Cockayne syndrome group A and B proteins convergeon transcription-linked resolution of non-B DNAMorten Scheibye-Knudsena,b,1, Anne Tsengb, Martin Borch Jensenc, Karsten Scheibye-Alsinga, Evandro Fei Fangb,Teruaki Iyamab, Sanjay Kumar Bhartib, Krisztina Marosid, Lynn Froetscherb, Henok Kassahune, David Mark Eckleyg,Robert W. Maulh, Paul Bastiang, Supriyo Deg, Soumita Ghoshi, Hilde Nilsene,f, Ilya G. Goldbergg, Mark P. Mattsond,David M. Wilson IIIb, Robert M. Brosh Jr.b, Myriam Gorospeg, and Vilhelm A. Bohrb,1

aCenter for Healthy Aging, Department of Cellular andMolecular Medicine, University of Copenhagen, 2300 Copenhagen, Denmark; bLaboratory of MolecularGerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224; cBuck Institute for Research on Aging, Novato, CA 94945;dLaboratory of Neuroscience, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224; eInstitute of Clinical Medicine, 0167 Lorenskog,Norway; fUniversity of Oslo and Akershus University Hospital, 0167 Lorenskog, Norway; gLaboratory of Genetics and Genomics, National Institute on Aging,National Institutes of Health, Baltimore, MD 21224; hLaboratory of Molecular Biology and Immunology, National Institute on Aging, National Institutes ofHealth, Baltimore, MD 21224; and iLaboratory of Clinical Investigation, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224

Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved September 19, 2016 (received for review June 23, 2016)

Cockayne syndrome is a neurodegenerative accelerated aging disor-der caused by mutations in the CSA or CSB genes. Although thepathogenesis of Cockayne syndrome has remained elusive, recentwork implicates mitochondrial dysfunction in the disease progression.Here, we present evidence that loss of CSA or CSB in a neuroblastomacell line converges on mitochondrial dysfunction caused by defectsin ribosomal DNA transcription and activation of the DNA damagesensor poly-ADP ribose polymerase 1 (PARP1). Indeed, inhibition ofribosomal DNA transcription leads to mitochondrial dysfunction in anumber of cell lines. Furthermore, machine-learning algorithms pre-dict that diseases with defects in ribosomal DNA (rDNA) transcriptionhave mitochondrial dysfunction, and, accordingly, this is found whenfactors involved in rDNA transcription are knocked down. Mechanis-tically, loss of CSA or CSB leads to polymerase stalling at non-B DNAin a neuroblastoma cell line, in particular at G-quadruplex structures,and recombinant CSB can melt G-quadruplex structures. Indeed,stabilization of G-quadruplex structures activates PARP1 and leadsto accelerated aging in Caenorhabditis elegans. In conclusion, thiswork supports a role for impaired ribosomal DNA transcription inCockayne syndrome and suggests that transcription-coupled resolu-tion of secondary structures may be a mechanism to repress spuriousactivation of a DNA damage response.

Cockayne syndrome | aging | polymerase I transcription | nucleolus | CSA |CSB

Cockayne syndrome (CS) is an early onset accelerated agingdisorder characterized by growth retardation, progressive

neurodegeneration, and typically death in the second decade of life(1, 2). The disease is caused by mutations in either the ERCC8(CSA) or ERCC6 (CSB) genes, which encode proteins that arethought to be involved in DNA repair, transcription, and chromatinremodeling (3–5). Recent work has demonstrated mitochondrialdysfunction in cell and animal models deficient in CSB (6–10). Thedisease mechanism may involve persistent activation of a DNAdamage response orchestrated by the enzyme poly-ADP ribose-polymerase 1 (PARP1) leading to loss of NAD+ and increasedlactate production (8, 11), features that are also observed in normalaging (12–14). However, it is unclear why PARP1 is activated uponloss of CSB or in normal aging and whether persistent PARP1activation also occurs in CSA deficiency. Our results support thenotion that the mitochondrial changes observed in CSA or CSBdeficiency are caused by stalled ribosomal DNA (rDNA) tran-scription and subsequent PARP1 activation. Accordingly, we findthat diseases with defects in rDNA transcription have dysfunctionalmitochondria whereas classic ribosomopathies do not appear tohave this defect. rDNA is prone to form secondary structures, suchas G-quadruplexes (G4), and loss of CSA or CSB leads to tran-scriptional pausing at these DNA conformations. G4-inducedtranscriptional stalling is also found in vivo in humans. CSB is able

to melt G4 structures in a process that involves annealing the twoDNA strands. Furthermore, stabilization of G4 structures leads toPARP1 activation and accelerated aging in Caenorhabditis elegans,which can be rescued by replenishment of NAD+. Combined, ourfindings suggest a role for CS proteins in transcription and may explainwhy PARP1 might be activated in this premature aging disorder.

