Resolution of Sister TelomereAssociation Is Required forProgression Through Mitosis

Jasmin N. Dynek and Susan Smith*

Cohesins keep sister chromatids associated from the time of their replicationin S phase until the onset of anaphase. In vertebrate cells, two distinct pathwaysdissociate cohesins, one acts on chromosome arms and the other on centro-meres. Here, we describe a third pathway that acts on telomeres. Knockdownof tankyrase 1, a telomeric poly(ADP-ribose) polymerase caused mitotic arrest.Chromosomes aligned normally on the metaphase plate but were unable tosegregate. Sister chromatids separated at centromeres and arms but remainedassociated at telomeres, apparently through proteinaceous bridges. Thus, telo-meres may require a unique tankyrase 1dependent mechanism for sisterchromatid resolution before anaphase.

Telomere length is regulated by theTTAGGG repeat binding protein TRF1 (1)and its associated factor tankyrase 1 (2), amember of the poly(ADP-ribose) polymer-ase (PARP) enzyme family (3). Tankyrase1 localizes to human telomeres (4 ), as wellother subcellular sites (5, 6 ). Tankyrase 1contains five discrete TRF1-binding sites(7 ) and controls TRF1 binding to telomericDNA through ADP-ribosylation (4 ). Whentankyrase 1 is overexpressed in humancells, TRF1 is released from telomeres anddegraded by the proteasome (8). Long-termoverexpression of tankyrase 1 induces

telomere lengthening in a reaction thatdepends on its catalytic PARP activity andon telomerase (2, 8, 9).

To inhibit tankyrase 1 expression, we trans-fected tankyrase 1 small interfering RNA(siRNA) duplexes (10) into asynchronouslygrowing HeLaI.2.11 cells (11). Tankyrase 1protein was lost at the earliest time point (24hours) and throughout the time course (Fig.1A). A similar knockdown was achieved with adifferent tankyrase 1 siRNA duplex (fig. S1).Cells treated with tankyrase 1 siRNA exhibiteda dramatic mitotic arrest, as measured by im-munofluorescence (Fig. 1B and fig. S2) and

fluorescence-activated cell sorting (FACS)analysis, which indicated a threefold increase inthe G2/M population (Fig. 1C). This phenotypewas observed with two different tankyrase 1siRNA duplexes, and it was observed in cellsindependent of their telomere length or telo-merase status (table S1).

Closer examination of the cells arrestedin mitosis indicated a predominance of ab-normal mitotic figures, which fell into threedistinct classes (Fig. 1D), each peaking innumber at a different point in the tankyrase1 siRNA time course (fig. S3). The earliestphenotype, fat, displayed a broad 4,6-diamidino-2-phenylindole (DAPI) stain onthe metaphase plate (Fig. 1D, b). The sec-ond had misaligned chromosomes (Fig.1D, c). And the third and terminal pheno-type, aberrant displayed a deterioratedspindle and a condensed and disorderedDAPI stain (Fig. 1D, d). Aberrant cells didnot reenter the cell cycle (fig. S4A) andwere not apoptotic (fig. S4B).

To determine whether tankyrase 1 ex-pression could rescue the abnormal mitoticphenotype, we cotransfected cells withtankyrase 1 siRNA and with wild-type(WT) or PARP-dead (HE/A) (9) tankyrase1 plasmids containing a single-base mis-match to the siRNA oligonucleotide, whichrendered them resistant to the siRNA-mediated knockdown (fig. S5). Analysis ofthe population of mitotic cells expressingtankyrase 1 indicated that wild-type (butnot PARP-dead) tankyrase 1 efficiently res-

