were washed with PBS 1x, blocked in 5% BSA in PBS for 1 hour and then incubated with Rabbit anti-H3K9me3 (1:1000 in 5% BSA PBS 1x) overnight at 4°C. The primary antibody was labeled with an anti-Rabbit secondary antibody conjugated to Alexa Fluor 488 (1:1000 in 5% BSA PBS 1x) for 1 hour at room temperature. Sequential immunolabeling was performed using Rabbit anti-H3 antibody (1:1,000 in 5% BSA PBS 1x for 1 hour) to counterstain chromatin followed by labeling with anti-Rabbit antibody conjugated to Alexa Fluor647 (1:1000 in 5% BSA PBS 1x) for 1 hour at room temperature. Chromatin fibers were imaged using a Leica ST5 confocal microscope equipped with an oil immersion 63× (HC PL APO CS2, NA 1.4) objective. H3K9me3 signal at EdU spots were quantified using ImageJ. Flow cytometric analysis (EdU/ γH2AX/DAPI) U2OS or HCT116 WT and degron cell lines were labeled with 10 μM EdU for 30 min, harvested by standard trypsinization and subsequently fixed for 10 min in 4% formaldehyde/PBS 1x. Cells were then washed twice and blocked over night at 4°C with 1% BSA/PBS 1x, pH 7.4. Next, they were permeabilized with 0.5% saponin/1% BSA/PBS 1x, and stained with primary mouse anti-γH2AX antibody diluted at 1:1000 in 0.5% saponin/1% BSA/PBS 1x for 2 hr. This was followed by incubation with a Goat anti-mouse Alexa 647 antibody diluted at 1:125 in 0.5% saponin/1% BSA/PBS 1x for 30 min. The incorporated EdU was labeled according to the manufacturer’s instructions. Total DNA was stained with 1 μg/mL DAPI dissolved in 1% BSA/PBS 1x. Samples were measured on an Attune NxT Flow Cytometer (Thermo Fisher) and analyzed using FlowJo software V.10.0.8 (FlowJo, LLC). Author contributions V.C. obtained new cell lines, assisted by D.G.-A., and performed most DNA and IF/PLA experiments, and all IPs, metaphase spreads, WB, FACS and EM experiments. In the lab of N.T., J. P. performed PLA and ChromStretch experiments on H3K9me3, while C. B. performed PLA experiments on G9a. S. A. and P.U.-C. performed most DNA fiber/WB experiments upon LAP2A downregulation. M. A. assisted in sample processing and image analysis for EM experiments. J.-P. G. performed a subset of WBs to detect global PAR levels, in the lab of G. G. P. M.L. designed and supervised the project, with crucial contributions of V.C. and N. T.. M. L. and V.C. wrote the manuscript, which was finalized with contributions of all coauthors. Competing interests N. T. holds an international patent for ChromStretch technology filed under PCT/NL2023/050120. No other authors have competing interests. Download figure Open in new tab Extended Data Figure 1. Related to Fig. 1. a. Step-wise representation of the 3D reconstruction of a representative HCT116 nucleus, where Lamin A/C proximity to nascent DNA (EdU) is detected by Lamin A/C:EdU PLA, Lamin A/C IF staining and confocal microscopy. First peripheral Lamin A/C was masked, followed by the PLA foci. Such image processing allows to conclude that PLA foci are detected throughout the nuclear volume (see also Extended Data Video 1 at the link below). Scale bar: 3 μm. b. Quantification of Lamin A/C PLA signals HCT116 nuclei upon Lamin A/C depletion by siRNA, from the experiment in Fig. 1c-e . Signal was qunatified in at least 100 EdU+ nuclei, in each of the 4 independent experiments. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values. Statistical analysis was applied on the median values, using one-way ANOVA test with Bonferroni’s post hoc correction. Residual PLA signal most likely reflects low residual levels of Lamin A/C upon siRNA downregulation. c. Experimental design for the IF/PLA experiment in d-e. d. Representative U2OS nuclei (DAPI) treated for 1h with 100 nM CPT and stained for DNA synthesis (EdU), Lamin A/C and its physical proximity to nascent DNA (Lamin A/C:EdU PLA), upon optional downregulation of LAP2A. e. Quantification of Lamin A/C PLA signals from c-d. Signal was quantified in at least 100 EdU+ nuclei, in each of the 3 independent experiments. