Supplemental Materials and Methods Supplemental References

Resolution of human rDNA occurs in anaphase, dependent on tankyrase 1, condensin II, and

topoisomerase II

Zharko Daniloski, Kamlesh Bisht, Brian McStay, and Susan Smith

List of Supplemental Material

Supplemental Materials and Methods

Supplemental References

Supplemental Figures S1-S7

1 Supplemental Materials and Methods

Plasmids CAP-D3 is full length human CAP-D3 with an N-terminal GFP tag cloned into pIRES-AcGFP (provided by Toru Hirota)(Abe et al. 2011). CAP-D3G10A was generated by substituting the glycine (G) at position 10 with alanine (A) using site-directed mutagenesis with the sense oligonucleotide 5’-

CTTGGTAGCGCCCTGCAGCCC-3’ and antisense oligonucleotide 5’-GCCCCGCAACGCCACCAT-3’. CAP- D3G524A was generated by substituting the glycine (G) at position 524 with alanine (A) using the sense oligonucleotide 5’-GAACCCTCAGCGGAGATCAAC-3’ and antisense oligonucleotide 5’- GGAACGGTTAGATGTCTG-3’. The double mutant CAP-D3GAA was generated by introducing G524C mutation in the CAP-D3G10A construct using the primer set listed above. Mutagenesis was performed using the Q5 site-directed mutagenesis kit (NEB) according to the manufacturer’s instructions.

Topoisomerase II is full length human topo II with an N-terminal 3XFlag tag cloned into a modified pcDNA3 plasmid (provided by Christine J. Farr)(Farr et al. 2014). The shRNA plasmids against CAP-D3 and

CAP-D2 were generated by cloning hairpins targeting the following sequences into the pLKO1.p puro vector: CAP-D3#1 against the coding sequence 5’-GCTCTGTTAGAACTGCCTGAA-3’; CAP-D3#2 against the 3’ UTR 5’-CCCATTCAGATAAGCTATAAT-3’; CAP-D2#1 against the coding sequence 5’- TTGCATCACTTTCGAAGTATA-3’; and CAP-D2#2 against the 3’ UTR 5’-CGAATTCTGTTTCCCTTGTAA-3’. Tankyrase 1 shRNA TNKS1-13 was described previously (Hsiao and Smith 2009). Tankyrase 1 is full length human tankyrase 1 with an N-terminal 3XFlag tag cloned into the pLSJH vector (pLSJH.TNKS1) that was rendered resistant to TNKS1-13 shRNA by site-directed mutagenesis (Kim and Smith 2014).

Cell lines HeLaI.2.11 (van Steensel et al. 1998), HTC75 (van Steensel and de Lange 1997), HEK293T (ATCC) BJ (ATCC), WI38 (ATCC), BJ-hTERT (ATCC), and U2OS (ATCC) cells were grown under standard conditions.

Cell synchronization and inhibitor treatments

Tankyrase inhibitor Ti8 (#8) (Haikarainen et al. 2013) ( MolPort-000-222-699) was added at 10 M final concentration for 16 hrs. Etoposide (Sigma, E1383) was added at 25 or 50 M final concentration and ICRF-193 (Sigma, I4659) was added at 10 M final concentration to asynchronous cells for 2 hr. For prometaphase arrest, nocodazole (Sigma, M1404) was added at 30 ng/ml final concentration for 16 hr. For anaphase analysis, nocodazole was at added at 30 ng/ml final concentration for 4.5 hr followed by wash

2 out with pre-warmed 1x PBS (3 times) and release in pre-warmed media for 45 min. For anaphase, only cells with separated centromeres were scored. For analysis of lagging rDNA, cells were arrested for 60 min in nocodazole at 30 ng/ml final concentration followed by wash out with pre-warmed 1x PBS (3 times) and release in pre-warmed media containing DMSO or etoposide at 50 M final concentration for 60 min.