Fig. 1. Knockdown of tankyrase 1 expressionresults in mitotic arrest. (A) Immunoblot anal-ysis of whole-cell extracts from HeLaI.2.11 cellstransfected without (mock) or with (TNKS1)tankyrase 1 siRNA. (B) Histogram showing thepercentage of cells in interphase or mitosisafter transfection with tankyrase 1 siRNA.About 1000 cells were scored by immunouo-rescence for each time point. (C) FACS analysisof HeLaI.2.11 cells 48 hours after transfectionwith tankyrase 1 siRNA. y axis, cell number. (D)Immunouorescence analysis of methanol-xed HeLaI.2.11 cells stained with DAPI (blue)and -tubulin antibody (green) after treatmentwith tankyrase 1 siRNA indicates a progressionof three classes of abnormal mitotic cells. Scalebar, 2 m. (E) Tankyrase 1 siRNA cells werecotransfected with siRNA-resistant tankyrase 1wild-type (WT) or HE/A plasmids. Histogramshows the ratio of normal to abnormaltankyrase 1expressing mitotic cells scored byimmunouorescence as described in table S2.(F and G) Immunouorescence of methanol-xed HeLaI.2.11 cells transfected withtankyrase 1 siRNA showing (F) measurementsof the DNA (DAPI) and spindle ( tubulin) or(G) centromere disposition by staining withDAPI (blue), -tubulin antibody (red), and an-tibody against centromeres (ACA) (green).Scale bar, 2 m.

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cued the abnormal mitotic phenotype (Fig.1E and table S2).

To gain insight into the mechanism ofthe arrest induced by tankyrase 1 siRNAtreatment, we focused on the earliest phe-notypes. Measurements of the fat andmisaligned metaphases indicated that theDAPI staining was much broader and thespindle much shorter than a wild-typemetaphase (Fig. 1F and fig. S6) and moreresembled an early anaphase figure. Con-sistent with anaphase entry, centromeresfrequently appeared separated (Fig. 1G, band c), which suggested that chromosomesaligned on the metaphase plate and under-went centromere separation, but then wereunable to proceed with anaphase.

Time-lapse analysis of mitosis in amock-transfected cell and a cell treatedwith tankyrase 1 siRNA showed that, inboth cases, chromosomes congressed nor-mally and by 38 min were aligned on themetaphase plate. However, although themock-transfected cell continued to progressthrough mitosis, the tankyrase 1 siRNA cellremained arrested in metaphase (Fig. 2A;Movies S1 and S2). The period of meta-phase arrest in tankyrase 1 siRNA cellsvaried. For example, one cell (Fig. 2B,arrowhead) remained in metaphase from 24to 188 min (164 min), whereas two otherflanking cells (Fig. 2B, arrows) remainedarrested in metaphase for the duration ofimaging (260 min) (Movie S3).

Progression through mitosis requiresubiquitin-mediated proteolysis of a numberof regulatory proteins by the anaphase-promoting complex (APC) (12). The APCsubstrates, cyclins A and B, were degraded infat mitotic figures (Fig. 2, C and D), whichindicated that the APC was active. Separationof sister chromatids also depends on theAPC, where the final, irreversible step iscleavage of the SCC1 cohesin subunit (13).The SCC1 subunit was cleaved in tankyrase 1siRNA mitotic cells (Fig. 2E). Thus, the ar-rest in mitosis was not due to a defect in theAPC. Finally, a negative immunostain for thehistone -H2AX (fig. S7) indicated that thearrest in mitosis was not due to DNA damage.

Cells transfected with tankyrase 1siRNA entered mitosis and proceeded tometaphase normally; chromosomes alignedon the metaphase plate and sister centro-meres separated. However, at that point, thecells were arrested and were delayed orunable to proceed through anaphase. Wewondered whether abnormal telomere asso-ciations were preventing sister chromatids

from separating and moving to the poles.Tankyrase 1 is a positive regulator of telo-mere length (2). Thus, we asked if therewas a gross defect in telomere replicationin tankyrase 1 siRNA cells by measuringDNA replication using 5-bromo-2-deoxyuridine (BrdU) incorporation. Telo-meric DNA from cells transfected withtankyrase 1 siRNA sedimented as heavy-light DNA, consistent with at least oneround of DNA replication and similar tomock-transfected cells (Fig. 3A). Thus,tankyrase 1 siRNA did not detectably in-hibit telomere replication.