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values. Statistical analysis was applied on the individual experiments, using Kruskal-Wallis test with Dunn’s post hoc correction. Similar results were observed in the three independent experiments. EdU-cells are used as negative control. Scale bar: 10 μm. Extended Data Video 1, related to Fig. 1 and Extended Data Fig. 1: https://www.dropbox.com/scl/fo/5bkx4lj2kh6ldpoef2ciz/AIRqKpGtXd6qi0XF4Y60RXI?rlkey=3v1rhv51utxr4awglcg9a5n4t&dl=0 Download figure Open in new tab Extended Data Figure 2. Related to Fig. 2. a. Graphical representation for the modification of the endogenous LMNA gene by mAID2-mClover in HCT116 mAID2-LMNA cells, allowing rapid Lamin A/C depletion upon 5-Ph-IIA addition to the culture media. b. Western blot displaying efficient depletion of mAID2-mClover-tagged Lamin A/C in clones 13 and 22 of HCT116 mAID2-mClover-LMNA cells, 24h after 5-Ph-IIA addition. H3 is used as loading control. c-d. FACS analysis of EdU incorporation and DNA damage response (DDR, ψH2AX) in HCT116 mAID2-mClover-LMNA cells (clones 13 and 22), 5h or 24h after 5-Ph-IIA addition. No marked cell cycle deregulation is observed at these time points, despite efficient Lamin A/C depletion at 24h (see b). e. FACS analysis of cell cycle distribution by DNA content (DAPI) in U2OS cells at different time points after transfection of siRNA targeting LMNA or LAP2A . While LAP2A downregulation does not lead to alteration of cell cycle profiles, a marked accumulation of cells in G2/M is observed 72h, but not 48h after siLMNA transfection. Given that efficient LMNA downregulation is already visibile at 48h (see Fig. 2h ), this time point was selected for further experiments, to avoid indirect effects due to cell cycle arrest. f. FACS analysis of EdU incorporation and DNA damage response (DDR, ψH2AX) in U2OS cells, 48h after transfection of siRNA targeting LMNA or LAP2A . At this time point we did not observe any significant reduction in the number of EdU+ cells, or any marked DDR activation. Download figure Open in new tab Extended Data Figure 3. Related to Fig. 3. a-c. DNA fiber analysis of U2OS cells upon treatment with 20 nM ETP and siRNA-mediated downregulation of LMNA and/or PRIMPOL . a. Western Blot analysis of Lamin A/C and PRIMPOL levels upon siRNA-mediated depletion for the experiment in a-b. H3 and actin are used as loading control. b. Schematic CldU/IdU pulse-labeling protocol used to evaluate fork progression upon 20 nM ETP. siRNAs were added 48h before the assay. c. IdU/CIdU ratio is plotted for a minimum of 100 forks from each of 3 independent experiments. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values. Statistical analysis was applied on the median values, using one-way ANOVA test with Bonferroni’s post hoc correction. d-f. DNA fiber analysis of U2OS cells upon treatment with 20 nM ETP and siRNA-mediated downregulation of LAP2A and/or PRIMPOL . d. Western Blot analysis of LAP2α and PRIMPOL levels upon siRNA-mediated depletion for the experiment in a-b. H3 and actin are used as loading control. e. Schematic CldU/IdU pulse-labeling protocol used to evaluate fork progression upon 20 nM ETP. siRNAs were added 48h before the assay. f. IdU/CIdU ratio is plotted for a minimum of 100 forks from each of 3 independent experiments. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values. Statistical analysis was applied on the median values, using one-way ANOVA test with Bonferroni’s post hoc correction. g. Table reporting the total number of analysed molecules (brackets) and the percentage of reversed forks (RF) observed in the two independent EM experiments in Fig. 3g-i . Download figure Open in new tab Extended Data Figure 4. Related to Fig. 4. a. Co-IP of Lamin A/C and PARP1 upon the indicate experimental conditions. See Methods for details. b. PAR analysis by WB in the indicated conditions, based on CHAPS extracts (see Methods). Two independent PAR/MAR antibodies were used for detection (E6F6A/96-10). c. PAR analysis by WB (detection by eAf1521). The Ponceau blot is provided as loading control. d. Densitometric analysis of PAR levels from 2 independent experiments as in c (eAf1521). The blot area used for quantification is depicted in the image, using the blot in c as representative example. e. Schematic CldU/IdU pulse-labeling protocol used to evaluate fork progression upon 20 nM ETP and/or PARGi treatment (PDD0017272, 1 μM), upon LAP2A downregulation. siRNA was added 48h before the assay, while PARGi was added 2h before. f. IdU/CIdU ratio is plotted for a minimum of 100 forks from each of 3 independent experiments. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values. Statistical analysis was applied on the median values, using one-way ANOVA test with Bonferroni’s post hoc correction. Download figure Open in new tab Extended Data Figure 5. Related to Figure 6. a. Model depicting the effect of Lamin A/C depletion on chromatin compaction and ADP-ribosylation, resulting in RECQ1-mediated unrestrained fork progression. Lamin A/C depletion leads to decreased H3K9me3 levels at replication factories, most likely reflecting deregulated access or activity of the H3K9me3 demethylase KDM3A. Decreased chromatin compaction in these conditions results in reduced PAR levels at replication sites, possibly reflecting increased PAR-glycohydrolase (PARG) activity. Decreased chromatin compaction and PARylation at replication forks, in turn, lead to deregulated access and/or activity of the RECQ1 helicase, which promotes premature reversed fork restart, unrestrained fork progression and genomic instability. Acknowledgments We are grateful to Jana Döhner, Nicolas Schilling, Flurin Sturzenegger and the whole Center for Microscopy of the University of Zurich for technical assistance with microscopy and image analysis. We also thank Nana Naetar, Lukas Muskalla and David Kubon for technical support. We are particularly grateful to Roland Foisner and Michael Hottiger for important reagents, technical and conceptual contributions, and critical reading of the manuscript. We are grateful to all members of the Lopes lab for useful discussions and suggestions on the manuscript. Work in the Lopes lab was supported by the SNF Project grants 310030_189206 and 310030_219393. V.C. received a State Scholarships Foundation “N.D. Chrisovergis” Bequest for Graduate studies abroad. Work in the Taneja lab was supported by the Oncode Institute, which is partly financed by the Dutch Cancer Society and Vidi funding (project no. 114122) and ERC funding (grant no. ChOReS, 101078750/ #114168). We thank the Josephine Nefkens Cancer Program for infrastructure support to the Taneja lab. References 1. ↵ Costa , A.A.B.A. da , Chowdhury , D ., Shapiro , G.I. , D’Andrea , A.D. , and Konstantinopoulos , P.A. ( 2023 ). Targeting replication stress in cancer therapy . Nat Rev Drug Discov 22 , 38 – 58 . doi: 10.1038/s41573-022-00558-5 . OpenUrl CrossRef 2. ↵ Cybulla , E. , and Vindigni , A . ( 2023 ). Leveraging the replication stress response to optimize cancer therapy . Nat Rev Cancer 23 , 6 – 24 . doi: 10.1038/s41568-022-00518-6 . OpenUrl CrossRef 3. ↵ Berti , M. , Cortez , D. , and Lopes , M . ( 2020 ). The plasticity of DNA replication forks in response to clinically relevant genotoxic stress . Nat Rev Mol Cell Biol 18 , 622 – 19 . doi: 10.1038/s41580-020-0257-5 . OpenUrl CrossRef 4. ↵ Neelsen , K.J. , and Lopes , M . ( 2015 ). Replication fork reversal in eukaryotes: from dead end to dynamic response . Nat Rev Mol Cell Biol 16 , 207 – 220 . doi: 10.1038/nrm3935 . OpenUrl CrossRef PubMed 5. ↵ Adolph , M.B. , and Cortez , D . ( 2024 ). Mechanisms and regulation of replication fork reversal . DNA Repair 141 , 103731 . doi: 10.1016/j.dnarep.2024.103731 . OpenUrl CrossRef PubMed 6. ↵ Zellweger , R. , Dalcher , D. , Mutreja , K. , Berti , M. , Schmid , J.A. , Herrador , R. , Vindigni , A. , and Lopes , M . ( 2015 ). Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells . J Cell Biol 208 , 563 – 579 . doi: 10.1083/jcb.201406099 . OpenUrl Abstract / FREE Full Text 7. ↵ Berti , M. , Chaudhuri , A.R. , Thangavel , S. , Gomathinayagam , S. , Kenig , S. , Vujanovic , M. , Odreman , F. , Glatter , T. , Graziano , S. , Mendoza-Maldonado , R. , et al. ( 2013 ). Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition . Nat Struct Mol Biol 20 , 347 – 354 . doi: 10.1038/nsmb.2501 . OpenUrl CrossRef PubMed 8. ↵ Flury , V. , and Groth , A . ( 2024 ). Safeguarding the epigenome through the cell cycle: a multitasking game . Curr. Opin. Genet. Dev . 85 , 102161 . doi: 10.1016/j.gde.2024.102161 . OpenUrl CrossRef PubMed 9. ↵ Karagyozova , T. , and Almouzni , G . ( 2024 ). Replicating chromatin in the nucleus: A histone variant perspective . Curr. Opin. Cell Biol . 89 , 102397 . doi: 10.1016/j.ceb.2024.102397 . OpenUrl CrossRef PubMed 10. ↵ Šviković , S. , and Sale , J.E . ( 2017 ). The Effects of Replication Stress on S Phase Histone Management and Epigenetic Memory . J. Mol. Biol . 429 , 2011 – 2029 . doi: 10.1016/j.jmb.2016.11.011 . OpenUrl CrossRef PubMed 11. ↵ Oberdoerffer , P. , and Miller , K.M . ( 2023 ). Histone H2A variants: Diversifying chromatin to ensure genome integrity . Semin. Cell Dev. Biol . 135 , 59 – 72 . doi: 10.1016/j.semcdb.2022.03.011 . OpenUrl CrossRef 12. ↵ Muñoz , S. , Barroso , S. , Badra-Fajardo , N. , Marqueta-Gracia , J.J. , García-Rubio , M.L. , Ubieto-Capella , P. , Méndez , J. , and Aguilera , A . ( 2024 ). SIN3A histone deacetylase action counteracts MUS81 to promote stalled fork stability . Cell Rep . 43 , 113778 . doi: 10.1016/j.celrep.2024.113778 . OpenUrl CrossRef PubMed 13. ↵ Gaggioli , V. , Lo , C.S.Y. , Reverón-Gómez , N. , Jasencakova , Z. , Domenech , H. , Nguyen , H. , Sidoli , S. , Tvardovskiy , A. , Uruci , S. , Slotman , J.A. , et al. ( 2023 ). Dynamic de novo heterochromatin assembly and disassembly at replication forks ensures fork stability . Nat. Cell Biol . 25 , 1017 – 1032 . doi: 10.1038/s41556-023-01167-z . OpenUrl CrossRef PubMed 14. ↵ Caridi , C.P. , D’Agostino , C. , Ryu , T. , Zapotoczny , G. , Delabaere , L. , Li , X. , Khodaverdian , V.Y. , Amaral , N. , Lin , E. , Rau , A.R. , et al. ( 2018 ). Nuclear F-actin and myosins drive relocalization of heterochromatic breaks . Nature 559 , 54 – 60 . doi: 10.1038/s41586-018-0242-8 . OpenUrl CrossRef PubMed 15. ↵ Schrank , B.R. , Aparicio , T. , Li , Y. , Chang , W. , Chait , B.T. , Gundersen , G.G. , Gottesman , M.E. , and Gautier , J . ( 2018 ). Nuclear Arp2/3 drives DNA break clustering for homology-directed repair . Nature 559 , 61 – 66 . doi: 10.1038/s41586-018-0237-5 . OpenUrl CrossRef PubMed 16. ↵ Lamm , N. , Read , M.N. , Nobis , M. , Ly , D.V. , Page , S.G. , Masamsetti , V.P. , Timpson , P. , Biro , M. , and Cesare , A.J . ( 2020 ). Nuclear F-actin counteracts nuclear deformation and promotes fork repair during replication stress . Nat Cell Biol 22 , 1460 – 1470 . doi: 10.1038/s41556-020-00605-6 . OpenUrl CrossRef PubMed 17. ↵ Shi , J. , Hauschulte , K. , Mikicic , I. , Maharjan , S. , Arz , V. , Strauch , T. , Heidelberger , J.B. , Schaefer , J.V. , Dreier , B. , Plückthun , A. , et al. ( 2023 ). Nuclear myosin VI maintains replication fork stability . Nat. Commun . 