Lentiviral infection

Lentiviruses were produced by transfection of 293FT (Invitrogen) packaging cells with a three-plasmid system as described previously (Naldini et al. 1996; Zufferey et al. 1997). 293FT cells were seeded in a 6- cm dish at 1.2 × 106 cells and 24 hr later were transfected with 1 µg lentiviral vector, 1 µg pCMVΔR.89 packaging plasmid, and 100 ng pMD.G envelope plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Lentiviral supernatants were collected at 48 hr after transfection, filtered with a 0.45-µm filter (Millipore), and frozen at −80°C. Twenty-four hr before infection, target cells were seeded at a density of 2.2 × 105. Target cells were infected for 48-72 hr with lentiviral supernatants supplemented with 8 µg/ml polybrene (Sigma-Aldrich). Infected cells were selected and propagated in media containing 2 µg/ml puromycin. Lentiviral infection was used to generate HelaI.2.11 cells stably expressing the following shRNAs: GFP, TNKS1-13, CAP-D2#1, CAP-D2#2, CAP-D3#1, or CAP-D3#2. Lentiviral infection was used to generate BJ-hTERT cells stably expressing GFP or TNKS1-13 shRNAs. HTC75 cells stably expressing GFP or TNKS1-13 shRNAs were described previously (Hsiao and Smith 2009). siRNA and plasmid transfection siRNA transfections were performed with Oligofectamine (Invitrogen) according to the manufacturer’s protocol for 48 hr or 72 hr for micronuclei analysis. The final concentration of siRNA was 100 nM. The following siRNAs (synthesized by Dharmacon Research Inc.) were used: TNKS1 (5′- CAAUUCACCGUCGUCCUCU-3′) described previously (6), SMC2 (5′-UGCUAUCACUGGCUUAAAU-3′) and siGFP Duplex I. For plasmids, cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturers protocol for 18 hr.

Chromosome specific FISH Mitotic cells were fixed and processed as described previously (Dynek and Smith 2004). Briefly, cells were isolated by mitotic shake-off, fixed twice in methanol:acetic acid (3:1) for 15 min, cytospun (Shandon

Cytospin) at 2000 rpm for 2 min onto slides, rehydrated in 2X SSC at 37C for 2 min, and dehydrated in an

3 ethanol series of 70%, 80%, and 95% for 2 min each. Cells were denatured at 75C for 2 min and hybridized overnight at 37oC with green centromere probes (15 cen and 13/21 cen) and red AcroP (rDNA) probes (Cytocell) or with green DJ or PJ probes and red rDNA-IGS probes(van Sluis et al. 2016). Cells were washed in 0.4X SSC at 72C for 2 min, and in 2X SSC with 0.05% Tween 20 at RT for 30 sec. DNA was stained with 0.2 µg/ml DAPI. Mitotic cells were scored as having rDNA, DJ, or PJ unresolved if 50% or more of their loci appeared as singlets, i.e. five out of ten, six out of eleven, etc. The DJ probe adjoins the rDNA on the telomere side and the PJ adjoins the rDNA on the centromere side, but the PJ probe signals are more complicated due to segmental duplication of their sequences(Floutsakou et al. 2013). Mitotic cells were scored as having stretched rDNA if they had 3 or more rDNA stretches. For micronuclei analysis, cells were trypsinized, fixed in methanol:acetic acid 3:1, and hybridized with the AcroP (rDNA) probe (as described above). Interphase cells were scored for the presence of one or more micronuclei with or without rDNA.

Aneuploidy analysis

For aneuploidy analysis asynchronous cells were fixed directly on coverslips in methanol-acetic acid and hybridized to centromere probes against chromosome 15, 10, 6, or 16 (Cytocell). Cells were scored as aneuploid when a chromosome number deviated from the norm, where the norm is defined by what the majority of the cells display for that particular chromosome. For HTC75 cells the norm was established as 3 copies for chromosome 15 and 4 copies for chromosome 10. For BJ-hTERT the norm was 2 copies for chromosomes 15, 10, 6, and 16. BJ-hTERT cells that displayed tetraploidy were not scored.