Loss of CSA or CSB Leads to Mitochondrial DysfunctionTo understand why a similar pathology is present in both com-plementation groups of CS, CSA and CSB, we generated stableknockdown of CSA or CSB in the neuroblastoma cell line SH-SY5Y using lentivirus (Fig. S1 A and B). In accordance with ourfindings for CSB (8), loss of CSA or CSB led to activation ofPARP1 and mitochondrial changes consisting of increased mito-chondrial membrane potential, superoxide production, and in-creased oxygen consumption rates (Fig. 1 A–D and Fig. S1C).Increased oxygen consumption rates were also found in C. elegansworms defective in csa-1 or csb-1 (Fig. 1E). Notably, microarrayanalysis showed very similar gene expression changes when eitherCSA or CSB were deficient (Fig. 1F and Dataset S1). ParametricAnalysis of Gene Set Enrichment showed significant up-regulationof Gene Ontology (GO) terms describing genes involved in ribo-some biogenesis and translation as well as in mitochondrial ATPproduction, likely reflecting the observed functional mitochondrialchanges (Fig. 1F). Down-regulated ontology terms included ubiq-uitin pathways and transcriptional regulation (Fig. 1F).

Significance

In this paper we describe a possible pathogenesis for theaccelerated aging disease Cockayne syndrome that entailsdefective transcription through DNA secondary structuresleading to activation of the DNA damage response enzymepoly-ADP-ribose polymerase 1 and downstream mitochondrialderangement. These findings are important because they signifya possible new role of transcription in the resolution of DNAstructures that form spontaneously and suggest a possiblepathogenesis for this accelerated aging disease.

Author contributions: M.S.-K., M.B.J., H.N., M.P.M., D.M.W., R.M.B., M.G., and V.A.B. designedresearch; M.S.-K., A.T., M.B.J., K.S.-A., E.F.F., T.I., S.K.B., K.M., L.F., H.K., D.M.E., R.W.M., P.B.,S.D., S.G., and I.G.G. performed research; M.S.-K., T.I., and V.A.B. contributed new reagents/analytic tools; M.S.-K., A.T., K.S.-A., E.F.F., T.I., S.K.B., K.M., L.F., H.K., D.M.E., R.W.M., P.B., S.D.,S.G., H.N., I.G.G., R.M.B., and M.G. analyzed data; and M.S.-K., L.F., M.P.M., D.M.W., and V.A.B.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence may be addressed. Email: mscheibye@sund.ku.dk or vbohr@nih.gov.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1610198113/-/DCSupplemental.

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Given that CSA and CSB have been implicated in transcriptiondriven by RNA polymerases I (15, 16), II (17), and III (18), wemeasured gene expression changes in unmodified SH-SY5Y cellsafter treatment with specific transcriptional inhibitors comparedwith controls (RNA polymerase I: CX5461; RNA polymerase I/II:triptolide; RNA polymerase II: α-amanitin; RNA polymerase I/II/III: actinomycin D; and RNA polymerase III: ML60218). Wetreated the cells with the inhibitors at several concentrations toavoid data bias that might occur when choosing only one con-centration. Additionally, we treated the knockdown cells with thePARP inhibitor PJ34. To validate the results, we included geneexpression array data from the cerebellum of human CS patientsand their controls from a recently published study (19). Notably,hierarchical clustering showed an association between the CS pa-tients and the transcription inhibitor treatments, despite batch andtissue differences. Intriguingly, clustering revealed close associa-tion between the loss of CSA or CSB and the inhibition of rDNAtranscription, and these changes were completely rescued byPARP inhibition (Fig. 1G). As a positive control, very low con-centrations of ML60218 (1 μM) or CX5461 (0.001 μM) led to veryfew significantly changed GO terms (Fig. 1G). Indeed, there wasno association between these two treatments and CSA or CSBknockdown in the hierarchical clustering (Fig. 1G). In summary,