14 , 3787 . doi: 10.1038/s41467-023-39517-y . OpenUrl CrossRef PubMed 18. ↵ Nieminuszczy , J. , Martin , P.R. , Broderick , R. , Krwawicz , J. , Kanellou , A. , Mocanu , C. , Bousgouni , V. , Smith , C. , Wen , K.-K. , Woodward , B.L. , et al. ( 2023 ). Actin nucleators safeguard replication forks by limiting nascent strand degradation . Nucleic Acids Res . 51 , 6337 – 6354 . doi: 10.1093/nar/gkad369 . OpenUrl CrossRef PubMed 19. ↵ Palumbieri , M.D. , Merigliano , C. , González-Acosta , D. , Kuster , D. , Krietsch , J. , Stoy , H. , Känel , T. von , Ulferts , S. , Welter , B. , Frey , J. , et al. ( 2023 ). Nuclear actin polymerization rapidly mediates replication fork remodeling upon stress by limiting PrimPol activity . Nat. Commun . 14 , 7819 . doi: 10.1038/s41467-023-43183-5 . OpenUrl CrossRef PubMed 20. ↵ Mutreja , K. , Krietsch , J. , Hess , J. , Ursich , S. , Berti , M. , Roessler , F.K. , Zellweger , R. , Patra , M. , Gasser , G. , and Lopes , M . ( 2018 ). ATR-Mediated Global Fork Slowing and Reversal Assist Fork Traverse and Prevent Chromosomal Breakage at DNA Interstrand Cross-Links . Cell Rep 24 , 2629 – 2642 .e5. doi: 10.1016/j.celrep.2018.08.019 . OpenUrl CrossRef PubMed 21. ↵ González-Acosta , D. , and Lopes , M . ( 2024 ). DNA replication and replication stress response in the context of nuclear architecture . Chromosoma 133 , 57 – 75 . doi: 10.1007/s00412-023-00813-7 . OpenUrl CrossRef PubMed 22. ↵ Emerson , D.J. , Zhao , P.A. , Cook , A.L. , Barnett , R.J. , Klein , K.N. , Saulebekova , D. , Ge , C. , Zhou , L. , Simandi , Z. , Minsk , M.K. , et al. ( 2022 ). Cohesin-mediated loop anchors confine the locations of human replication origins . Nature 606 , 812 – 819 . doi: 10.1038/s41586-022-04803-0 . OpenUrl CrossRef PubMed 23. ↵ Schie, J.J.M. van, and Lange, J. de ( 2021 ). The Interplay of Cohesin and the Replisome at Processive and Stressed DNA Replication Forks . Cells 10 , 3455 . doi: 10.3390/cells10123455 . OpenUrl CrossRef 24. ↵ Buxboim , A. , Kronenberg-Tenga , R. , Salajkova , S. , Avidan , N. , Shahak , H. , Thurston , A. , and Medalia , O . ( 2023 ). Scaffold, mechanics and functions of nuclear lamins . FEBS Lett . 597 , 2791 – 2805 . doi: 10.1002/1873-3468.14750 . OpenUrl CrossRef PubMed 25. ↵ Malashicheva , A. , and Perepelina , K . ( 2021 ). Diversity of Nuclear Lamin A/C Action as a Key to Tissue-Specific Regulation of Cellular Identity in Health and Disease . Front. Cell Dev. Biol . 9 , 761469 . doi: 10.3389/fcell.2021.761469 . OpenUrl CrossRef PubMed 26. ↵ Moir , R.D. , Spann , T.P. , Lopez-Soler , R.I. , Yoon , M. , Goldman , A.E. , Khuon , S. , and Goldman , R.D . ( 2000 ). Review: The Dynamics of the Nuclear Lamins during the Cell Cycle— Relationship between Structure and Function . J. Struct. Biol . 129 , 324 – 334 . doi: 10.1006/jsbi.2000.4251 . OpenUrl CrossRef PubMed Web of Science 27. ↵ Naetar , N. , Ferraioli , S. , and Foisner , R . ( 2017 ). Lamins in the nuclear interior − life outside the lamina . J Cell Sci 130 , 2087 – 2096 . doi: 10.1242/jcs.203430 . OpenUrl Abstract / FREE Full Text 28. ↵ Naetar , N. , Georgiou , K. , Knapp , C. , Bronshtein , I. , Zier , E. , Fichtinger , P. , Dechat , T. , Garini , Y. , and Foisner , R . ( 2021 ). LAP2alpha maintains a mobile and low assembly state of A-type lamins in the nuclear interior . Elife 10 , e63476 . doi: 10.7554/elife.63476 . OpenUrl CrossRef PubMed 29. ↵ Liu , B. , Wang , Z. , Zhang , L. , Ghosh , S. , Zheng , H. , and Zhou , Z . ( 2013 ). Depleting the methyltransferase Suv39h1 improves DNA repair and extends lifespan in a progeria mouse model . Nat. Commun . 4 , 1868 . doi: 10.1038/ncomms2885 . OpenUrl CrossRef PubMed 30. ↵ Singh , M. , Hunt , C.R. , Pandita , R.K. , Kumar , R. , Yang , C.