Cell extracts

Cells were resuspended in 4 volumes of TNE buffer [10 mM Tris (pH7.8), 1% Nonidet P-40, 0.15 M NaCl, 1 mM EDTA, and 2.5% protease inhibitor cocktail (PIC) (Sigma)] and incubated for 1 hr on ice. Suspensions were pelleted at 8000xg for 15 min. Equal amounts of supernatant proteins (determined by Bio-Rad protein assay) were fractionated by SDS-PAGE and analyzed by immunoblotting.

Immunoblot analysis

Immunoblots were incubated separately with the following primary antibodies: rabbit anti-SMC2 (0.4 g/ml) (Betyl, A300-058); rabbit anti-TNKS1 465 (1 g/ml)(Smith et al. 1998), rabbit anti-TNKS1 762 (1

g/ml)(Scherthan et al. 2000); rabbit anti-CAP-D3 (0.1g/ml) (Bethyl, A300-604A); rabbit anti-CAP-D2 serum

(1:2000) (Upstate, 07-701); rabbit anti-CAP-H (0.1g/ml) (Bethyl, A300-603A); anti-CAP-H2 (0.8g/ml)

4 (Santa Cruz SC393333); mouse anti-topoisomerase II (0.4 g/ml) (SantaCruz, sc-166934); and mouse anti- α-tubulin ascites (0.6 g/ml) (Sigma Aldrich, T5168), followed by horseradish peroxidase-conjugated donkey anti-rabbit or anti-mouse IgG (Amersham). Bound antibody was detected with Super Signal West Pico (Thermo Scientific).

Immunoprecipitation

For each immunoprecipitation, cells from one 10cm dish (at 85% confluency) were resuspended in 0.7 ml TNE buffer with protease inhibitors as described above. Supernatants were precleared with protein G- Sepharose rotating at 4C for 30 min. Nonspecific protein aggregates were removed by centrifugation and the supernatant was used for immunoprecipitation analysis or fractionated directly on SDS-PAGE. Equal amounts of supernatant proteins (determined by Bio-Rad protein assay) were used as starting material. For immunoprecipitation of endogenous tankyrase, HeLaI.2.11 cell lysates were incubated with 2 µg of either rabbit IgG (Cell Signaling, 2729S) or rabbit-TNKS1 762 antibody(Scherthan et al. 2000) for 4 hr. Protein G beads were added for 2 hr followed by washing five times with 1 ml TNE buffer. For immunoprecipitation of GFP-tagged proteins, U2OS cells were transiently transfected with GFP-vector, GFP-CAP-D3WT or GFP- CAP-D3GAA for 18 hr. PARG inhibitor (Sigma, PDD00017273) was added at final concentration of 5 µM for 6 hr prior to harvest, Cells were collected and lysed in TNE buffer containing protease inhibitors and PARG inhibitor (5 µM) and processed as described above. For immunoprecipitation of GFP-tagged proteins, U2OS cell lysates were incubated with 2 µg of rabbit anti-GFP (Abcam, ab290) each. Samples were fractionated by SDS-PAGE and analyzed by immunoblotting.

Indirect immunofluorescence

For γH2AX and UBF staining, cells were fixed in 3.7% formaldehyde in PBS for 10 min, permeabilized in

0.5% NP-40/PBS, blocked in 1% BSA in PBS and incubated with mouse anti-γH2AX (2g/ml) (Millipore, 05- 636) and sheep anti-UBF (0.1g/ml)(van Sluis et al. 2016).

Image Acquisition Images were acquired using a microscope (Axioplan 2; Carl Zeiss, Inc.) with a Plan Apochrome 63x NA 1.4 oil immersion lens (Carl Zeiss, Inc.) and a digital camera (C4742-95; Hamamatsu Photonics). Images were acquired and processed using Openlab software (Perkin Elmer). When FISH signals were not in a single plane of focus, multiple planes were captured and merged. For deconvolution, FISH images were collected

5 as optical sections at every 0.24 μm depth Z-stacks and deconvolved with the nearest-neighbor deconvolution algorithm, using Openlab digital deconvolution software (Perkin Elmer).