these data suggest that CSA and CSB deficiency leads to decreasedrDNA transcription.To address this hypothesis, rDNA transcription was measured

and found to be lower in short hairpin (sh)CSA and shCSB celllines, as demonstrated by the loss of 47S pre-rRNA by RT-qPCRas well as by loss of nucleolar ethynyl uridine (EU) incorporation(Fig. 1 H and I and Fig. S1D). Loss of rDNA transcription was alsofound in fibroblasts from CSA- and from CSB-deficient patientscompared with isogenic controls transfected with wild-type (WT)CSA or CSB (Fig. S1 E–G). Because microarray analysis showedup-regulation of genes involved in translation and mitochondrialpathways, we postulate that this outcome reflects compensationdue to loss of rDNA transcription. Indeed, inhibition of rDNAtranscription using CX5461 led to a significant increase in mito-chondrial membrane potential, content (by mitotracker greenstaining), and superoxide production (Fig. 1J and Fig. S1 H and I).Furthermore, there was no additive effect between loss of CSA orCSB and the CX5461 treatment, suggesting that the mitochondrialsignaling comes from the same pathway (Fig. 1J and Fig. S1 Hand I). In summary, loss of rDNA transcription may be drivingmitochondrial changes in CS.

Loss of rDNA Transcription Leads to MitochondrialDysfunctionWe next asked whether inhibition of transcription produced amitochondrial response across different cell lines. We treated SH-SY5Y, HeLa (WT and PARP1 null), HEK293T, WI38, primaryMEFs, U2OS, and HCT116 (WT and p53 null) with differentconcentrations of inhibitors of RNA polymerase I (CX-5461 andtriptolide), II (α-amanitin and triptolide), or III (ML-60218) andmeasured mitochondrial changes. Interestingly, RNA polymerase Iinhibition led to a mitochondrial phenotype similar to what is seenin CS, with significantly increased mitochondrial membrane po-tential, content, and mitochondrial superoxide production in allcell lines tested (Fig. 2A; see Fig. S2A for raw values of control celllines). Notably, the effect of RNA polymerase I inhibition on mi-tochondrial function did not appear to be dependent on p53 be-cause HCT116 WT cells overall showed less of a response to theinhibitors than the p53−/− cells. Immortalization did not appear toaffect the response either. However, HeLa cells where PARP1 wasdeleted (PARP1−/−) showed a significantly attenuated response toRNA polymerase I inhibition compared with the parental HeLacell line (Fig. 2A). To further test whether these changes dependedon PARP1 activation, we treated HeLa cells with RNA polymeraseI inhibitor alone or in combination with the PARP1 inhibitor PJ34.As shown, the mitochondrial changes that are caused by inhibitionof RNA polymerase I were completely reversed by treatment withPJ34 (Fig. 2B and Fig. S2 B–D). To test the effect of the inhibitorsat an organismal level independent of CS, C. elegans were treatedwith RNA polymerase I, II, or III inhibitors and examined formitochondrial changes. In agreement with the cellular data, RNApolymerase inhibitors increased oxygen consumption rates, withRNA polymerase I inhibition having the greatest effect (Fig. 2C).Thus, there appears to be a direct link between transcriptionalefficiency, particularly of rDNA, and mitochondrial function.

Ribosomal Defects Do Not Cause Mitochondrial DysfunctionA number of diseases have been described with defects in rDNAtranscription [spinocerebellar ataxia 17 (SCA17) and hypomyeli-nating leukodystrophy 7 (HLD7)], pre-rRNA processing [NorthAmerican Indian Childhood Cirrhosis (NAIC) and TreacherColins syndrome (TCOF)], and ribosome assembly [Diamond-Blackfan anemia (DBA)] (20–25). To understand the mecha-nism of how transcriptional defects generate mitochondrialchanges, we used our recently developed in silico algorithms topredict mitochondrial involvement in the above-listed disordersbased on clinical features (11, 26). Considering the clinical spectrumof these diseases, hierarchical clustering showed an association