An alternative explanation for the mitot-

ic arrest phenotype in tankyrase 1 siRNAcells could be covalent ligation of chromo-some ends. Inhibition of the telomere repeatbinding protein TRF2 results in loss of the3 G-strand telomere tail, ligation of telo-mere restriction fragments, and end-to-endfusions as observed in metaphase spreads(11). We performed similar analyses to ad-dress this question in cells transfected withtankyrase 1 siRNA, and there was no indi-cation of loss of G-strand tails or telomericfusion products (Fig. 3B).

We further assayed for covalent ligationof telomeres in metaphase spreads usingstandard procedures, which include hypo-

Skirball Institute of Biomolecular Medicine, New YorkUniversity School of Medicine, 540 First Avenue, NewYork, NY 10016, USA.

*To whom correspondence should be addressed. E-mail: smithsu@saturn.med.nyu.edu

Fig. 2. Characterization of the mitotic arrest in tankyrase 1 siRNA cells. (A and B) Time-lapse videolive-cell imaging of a HeLa cell line expressing histone H2B tagged with green uorescent protein(HeLa-H2B-GFP cells) (24 ) 36 hours after transfection with tankyrase 1 siRNA. (A) Progression ofmitosis in a mock-transfected cell versus a tankyrase 1 siRNA cell for 108 min. Scale bar, 10 m.(B) Larger eld which includes the tankyrase 1 siRNA cell shown in (A), (arrowhead) and twoanking cells (arrows) that remain in metaphase for 260 min. Scale bar, 10 m. (C and D)Immunouorescence analysis of mitotic cyclins in methanol-xed HeLaI.2.11 cells stained withDAPI (blue) and cyclin A or B (red) 20 hours after transfection with tankyrase 1 siRNA. Scale bar,5 m. (E) Immunoblot analysis of whole-cell extracts from HeLa-SCC1-myc cells 40 hours aftertreatment with tankyrase 1 siRNA. TNKS1 cells were isolated by mitotic shake-off. Arrow indicatesthe SCC1myc cleavage product.

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tonic treatment. Note that this treatment,which was developed to allow visualizationof separated chromosome arms, may re-lease chromosomal proteins (14 ). Typical-ly, in these preparations, sister chromatidsremain associated at their centromeres be-cause of tenacious protein-protein interac-tions (Fig. 3C). Because centromeres wereno longer associated in cells transfectedwith tankyrase 1 siRNA (Fig. 1G and seeFig. 4B, below), chromosomes appeared asseparated chromatids, and end-to-end fu-sions were not observed (Fig. 3C).

Alternatively, sister telomeres could as-sociate through proteinaceous bridges,which may not survive the hypotonic swell-ing used in metaphase spread preparations.

To address this question, we turned to afluorescent in situ hybridization (FISH)replication timing assay (15), where cellswere isolated by mitotic shake-off andfixed directly (without hypotonic treat-ment) to avoid artifactual separation of sis-ter chromatids. In control mitotic cells, tel-omeric regions generally appeared as twodoublets, which indicated that telomereshad replicated and separated (Fig. 4A; tableS3). In contrast, in cells transfected withtankyrase 1 siRNA, two singlets were fre-quently observed, which indicated that thetelomeric regions had not separated (Fig.4A; table S3). Centromere probes appearedas two doublets in both mock-transfectedcells and cells transfected with tankyrase 1

siRNA, but showed greater separation inthe latter (Fig. 4B).

In contrast to telomere-specific probes,arm probes showed no difference betweencells treated with tankyrase 1 siRNA andmock-transfected cells and generally ap-peared as two doublets (Fig. 4C; table S3).Thus, unlike telomeres, chromosome armshad separated. Finally, we performed doubleFISH using arm- and telomere-specificprobes from the same chromosome simulta-neously. In tankyrase 1 siRNA cells, chromo-some arms appeared as two doublets, where-as, in the same cell, telomeres appeared astwo singlets (Fig. 4D). Thus, in tankyrase 1siRNA cells, chromosome arms and centro-meres had replicated and separated, but telo-meres [although replicated (Fig. 3A)] re-mained associated.