-R. , Horikoshi , N. , Bachoo , R. , Serag , S. , Story , M.D. , Shay , J.W. , et al. ( 2013 ). Lamin A/C depletion enhances DNA damage-induced stalled replication fork arrest . Mol Cell Biol 33 , 1210 – 1222 . doi: 10.1128/mcb.01676-12 . OpenUrl Abstract / FREE Full Text 31. ↵ Graziano , S. , Coll-Bonfill , N. , Teodoro-Castro , B. , Kuppa , S. , Jackson , J. , Shashkova , E. , Mahajan , U. , Vindigni , A. , Antony , E. , and Gonzalo , S . ( 2021 ). Lamin A/C recruits ssDNA protective proteins RPA and RAD51 to stalled replication forks to maintain fork stability . J Biological Chem 297 , 101301 . doi: 10.1016/j.jbc.2021.101301 . OpenUrl CrossRef PubMed 32. ↵ Bao , K. , Zhang , Q. , Liu , S. , Song , N. , Guo , Q. , Liu , L. , Tian , S. , Hao , J. , Zhu , Y. , Zhang , K. , et al. ( 2022 ). LAP2α preserves genome integrity through assisting RPA deposition on damaged chromatin . Genome Biol . 23 , 64 . doi: 10.1186/s13059-022-02638-6 . OpenUrl CrossRef PubMed 33. ↵ Mosler , T. , Baymaz , H.I. , Gräf , J.F. , Mikicic , I. , Blattner , G. , Bartlett , E. , Ostermaier , M. , Piccinno , R. , Yang , J. , Voigt , A. , et al. ( 2022 ). PARP1 proximity proteomics reveals interaction partners at stressed replication forks . Nucleic Acids Res . 50 , 11600 – 11618 . doi: 10.1093/nar/gkac948 . OpenUrl CrossRef PubMed 34. ↵ Vaitsiankova , A. , Burdova , K. , Sobol , M. , Gautam , A. , Benada , O. , Hanzlikova , H. , and Caldecott , K.W . ( 2022 ). PARP inhibition impedes the maturation of nascent DNA strands during DNA replication . Nat Struct Mol Biol 29 , 329 – 338 . doi: 10.1038/s41594-022-00747-1 . OpenUrl CrossRef PubMed 35. ↵ Hanzlikova , H. , Kalasova , I. , Demin , A.A. , Pennicott , L.E. , Cihlarova , Z. , and Caldecott , K.W . ( 2018 ). The Importance of Poly(ADP-Ribose) Polymerase as a Sensor of Unligated Okazaki Fragments during DNA Replication . Mol Cell 71 , 319 – 331 .e3. doi: 10.1016/j.molcel.2018.06.004 . OpenUrl CrossRef PubMed 36. ↵ Liu , W. , Saito , Y. , Jackson , J. , Bhowmick , R. , Kanemaki , M.T. , Vindigni , A. , and Cortez , D . ( 2023 ). RAD51 bypasses the CMG helicase to promote replication fork reversal . Science 380 , 382 – 387 . doi: 10.1126/science.add7328 . OpenUrl CrossRef PubMed 37. ↵ Yesbolatova , A. , Saito , Y. , Kitamoto , N. , Makino-Itou , H. , Ajima , R. , Nakano , R. , Nakaoka , H. , Fukui , K. , Gamo , K. , Tominari , Y. , et al. ( 2020 ). The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice . Nat Commun 11 , 5701 . doi: 10.1038/s41467-020-19532-z . OpenUrl CrossRef PubMed 38. ↵ Jackson , D.A. , and Pombo , A . ( 1998 ). Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells . J Cell Biol 140 , 1285 – 1295 . OpenUrl Abstract / FREE Full Text 39. ↵ Bai , G. , Kermi , C. , Stoy , H. , Schiltz , C.J. , Bacal , J. , Zaino , A.M. , Hadden , M.K. , Eichman , B.F. , Lopes , M. , and Cimprich , K.A . ( 2020 ). HLTF Promotes Fork Reversal, Limiting Replication Stress Resistance and Preventing Multiple Mechanisms of Unrestrained DNA Synthesis . Mol Cell . doi: 10.1016/j.molcel.2020.04.031 . OpenUrl CrossRef PubMed 40. Quinet , A. , Tirman , S. , Jackson , J. , Šviković , S. , Lemacon , D. , Carvajal-Maldonado , D. , González-Acosta , D. , Vessoni , A.T. , Cybulla , E. , Wood , M. , et al. ( 2020 ). PRIMPOL-Mediated Adaptive Response Suppresses Replication Fork Reversal in BRCA-Deficient Cells . Mol Cell 77 , 461 – 474 .e9. doi: 10.1016/j.molcel.2019.10.008 . OpenUrl CrossRef 41. ↵ Vallerga , M.B. , Mansilla , S.F. , Federico , M.B. , Bertolin , A.P. , and Gottifredi , V . ( 2015 ). Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation . Proc Natl Acad Sci U S A 112 , E6624 – 33 . doi: 10.1073/pnas.1508543112 . OpenUrl Abstract / FREE Full Text 42. ↵ Zellweger , R. , and Lopes , M . ( 2017 ). Genome Instability 2nd ed . M. Muzi-Falconi and G. W. Brown , eds. ( Springer New York ) doi: 10.1007/978-1-4939-7306-4_19 . OpenUrl CrossRef 43. ↵ Stoy , H. , Luethi , J. , Roessler , F.K. , Riemann , J. , Kaech , A. , and Lopes , M . ( 2022 ). R-Loops , Methods and Protocols. Methods Mol Biology 2528 , 1 – 20 . doi: 10.1007/978-1-0716-2477-7_1 . OpenUrl CrossRef 44. ↵ Weixler , L. , Ikenga , N.J. , Voorneveld , J. , Aydin , G. , Bolte , T.M. , Momoh , J. , Bütepage , M. , Golzmann , A. , Lüscher , B. , Filippov , D.V. , et al. ( 2022 ). Protein and RNA ADP-ribosylation detection is influenced by sample preparation and reagents used . Life Sci. Alliance 6 , e202201455 . doi: 10.26508/lsa.202201455 . OpenUrl Abstract / FREE Full Text 45. ↵ Nowak , K. , Rosenthal , F. , Karlberg , T. , Bütepage , M. , Thorsell , A.-G. , Dreier , B. , Grossmann , J. , Sobek , J. , Imhof , R. , Lüscher , B. , et al. ( 2020 ). Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins . Nat. Commun . 11 , 5199 . doi: 10.1038/s41467-020-18981-w . OpenUrl CrossRef PubMed 46. ↵ Bronshtein , I. , Kepten , E. , Kanter , I. , Berezin , S. , Lindner , M. , Redwood , A.B. , Mai , S. , Gonzalo , S. , Foisner , R. , Shav-Tal , Y. , et al. ( 2015 ). Loss of lamin A function increases chromatin dynamics in the nuclear interior . Nat. Commun . 6 , 8044 . doi: 10.1038/ncomms9044 . OpenUrl CrossRef PubMed 47. ↵ Wootton , J. , and Soutoglou , E . ( 2021 ). Chromatin and Nuclear Dynamics in the Maintenance of Replication Fork Integrity . Frontiers Genetics 12 , 773426 . doi: 10.3389/fgene.2021.773426 . OpenUrl CrossRef PubMed 48. ↵ Marjanović , M.P. , Crawford , K. , and Ahel , I . ( 2017 ). PARP, transcription and chromatin modeling . Semin. Cell Dev. Biol . 63 , 102 – 113 . doi: 10.1016/j.semcdb.2016.09.014 . OpenUrl CrossRef PubMed 49. ↵ Chappidi , N. , Quail , T. , Doll , S. , Vogel , L.T. , Aleksandrov , R. , Felekyan , S. , Kühnemuth , R. , Stoynov , S. , Seidel , C.A.M. , Brugués , J. , et al. ( 2024 ). PARP1-DNA co-condensation drives DNA repair site assembly to prevent disjunction of broken DNA ends . Cell 187 , 945 – 961 .e18. doi: 10.1016/j.cell.2024.01.015 . OpenUrl CrossRef PubMed 50. ↵ Shinkai , Y. , and Tachibana , M . ( 2011 ). H3K9 methyltransferase G9a and the related molecule GLP . Genes Dev . 25 , 781 – 788 . doi: 10.1101/gad.2027411 . OpenUrl Abstract / FREE Full Text 51. ↵ Cao , Y. , Sun , J. , Li , M. , Dong , Y. , Zhang , Y. , Yan , J. , Huang , R. , and Yan , X . ( 2019 ). Inhibition of G9a by a small molecule inhibitor, UNC0642, induces apoptosis of human bladder cancer cells . Acta Pharmacol. Sin . 40 , 1076 – 1084 . doi: 10.1038/s41401-018-0205-5 . OpenUrl CrossRef PubMed 52. ↵ Sobo , J.M. , Alagna , N.S. , Sun , S.X. , Wilson , K.L. , and Reddy , K.L . ( 2024 ). Lamins: The backbone of the nucleocytoskeleton interface . Curr. Opin. Cell Biol . 86 , 102313 . doi: 10.1016/j.ceb.2023.102313 . OpenUrl CrossRef PubMed 53. ↵ Whalen , J.M. , and Freudenreich , C.H . ( 2020 ). Location, Location, Location: The Role of Nuclear Positioning in the Repair of Collapsed Forks and Protection of Genome Stability . Genes 11 , 635 . doi: 10.3390/genes11060635 . OpenUrl CrossRef 54. ↵ Maya-Mendoza , A. , Moudry , P. , Merchut-Maya , J.M. , Lee , M. , Strauss , R. , and Bartek , J . ( 2018 ). High speed of fork progression induces DNA replication stress and genomic instability . Nature 559 , 279 – 284 . doi: 10.1038/s41586-018-0261-5 . OpenUrl CrossRef PubMed 55. ↵ Maynard , S. , Hall , A. , Galanos , P. , Rizza , S. , Yamamoto , T. , Gram , H.H. , Munk , S.H.N. , Shoaib , M. , Sørensen , C.S. , Bohr , V.A. , et al. ( 2022 ). Lamin A/C impairments cause mitochondrial dysfunction by attenuating PGC1α and the NAMPT-NAD+ pathway . Nucleic Acids Res . 