Fig. 1. Loss of rDNA transcription leads to mitochondrial dysfunction inmodels of Cockayne syndrome. (A) Western blots of SH-SY5Y cells subjectedto stable knockdown of CSA (shCSA) and CSB (shCSB). (Magnification: 60×.)(B) Mitochondrial membrane potential in the SH-SY5Y cells measured usingtetramethylrhodamine methyl ester (TMRM) (mean ± SEM, n = 5). (C) Mi-tochondrial superoxide production (mitosox fluorescence) in the SH-SY5Ycells (mean ± SEM, n = 5). (D) OCR in SH-SY5Y cells (mean ± SEM, n = 3).(E) OCR in WT (N2), csa-1–, and csb-1–deficient worms (mean ± SEM, n = 3).(F and G) Altered GO terms, hierarchical clustering, and heat map of alteredGO terms in SH-SY5Y cells after knockdown of CSA or CSB or indicatedtreatmets. (H) Quantitative PCR (qPCR) of 47S rRNA levels in SH-SY5Y cells.(I) Confocal microscope images of the SH-SY5Y cells after short-term treat-ment with EU and staining with antibody against nucleolin. (J) Mitochon-drial superoxide production in the SH-SY5Y cells treated with 1 μM CX5461or vehicle (veh) (mean ± SEM, n = 3–5).

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between defects in rDNA transcription (SCA17 and HLD7) andmitochondrial disorders, whereas NAIC, TCOF, and DBA did notshow this relationship (Fig. 2D). A network algorithm corrobo-rated these findings where close associations were observedbetween SCA17 and HLD7 and mitochondrial diseases (seeinteractive figure on www.mitodb.com/network.html or Fig. S3).Furthermore, a machine-learning algorithm and a mitochondrial-scoring algorithm predicted mitochondrial involvement in SCA17and HLD7, but not in NAIC, TCOF, or DBA (Fig. 2 E and F).Indeed, siRNA knockdown of TBP (mutated in SCA17) orPOLR3A (mutated in HLD7) led to changes of mitochondrialmembrane potential similar to those observed in CSA and CSBknockdown cells, whereas knockdown of CIRH1A (mutated inNAIC), TCOF1 (mutated in TCOF), or Rps19 (mutated in DBA)did not result in mitochondrial membrane potential alterations(Fig. 2G). Indeed, TCOF1 has recently been shown to be down-stream of rDNA transcription (24) as well as of PARP1 re-cruitment to rDNA (27). Notably, the mitochondrial membranepotential changes associated with TBP or POLR3A knockdownwere not observed in PARP1-null cells (Fig. 2G). In support of theobservation that general translation defects did not lead to mito-chondrial changes, inhibition of translation by treatment with

cycloheximide did not result in a mitochondrial phenotype (Fig.S2 E and F). Likewise, only modest changes in translation rateswere seen by measuring incorporation of 35S-labeled methio-nine and cysteine and by assessing polysome profiles in CSA- orCSB-deficient SH-SY5Y cells as well as in SH-SY5Y cellstreated with transcriptional inhibitors (Fig. S2 G and H). Insummary, loss of rDNA transcription, but not translation, leadsto mitochondrial dysfunction in Cockayne syndrome, and amitochondrial etiology mediated by PARP1 may underlie thepathology observed in SCA17 and HLD7.

Loss of CSA or CSB Leads to Transcriptional Stalling at Non-BDNABecause inhibition or loss of PARP1 attenuated the mitochondrialphenotype caused by defective rDNA transcription, we asked ifPARP1 might be activated in the nucleolus of cells with reducedCSA or CSB. Using immunocytochemistry, we found increasedlevels of poly-ADP ribose (PAR) in the nucleolus (marked bynucleolin) of cells deficient in CSA or CSB (Fig. 3A). Notably,nucleolin levels appeared to be increased in the CSA- or CSB-deficient cells (Fig. 3A). Nucleolin binds DNA secondary structuressuch as G4 structures (28), and these structures have been pro-posed to activate PARP1 (29, 30). We therefore asked if CSA- or