Knockdown of tankyrase 1 expressionblocked cells from completing mitotic an-aphase. Although sister chromatids separatedat their centromeres and along their arms,they remained associated at their telomeres.Thus, the inability of sister chromatids tosegregate to daughter poles appeared to bedue to a persistent telomere association. Arole for telomeres in sister chromatid separa-tion during mitosis has been suggested inTetrahymena (16 ), Schizosaccharomycespombe (17), and Drosophila (18).

Our results are consistent with the hypoth-esis that telomeres of sister chromatids nor-mally associate and that resolution of thisassociation is required for progressionthrough anaphase. Our finding that telomereresolution is blocked, whereas centromereand arm resolution appears to occur nor-mally, indicates a unique mechanism fortelomeres which is distinct from arms andcentromeres. In vertebrates, cohesins are re-leased from chromosomes by two distinctposttranslational mechanisms (13, 19). Thebulk of cohesins are released during prophaseby a phosphorylation-mediated mechanism(20, 21), whereas the remaining centromeric

Fig. 3. Telomeres undergoDNA replication and arenot covalently fused intankyrase 1 siRNA cells.(A) Telomeric DNA wasisolated from HelaI.2.11cells after 48 hours of co-treatment with tankyrase1 siRNA and BrdU, andfractionated on CsCl den-sity gradients. TelomericDNA was measured bydot-blot analysis using a32P-labeled telomeric probe.The positions of light-light(LL), heavy-light (HL), andheavy-heavy (HH) DNA areindicated. (B) Southern blotanalysis of telomere restric-tion fragments isolated fromHeLaI.2.11 cells after 48hours of treatment withtankyrase 1 siRNA, and frac-tionated by pulsed-eld gelelectrophoresis. The gel washybridized to a 32P end-labeled (CCCTAA)4 oligo-nucleotide under native ordenatured conditions. (C) Telomeric PNA FISH analysis (FISH using peptide nucleic acid probes) ofmetaphase spreads of HeLaI.2.11 cells collected 48 hours after transfection with tankyrase 1 siRNA,swollen in hypotonic buffer, and xed in methanol-acetic acid. Telomeric repeats were detected byusing a Cy3-(CCCTAA)3 PNA probe (red). DNA was stained with DAPI (blue). Scale bar, 5 m.

Fig. 4. Sister telomeres (unlike arms and centromeres) remain associatedin cells treated with tankyrase 1 siRNA. (A to D) Chromosome-specic FISH analysis of HeLaI.2.11 cells collected by mitotic shake-off 48 hours after treatment with tankyrase 1 siRNA. Cells were

xed directly in methanol-acetic acid without hypotonic swelling andhybridized to chromosome-specic uorescently labeled probes: telo-meres (green), arms (red), and centromeres (red). DNA was stained withDAPI (blue). Scale bar, 2 m.

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cohesins are released at metaphase by proteo-lytic cleavage (22, 23). Our finding thattankyrase 1 PARP activity is required to res-cue the abnormal mitotic phenotype impli-cates a third posttranslational mechanism,poly(ADP-ribosyl)ation, in sister chromatidresolution. Whether telomeres are held to-gether by cohesins or by telomere-specificproteins, such as TRF1 and its associatedfactors, remains to be determined.

References and Notes1. B. van Steensel, T. de Lange, Nature 385, 740 (1997).2. S. Smith, T. de Lange, Curr. Biol. 10, 1299 (2000).3. S. Smith, Trends Biochem. Sci. 26, 174 (2001).4. S. Smith, I. Giriat, A. Schmitt, T. de Lange, Science

282, 1484 (1998).5. S. Smith, T. de Lange, J. Cell Sci. 112, 3649 (1999).6. N. W. Chi, H. F. Lodish, J. Biol. Chem. 275, 38437

(2000).7. H. Seimiya, S. Smith, J. Biol. Chem. 277, 14116

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8. W. Chang, J. N. Dynek, S. Smith, Genes Dev. 17, 1328(2003).