Statistical Analysis Statistical analysis was performed using Prism 7 software. Student’s unpaired t-test was applied. Data are shown as mean ±SEM (standard error of the mean); P<0.05 values were considered significant.

6 Supplemental References

Abe S, Nagasaka K, Hirayama Y, Kozuka-Hata H, Oyama M, Aoyagi Y, Obuse C, Hirota T. 2011. The initial phase of chromosome condensation requires Cdk1-mediated phosphorylation of the CAP-D3 subunit of condensin II. Genes Dev 25: 863-874. Dynek JN, Smith S. 2004. Resolution of sister telomere association is required for progression through mitosis. Science 304: 97-100. Farr CJ, Antoniou-Kourounioti M, Mimmack ML, Volkov A, Porter AC. 2014. The alpha isoform of topoisomerase II is required for hypercompaction of mitotic chromosomes in human cells. Nucleic Acids Res 42: 4414-4426. Floutsakou I, Agrawal S, Nguyen TT, Seoighe C, Ganley AR, McStay B. 2013. The shared genomic architecture of human nucleolar organizer regions. Genome Res 23: 2003-2012. Haikarainen T, Koivunen J, Narwal M, Venkannagari H, Obaji E, Joensuu P, Pihlajaniemi T, Lehtio L. 2013. para-Substituted 2-phenyl-3,4-dihydroquinazolin-4-ones as potent and selective tankyrase inhibitors. ChemMedChem 8: 1978-1985. Hsiao SJ, Smith S. 2009. Sister telomeres rendered dysfunctional by persistent cohesion are fused by NHEJ. J Cell Biol 184: 515-526. Kim MK, Smith S. 2014. Persistent telomere cohesion triggers a prolonged anaphase. Mol Biol Cell 25: 30- 40. Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D. 1996. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272: 263-267. Scherthan H, Jerratsch M, Li B, Smith S, Hulten M, Lock T, de Lange T. 2000. Mammalian meiotic telomeres: protein composition and redistribution in relation to nuclear pores. Mol Biol Cell 11: 4189-4203. Smith S, Giriat I, Schmitt A, de Lange T. 1998. Tankyrase, a poly(ADP-ribose) polymerase at human telomeres [see comments]. Science 282: 1484-1487. van Sluis M, van Vuuren C, McStay B. 2016. The Relationship Between Human Nucleolar Organizer Regions and Nucleoli, Probed by 3D-ImmunoFISH. Methods Mol Biol 1455: 3-14. van Steensel B, de Lange T. 1997. Control of telomere length by the human telomeric protein TRF1. Nature 385: 740-743. van Steensel B, Smogorzewska A, de Lange T. 1998. TRF2 protects human telomeres from end-to-end fusions. Cell 92: 401-413. Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D. 1997. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nature biotechnology 15: 871-875.

7 A

rDNA DJ Merge B

rDNA PJ Merge

C D BJ cells 80 *

rDNA DJ Merge 40

locus uresolved (% cells) 0 probe: rDNA DJ

Figure S1. FISH analysis with DJ and PJ probes. (A-B) The DJ and PJ appear as singlets in interphase. FISH analysis of interphase cells probed with (A) DJ (green) or (B) PJ (green) and rDNA (red). (C-D) rDNA is unresolved and DJ is resolved in mitotic BJ fibroblasts as in mitotic HeLaI.2.11 cells. (C) FISH analysis of mitotic cells isolated by shake-off from asynchronously growing BJ cells and probed for rDNA (red) and DJ (green) and (D) quantified. Mitotic cells were scored as having rDNA and DJ unresolved if 50% or more of their loci appeared as singlets. Average of two independent experiments (n=32-35 cells each) ± SEM. *p 0.05, students unpaired t-test. (A, B, and C) DNA was stained with DAPI (blue). Scale bar, 2 m. A HeLa cells B GFP siRNA C HeLa cells 50 siRNA: GFP TNKS1 TNKS1- -170 40