Fig. 2. Loss of rDNA transcription and not ribosome dysfunction leadsto mitochondrial dysfunction. (A) Heatmap of changes in mitochondrialmembrane potential (TMRM), mitochondrial content (mitotracker green),mitochondrial superoxide production (mitosox), and whole-cell superoxideproduction [dihydroethedium (DHE)] (each square represents the mean ofthree independent experiments normalized to vehicle). (B) Mitochondrialmembrane potential in HeLa cells treated for 24 h with vehicle (veh), 1 μMCX5461, 10 μM PJ34, or 1 μM CX5461 and 10 μM PJ34 (mean ± SEM, n = 3–6).(C) OCR in worms treated with CX5461, ML60218, and α-amanitin for 24 h(mean ± SEM, n = 3). (D) Hierarchical clustering of diseases based on theirclinical parameters (red: mitochondrial diseases; green: nonmitochondrialdiseases; blue: diseases with transcription defects; purple: classic ribosomo-pathies). (E and F) Mitochondrial and support vector machine scoring forlikely mitochondrial involvement. (G) Mitochondrial membrane potential inWT or PARP1−/− HeLa cells subjected to knockdown of the indicated genes.

Fig. 3. CSA and CSB mediate transcription through secondary DNA struc-tures. (A) Representative immunocytochemistry micrograph of cells stainedwith antibody against poly-ADP ribose or nucleolin and quantification of thedata (mean ± SEM, n = 8–15). (B) Change in coverage in the vicinity of a G4structure. Data represent the mean of ∼10,000 transcripts. (C) Representa-tive Sybr gold-stained gel of recombinant CSB incubated with G4 DNA andcomplementary oligo (rc) with a 3′ prime or 5′ prime overhang. (Magnifi-cation: 60×.) (D) Quantification of C (mean ± SEM, n = 3). (E) Quantified flowcytometry data of CS3BE cells stably expressing GFP-tagged CSA, CSB, nu-cleolar targeted CSB (NOLS-CSB), or GFP and stained with mitosox (mean ±SEM, n = 3). (F) OCR in CS3BE cells stably expressing GFP, GFP-CSA. GFP-CSB,or GFP-tagged nucleolar targeted CSB (mean ± SEM, n = 3–5). (G) ClonogenicUV-survival of cells described in E (mean ± SD, n = 3).

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CSB-deficient SH-SY5Y cells might show defects in transcriptionthrough G4 DNA sequences or other DNA secondary structures.Toward this aim, we performed RNA sequencing (RNA-seq) todetermine whether certain DNA regions might influence thetranscription efficiency in CSA- or CSB-deficient cells. Alignedreads were normalized to the control to minimize possible se-quencing artifacts. After alignment, we developed a custom scriptto look at the normalized RNA-seq coverage in the 400 nucleotidespreceding and following predicted DNA secondary structures inthe genome. Inverted-, direct-, or mirror repeats, z-DNA se-quences, or short tandem repeats only led to minor pausing of theRNA polymerase in the CSA- or CSB-deficient cells, and therewas, as expected, no strand bias, whereas A-phased repeats in thenontranscribed strand led to moderate stalling (Fig. S4). However,the strongest stalling of the polymerase was seen with G4-formingsequences as evidenced by increased coverage approaching the G4structure. Loss of coverage was observed in the nucleotides aroundthe G4 structure, likely representing alignment artifacts due to theG-rich sequence. Interestingly, polymerase stalling displayed strandbias in the CSA- or CSB-deficient cells, with particularly prominentstalling occurring when the G4 is in the transcribed strand (Fig.3B). Furthermore, the amount of stalling depended on the amountof guanines in the sequence and thus the likelihood of the for-mation of a G4 (Fig. 3B). Notably, there was no loss of signaldownstream of the structures, suggesting that the polymeraseeventually makes it through.Obstructive DNA structures might lead to less transcription in

the distal compared with proximal part of genes. To test this, weperformed nuclear run-on experiments on a few genes that showedaltered expression in the microarray experiment. Although loss oftranscription elongation was found in CSA- or CSB-deficient SH-SY5Y cells as previously reported (4, 17), there was no overall lossof transcription when we compared the signal from the 5′ end withthe 3′ end of the genes (Fig. S5 A and B). Furthermore, the RNA-seq data did not show loss of transcription in CSA- or CSB-deficientcells relative to the control when investigating loss of coverage as afunction of gene size across the entire transcriptome (Fig. S5C). Inaddition, we did not find a generalized change in mitochondrialtranscription rates in CSA- or CSB-deficient cells that could explainalterations in mitochondrial function (Fig. S5D).We next studied whether changes in transcription through G4