9. B. D. Cook, J. N. Dynek, W. Chang, G. Shostak, S.Smith, Mol. Cell. Biol. 22, 332 (2002).

10. S. M. Elbashir et al., Nature 411, 494 (2001).11. B. van Steensel, A. Smogorzewska, T. de Lange, Cell

92, 401 (1998).12. J. M. Peters, Mol. Cell 9, 931 (2002).13. I. C. Waizenegger, S. Hauf, A. Meinke, J. M. Peters, Cell

103, 399 (2000).14. Y. Ohnuki, Chromosoma 25, 402 (1968).15. R. Or et al., Chromosoma 111, 147 (2002).16. K. E. Kirk, B. P. Harmon, I. K. Reichardt, J. W. Sedat,

E. H. Blackburn, Science 275, 1478 (1997).17. H. Funabiki, I. Hagan, S. Uzawa, M. Yanagida, J. Cell

Biol. 121, 961 (1993).18. M. M. Donaldson, A. A. Tavares, H. Ohkura, P. Deak,

D. M. Glover, J. Cell Biol. 153, 663 (2001).19. A. Losada, M. Hirano, T. Hirano, Genes Dev. 12, 1986

(1998).20. I. Sumara et al., Mol. Cell 9, 515 (2002).21. A. Losada, T. Yokochi, R. Kobayashi, T. Hirano, J. Cell

Biol. 150, 405 (2000).22. S. Hauf, I. C. Waizenegger, J. M. Peters, Science 293,

1320 (2001).

23. F. Uhlmann, F. Lottspeich, K. Nasmyth, Nature 400,37 (1999).

24. T. Kanda, K. F. Sullivan, G. M. Wahl, Curr. Biol. 8, 377(1998).

25. We thank G. Wahl for H2B-GFP-HeLa cells, J. M.Peters for SCC1-mycHeLa cells; M. Pagano for anti-body against cyclin A; A. J. North (Bio-Imaging Re-source Center, Rockefeller University) for assistancewith live imaging; and D. Roth, T. Meier, and mem-bers of the Smith laboratory for comments on themanuscript. J.N.D. was supported by an NIH Predoc-toral Training Program in Cell and Molecular Biology(GM07238-28). This work was supported by grants toS.S. from the Edward Mallinckrodt, Jr., Foundation,the New York City Council Speakers Fund for Bio-medical Research, and the NIH (R01 CA95099-01).

Supporting Online Materialwww.sciencemag.org/cgi/content/full/304/5667/97/DCMaterials and MethodsFigs. S1 to S7Tables S1 to S3ReferencesMovies S1 to S3

16 December 2003; accepted 2 February 2004

SUMO Modication ofHuntingtin and Huntingtons

Disease PathologyJoan S. Steffan,1 Namita Agrawal,2 Judit Pallos,2

Erica Rockabrand,1 Lloyd C. Trotman,3 Natalia Slepko,2

Katalin Illes,1 Tamas Lukacsovich,2 Ya-Zhen Zhu,1

Elena Cattaneo,4 Pier Paolo Pandol,3

Leslie Michels Thompson,1,5* J. Lawrence Marsh2*

Huntingtons disease (HD) is characterized by the accumulation of a pathogenicprotein, Huntingtin (Htt), that contains an abnormal polyglutamine expansion.Here, we report that a pathogenic fragment of Htt (Httex1p) can be modiedeither by small ubiquitin-like modier (SUMO)1 or by ubiquitin on identicallysine residues. In cultured cells, SUMOylation stabilizes Httex1p, reduces itsability to form aggregates, and promotes its capacity to repress transcription.In a Drosophila model of HD, SUMOylation of Httex1p exacerbates neuro-degeneration, whereas ubiquitination of Httex1p abrogates neurodegeneration.Lysinemutations that prevent both SUMOylation and ubiquitination of Httex1preduce HD pathology, indicating that the contribution of SUMOylation to HDpathology extends beyond preventing Htt ubiquitination and degradation.