50 , 9948 – 9965 . doi: 10.1093/nar/gkac741 . OpenUrl CrossRef PubMed 56. ↵ Vignier , N. , Chatzifrangkeskou , M. , Rodriguez , B.M. , Mericskay , M. , Mougenot , N. , Wahbi , K. , Bonne , G. , and Muchir , A . ( 2018 ). Rescue of biosynthesis of nicotinamide adenine dinucleotide protects the heart in cardiomyopathy caused by lamin A/C gene mutation . Hum. Mol. Genet . 27 , 3870 – 3880 . doi: 10.1093/hmg/ddy278 . OpenUrl CrossRef PubMed 57. ↵ Mattioli , E. , Andrenacci , D. , Garofalo , C. , Prencipe , S. , Scotlandi , K. , Remondini , D. , Gentilini , D. , Blasio , A.M.D. , Valente , S. , Scarano , E. , et al. ( 2018 ). Altered modulation of lamin A/C-HDAC2 interaction and p21 expression during oxidative stress response in HGPS . Aging Cell 17 , e12824 . doi: 10.1111/acel.12824 . OpenUrl CrossRef 58. ↵ Ghosh , S. , Liu , B. , Wang , Y. , Hao , Q. , and Zhou , Z . ( 2015 ). Lamin A Is an Endogenous SIRT6 Activator and Promotes SIRT6-Mediated DNA Repair . Cell Rep . 13 , 1396 – 1406 . doi: 10.1016/j.celrep.2015.10.006 . OpenUrl CrossRef PubMed 59. ↵ Schvartzman , J.-M. , Forsyth , G. , Walch , H. , Chatila , W. , Taglialatela , A. , Lee , B.J. , Zhu , X. , Gershik , S. , Cimino , F.V. , Santella , A. , et al. ( 2023 ). Oncogenic IDH mutations increase heterochromatin-related replication stress without impacting homologous recombination . Mol. Cell 83 , 2347 – 2356 .e8. doi: 10.1016/j.molcel.2023.05.026 . OpenUrl CrossRef PubMed 60. ↵ Zou , L . ( 2023 ). Heterochromatin-Dependent Replication Stress: A Lesson from IDH1/2 Mutants . Cancer Res . 83 , 2987 – 2989 . doi: 10.1158/0008-5472.can-23-2015 . OpenUrl CrossRef PubMed 61. ↵ Muthurajan , U.M. , Hepler , M.R.D. , Hieb , A.R. , Clark , N.J. , Kramer , M. , Yao , T. , and Luger , K . ( 2014 ). Automodification switches PARP-1 function from chromatin architectural protein to histone chaperone . Proc. Natl. Acad. Sci . 111 , 12752 – 12757 . doi: 10.1073/pnas.1405005111 . OpenUrl Abstract / FREE Full Text 62. ↵ Wacker , D.A. , Ruhl , D.D. , Balagamwala , E.H. , Hope , K.M. , Zhang , T. , and Kraus , W.L . ( 2007 ). The DNA Binding and Catalytic Domains of Poly(ADP-Ribose) Polymerase 1 Cooperate in the Regulation of Chromatin Structure and Transcription . Mol. Cell. Biol . 27 , 7475 – 7485 . doi: 10.1128/mcb.01314-07 . OpenUrl Abstract / FREE Full Text 63. ↵ Chou , D.M. , Adamson , B. , Dephoure , N.E. , Tan , X. , Nottke , A.C. , Hurov , K.E. , Gygi , S.P. , Colaiácovo , M.P. , and Elledge , S.J . ( 2010 ). A chromatin localization screen reveals poly (ADP ribose)-regulated recruitment of the repressive polycomb and NuRD complexes to sites of DNA damage . Proc. Natl. Acad. Sci . 107 , 18475 – 18480 . doi: 10.1073/pnas.1012946107 . OpenUrl Abstract / FREE Full Text 64. ↵ Ahel , D. , Hořejší , Z. , Wiechens , N. , Polo , S.E. , Garcia-Wilson , E. , Ahel , I. , Flynn , H. , Skehel , M. , West , S.C. , Jackson , S.P. , et al. ( 2009 ). Poly(ADP-ribose)–Dependent Regulation of DNA Repair by the Chromatin Remodeling Enzyme ALC1 . Science 325 , 1240 – 1243 . doi: 10.1126/science.1177321 . OpenUrl Abstract / FREE Full Text 65. ↵ Joo , Y.K. , Black , E.M. , Trier , I. , Haakma , W. , Zou , L. , and Kabeche , L . ( 2023 ). ATR promotes clearance of damaged DNA and damaged cells by rupturing micronuclei . Mol. Cell 83 , 3642 – 3658 .e4. doi: 10.1016/j.molcel.2023.09.003 . OpenUrl CrossRef 66. ↵ Kovacs , M.T. , Vallette , M. , Wiertsema , P. , Dingli , F. , Loew , D. , Nader , G.P. de F. , Piel , M. , and Ceccaldi , R. ( 2023 ). DNA damage induces nuclear envelope rupture through ATR-mediated phosphorylation of lamin A/C . Mol. Cell 83 , 3659 – 3668.e10 . doi: 10.1016/j.molcel.2023.09.023 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted January 18, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. 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