quadruplex structures are involved in normal aging by analyzing apublished RNA-seq dataset of cells harvested from the iliac crest ofyoung and elderly women (Gene Expression Omnibus accessionno. GSE72815). Although we found strand bias and transcriptionalpausing at G4 sequences, we did not find increased pausing withaging (Fig. S6A). However, we did find increased variability intranscriptional rates with age around G4 regions, perhaps sug-gesting alterations in the way G4 structures are handled (Fig. S6A).If G4 structures have to be resolved for transcription to occur, onewould expect that regions that show less transcription would havemore stalling compared with highly transcribed regions. Indeed,that appears to be the case in vivo when we measured the amountof stalling as a function of coverage in the G4-containing region.Here we found that regions with low coverage have greater stallingthan regions with high coverage, and this effect is only seen whenthe G4 is on the transcribed strand (Fig. S6B). In summary, tran-scriptional stalling occurs at DNA secondary structures, and thereappears to be transcription-coupled resolution of DNA secondarystructures involving CSA and CSB.

CSB Is Able to Resolve G4 StructuresBecause CSB contains a helicase domain and is able to annealcDNA strands, we asked whether recombinant CSB can resolve G4structures and thereby facilitate transcription through them. Wefound that CSB is unable to unwind bimolecular and tetramo-lecular G4s (Fig. S7 A–C). However, unimolecular G4s that con-tained either a 3′ or a 5′ oligonucleotide tail were efficiently melted

by CSB in the presence of cDNA, leading to annealing of thetwo oligonucleotides (Fig. 3 C and D and Fig. S7D). This ac-tivity was independent of ATP hydrolysis because the reactionoccurred equally efficiently with or without ATP. The anneal-ing activity of CSB was contingent upon either a 3′ or a 5′ tailbecause CSB was unable to anneal the G4 structure with apeptide nucleic acid complementary to only the G4 sequence.FANCJ, a known G4 DNA helicase, however, was able to re-solve this structure (Fig. S7E). Because CSB appeared to beindependently able to melt G4 structures, we asked if we couldrescue mitochondrial features in CSA-deficient CS3BE cellsby overexpressing CSB targeted to the nucleolus (Fig. S8A).Indeed, that appeared to be the case, as assessed by mito-chondrial membrane potential, ROS production, and oxygenconsumption rates (Fig. 3 E and F and Fig. S8B). Interestingly,only CSA overexpression and not CSB was able to rescue UVsensitivity in CS3BE cells, suggesting a decoupling between UVsensitivity and mitochondrial phenotypes as recently also sug-gested from clinical data (31) (Fig. 3G).

Stabilization of G4 Structures Leads to Accelerated AgingWe next asked if rDNA G4 structures could activate PARP1. In-deed, recombinant PARP1 was activated by single-stranded rDNAand rRNA that contained G4-forming sequences whereas thecomplementary controls did not activate PARP1 (Fig. S9A). Toavoid the confounding effects of G4 stabilization on replication, weperformed a number of experiments on primary, fully differenti-ated, nondividing rat cortical neurons. Treatment of these cellswith the G4-stabilizing drug pyridostatin or the RNA polymerase Iinhibitor CX5461 led to PARP1 activation (Fig. 4A). Notably,treatment with these drugs did not lead to histone H2AX phos-phorylation, suggesting that strand breaks are not driving the ac-tivation of PARP1 (Fig. 4A).PARP1 activation may lead to loss of NAD+ and increased

nicotinamide (NAM). We therefore measured the levels of thesemetabolites in the neurons treated with CX5461 or pyridostatin andfound a significant decrease in NAD+/NADH and a trend to in-creased NAM (Fig. 4 B andC). Loss of NAD+ will lead to increasedshunting of pyruvate to lactate. Consistently, by investigating oxygenconsumption (OCR) and extracellular acidification (ECAR), wefound loss of OCR/ECAR ratios in primary neurons treated withCX5461, pyridostatin, or another G4 stabilizer, TMPYP4, and thesechanges were rescued by treatment with the PARP1 inhibitor PJ34(Fig. 4D and raw data in Fig. S9 B and C). In addition, treatment ofneurons with pyridostatin led to a dose-dependent increase in mi-tochondrial membrane potential (Fig. S9D). Furthermore, treat-ment of HeLa cells with pyridostatin or CX5461 led to a similarmitochondrial profile as observed in CSA- or CSB-deficient cells,and this effect was nonadditive (Fig. 4E and Fig. S9 E–G). In ad-dition, pyridostatin treatment led to loss of rDNA transcription asevidenced by decreased levels of 47S rRNA (Fig. 4F).To further understand if defects in transcription through DNA