HD is a dominant neurodegenerative disordercaused by the expansion of a polyglutamine[poly(Q)] repeat in Htt (1, 2). In HD and

other poly(Q) diseases, mutant proteins orpathogenic poly(Q) peptides produced byproteolytic processing aggregate into nuclearand/or cytosolic inclusions in neurons and inneuronal processes. These aggregates alsocontain other cellular proteins includingtranscription-regulating proteins, chaperones,proteasome subunits, and ubiquitin (35).

Pathogenic poly(Q) proteins can bemodified in ways that change their cellularfunction or fate. Htt is subject to ubiquiti-nation, which normally targets proteins fordegradation (6, 7 ). Mutations in ubiquitinligases enhance poly(Q) toxicity in Dro-sophila, mouse, and cell models (810),whereas overexpression of Parkin, an E3ubiquitin ligase, can reduce poly(Q) aggre-

gation and suppress cytotoxicity (11).Thus, ubiquitination appears to reducepoly(Q) toxicity, presumably by promotingthe degradation of toxic proteins.

SUMOylationthe covalent attachmentof SUMO-1 to lysine residuesis a post-translational modification process [for re-views, see (12, 13)] that is biochemicallysimilar to, but functionally distinct from,ubiquitination. SUMOylation can alter thefunction or subcellular localization of pro-teins, and competition between SUMO-1and ubiquitin for identical target lysinescan protect some proteins from degradation(1416 ). The majority of SUMO-modifiedproteins are located in the nucleus (17 ), andSUMOylation can have a direct effect onnucleocytoplasmic transport. Here, we in-vestigate how SUMOylation of Htt mightaffect HD pathogenesis.

Truncated Htt [Httex1p 97QP (Fig. 1A)(18)] and HISSUMO-1 colocalize whentransfected into immortalized striatal nervecells from cell line 12.7 (19) (Fig. 1B), re-flecting either direct modification of Htt bySUMO-1 or colocalization of Htt with otherSUMOylated proteins. To identify possiblemodifications of mutant Httex1p (7, 20),which contains only three lysine residues[K6, K9, and K15 (Fig. 1A)], HISSUMO-1or HIS-ubiquitin was coexpressed withHttex1p either intact or with the lysines mu-tated to arginine [K63R6 (K6R), K9R, andK15R] in both striatal cells and HeLa cells.We also compared Htt fragments either withor without (97QP and 103Q, respectively) theproline-rich domain immediately followingthe poly(Q) region (Fig. 1A). Both proteinscan be SUMOylated or ubiquitinated (Fig.1C, lower panel) and a single primarySUMOylated species predominates, althoughmore complex SUMO or ubiquitin modifica-tions can be seen [Fig. 1D and supporting

1Department of Psychiatry and Human Behavior, Gillespie2121, University of California, Irvine, CA 92697, USA. 2De-partment of Developmental and Cell Biology, 4444 Mc-Gaugh Hall, University of California, Irvine, CA 92697, USA.3Molecular Biology Program and Department of Pathology,Sloan-Kettering Institute, Memorial Sloan-Kettering CancerCenter, 1275 York Avenue, New York, NY 10021, USA.4Department of Pharmacological Sciences, Center of Excel-lence onNeurodegenerative Diseases, University ofMilano,Via Balzaretti 9, 20133 Milano, Italy. 5Department of Bio-logical Chemistry, D240 Medical Sciences I, University ofCalifornia, Irvine, CA 92697, USA.

*These authors contributed equally to this work.To whom correspondence should be addressed. E-mail: jlmarsh@uci.edu

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