secondary structures could lead to accelerated aging, we treatedC. elegans (the somatic cells of which are all postmitotic) withpyridostatin as well as with the rDNA transcriptional inhibitorCX-5461. These treatments led to decreased pharyngeal pumping,loss of mobility, and shortened life span, all hallmarks ofaccelerated aging (Fig. 4 G–I). Accelerated aging was also foundwhen using a machine classifier to quantify morphological changesthat occur with aging in the head of nematodes (32–34) (Fig. 4J).Strikingly, the accelerated aging changes could be rescued bytreatment with the NAD+-precursor nicotinamide riboside (NR),although at higher pyridostatin or CX5461 concentrations life-span shortening could not be rescued (Fig. 4 G–J and Fig. S9H).In summary, our results suggest that loss of transcription-coupledresolution of DNA secondary structures leads to accelerated agingthrough G4 formation, PARP1 activation, NAD+ depletion, andmitochondrial dysfunction.

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DiscussionPARP1 activity increases with normal aging and in acceleratedaging diseases, such as xeroderma pigmentosum group A, ATM,and Cockayne syndrome (8, 11). We present evidence that PARP1activation in Cockayne syndrome is caused by stalled transcriptionat DNA secondary structures that are enriched in rDNA. Using insilico and in vivo techniques, we find that transcription per serather than downstream translational defects lead to mitochondrialpathology, a conclusion partly based on the discovery of mito-chondrial dysfunction in the diseases hypomyelinating leukodys-trophy 7 and spinocerebellar ataxia 17. Interestingly, the observedmitochondrial changes with increased mitochondrial membranepotential and oxygen consumption suggest that the changes are notcaused by primary mitochondrial dysfunction but rather by a sec-ondary compensatory response due to nuclear changes, as pro-posed previously (35, 36). This is in agreement with a number ofprevious studies showing similar mitochondrial changes as a re-sponse to nuclear DNA damage whereas studies in nonisogeniccell lines in the context of Cockayne syndrome have been in-conclusive (7, 10, 37–41). Notably, G4-forming DNA sequences,which are abundant in rDNA, appear to be potent obstacles totranscription in CSA- or CSB-deficient cells, and these structures

activate PARP1 in vitro and in vivo. Recombinant CSB is able toresolve G4-forming DNA structures through an ATP-independentDNA-annealing event, supporting the hypothesis that an accu-mulation of irresolvable G4 structures in Cockayne syndrome un-derlies the PARP1 activation in this disease. Finally, acceleratedaging caused by G4 stabilization in nematodes can be rescuedthrough replenishment of the central bioenergetics moleculeNAD+. Although we believe that the most likely explanation forPARP1 activation is through direct activation by G4 structures,alternatively, PARP1 activation could occur through signaling fromposttranslational modification of the stalled RNA polymerase orfrom other chromatin-associated proteins. Combined, our findingssuggest an explanation for the PARP1 activation and mitochon-drial alterations observed in Cockayne syndrome (Fig. S9I).Previous findings implicate both CSA and CSB in rDNA

transcription. Our data support this hypothesis and furtherunderscore the role of rDNA in the aging process (15, 16, 18).Indeed, an expansion of extrachromosomal rDNA circles(ERCs) were among the first proposed causes of aging in eu-karyotes (42). Although ERC accumulation appears to be anage-associated phenomenon particular to yeast, recent datahave shown increased rDNA content in the brains of patientssuffering from of Alzheimer’s disease or Lewy body dementia(43, 44). Notably, PARP1 is activated in these diseases (45, 46).Perhaps age-associated PARP1 activation and NAD+ depletioncould be caused by rDNA accumulation leading to loss of activityof NAD+-dependent enzymes, such as sirtuins, and consequentmitochondrial dysfunction.G4 structures have been implicated in both replication and

transcription regulation (47–50). Here we provide in vivo evidencefor transcriptional pausing at DNA sequences that can form G4structures. In Cockayne syndrome cells, this pausing is exacerbated,leading to mitochondrial dysfunction. Although our study showsthat CSB can biochemically resolve G4 structures, the role of CSAin this process is unknown. CSA is part of the DDB1-Cul4-Rbxcomplex that is involved in ubiquitination of various substrates. Itis possible that CSA ubiquitinates proteins involved in the sta-bilization or resolution of G4 structures. Loss of CSA could thuslead to accumulation of G4 structures and subsequent PARP1activation.G4 structures occur at a high frequency in G-rich regions

such as the rDNA loci; however, this particular DNA structureis also observed in a number of other regions in the genome. Itis possible that the effect that we see when inhibiting RNApolymerase I is due to the large amount of G4 structures in thisregion, and it is also possible that there are other propertiesinherently different about the rDNA. Indeed, inhibition ofRNA polymerase II also leads to changes in mitochondrialfunction although these changes are much smaller than ob-served after RNA polymerase I inhibition. Notably, althoughwe find transcriptional pausing at G4 structures in cells fromnormal humans, aging does not exacerbate this pausing. Thisobservation suggests that a possible PARP1 activation in nor-mal aging does not depend on decreased transcription-coupledresolution of DNA secondary structures.In conclusion, this work represents a mechanistic explanation

for the pathogenesis of Cockayne syndrome through a con-vergence of the CSA and CSB pathways and describes a role oftranscription-coupled resolution of secondary DNA structures.

Materials and MethodsAll cell culture and nematode work was performed according to standardprocedures. Western blot, RT-PCR, flow cytometry, Seahorse XF analyses, andnuclear run-ons were performed according to standard procedures. In silicoanalyses of diseases was done as previously described (26) using the prevalenceof clinical features in each disease. RNA-seq was performed by the NationalCancer Institute sequencing core. DNA helicase and DNA-annealing assayswere done with gel-purified oligonucleotides purchased from Lofstrand Labs.

Fig. 4. G4 structure stabilization impairs neuronal bioenergetics and acceler-ates aging. (A) Western blot of primary rat cortical neurons treated with 5 μg/mLα-amanitin, 1 μM CX5461, 1 μM pyridostatin, 10 μM PJ34, or 20 μM menadionefor 2 h. (Magnification: 60×.) (B) NAD+/NADH in primary rat cortical neuronstreated with 1 μM pyridostatin or CX5461 for 2 h (mean ± SEM, n = 3). (C) NAMlevels in primary rat cortical neurons treated with 1 μM pyridostatin or CX5461for 2 h (mean ± SEM, n = 2–3). (D) OCR/ECAR in primary neurons treated with G4stabilizers, pyridostatin and TMPYP4, and RNA polymerase I inhibitor CX5461in combination with the PARP1 inhibitor PJ34 (mean ± SEM, n = 3). (E) Mito-chondrial membrane potential in HeLa cells treated with 1 μMpyridostatin, 1 μMCX5461, or 1 μM pyridostain and 1 μM CX5461 for 24 h (mean ± SEM, n = 3–6).(F) qPCR of 47S rRNA in SH-SY5Y cells treated with 1 μM pyridostatin for 24 h(mean ± SEM, n = 3). (G) Pharyngeal pumpting rates of nematodes treated withpyridostatin, CX5461, and/or NR. (H) Swimming ability of nematodes treated asin f. (I) Life-span measurements of nematodes treated as in F. (J) Age-state scorefor pharynx terminal bulb of nematodes treated as in F.

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One-way ANOVA with Tukey’s posttest was used to determine significantdifference across multiple samples. Two-tailed t tests were used to comparesingle groups. Statistical analyses were done with GraphPad Prism (GraphPadSoftware, Inc.) or R. For detailed materials and methods, see SI Materialsand Methods).

ACKNOWLEDGMENTS. We thank Jennifer L. Martindale and Dr. Ruin Moaddelfor technical assistance and Elayne Fivenson and Mustafa Okur for feedback onthe manuscript. H.N. is supported by funding from the South East RegionalHealth Authority. This research was supported by the Intramural Research Pro-gram of the NIH, National Institute on Aging.

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