30 α-tubulin- -55 rDNA-IGS TNKS1 siRNA 20

stretched rDNA (% cells) stretched rDNA 0 siRNA: GFP TNKS1 rDNA-IGS

D E F DMSO G DMSO HeLa cells BJ cells 40 12

30 8 rDNA rDNA 20 Ti8 Ti8 4 10 stretched rDNA (% cells) stretched rDNA stretched rDNA (% cells) stretched rDNA 0 0 DMSO Ti8 DMSO Ti8 rDNA rDNA

H I WI38 cells J WT K DMSO HEK293T cells 10 * 32

8 24 rDNA 6 rDNA 16 Ti8 4 TNKS1 KO #13 8 2 stretched rDNA (% cells) stretched rDNA stretched rDNA (% cells) stretched rDNA 0 0 DMSO Ti8 WT #13 #15 rDNA rDNA TNKS1KO

Figure S2. Tankyrase depletion by different methods leads to rDNA stretching across multiple cell types. (A-C) HeLaI.2.11 cells were transfected with GFP or TNKS1 siRNA for 48 hr and analyzed by (A) immunoblot or (B) isolated by mitotic shake-off and analyzed by FISH using an rDNA probe to the non-transcribed intergenic sequence (rDNA-IGS) (red) and (C) quantified. (n=94-102 cells). (D-I) rDNA stretching is induced by a tankyrase small molecule inhibitor Ti8. (D) HeLaI.2.11, (F) BJ, or (H) WI38 cells were treated with DMSO (vehicle) or Ti8 for 16 hr, isolated by mitotic shake-off, and analyzed by FISH using an rDNA probe (red). (E, G, and I) Quantification of results in D, F, and H respectively. (E) (n=62-72 cells). (G) (n=56 cells). (I) Average of two independent experiments (n=43-55 cells each) ± SEM. *p 0.05, students unpaired t-test. (B, D, F, and H) Mitotic cells with 3 stretched rDNA lines were scored as stretched rDNA. (J-K) rDNA stretching is detected in HEK293T TNKS1 knockout (KO) cell lines. HEK293T WT or TNKS1 KO clones #13 and #15 were isolated by mitotic shake-off and analyzed by (J) FISH using an rDNA probe (red) and (K) quantified. Mitotic cells with one or more stretched rDNA lines were scored as stretched rDNA. (n=56-60 cells). (B, D, F, H, and J) DNA was stained with DAPI (blue). Scale bar, 2 m. IP antibody Input x IgG D3 D2

CAP-D3- -170 -170 CAP-D2-

CAP-H2- -96

CAP-H- -96 -170 SMC2-

IgG- -55

Figure S3. Immunoblot analysis showing that the condensin I and II complexes are intact. Immunoblot analysis of HeLa cells immunoprecipitated with IgG, CAP-D3, or CAP-D2 antibodies and probed with the indicated antibodies. A GFP shRNA GFP DJ rDNA Merge

TNKS1 shRNA DJ rDNA Merge

CAP-D3 shRNA DJ rDNA Merge

B GFP shRNA GFP PJ rDNA Merge

TNKS1 shRNA PJ rDNA Merge

CAP-D3 shRNA PJ rDNA Merge

Figure S4. (A-B) DJ and PJ sequences do not appear stretched in TNKS1 or CAP-D3 depleted anaphase cells. HeLa cell lines stably expressing GFP, TNKS1, or CAP-D3 shRNAs were isolated by mitotic shake-off following a 4.5 hr incubation in nocodazole and 45 min release into anaphase and analyzed by FISH using rDNA (red) and (A) DJ or (B) PJ (green) probes. DNA was stained with DAPI (blue). Scale bar, 2 m.

Merge 15

0 stretched 13/21 cen (% cells) DMSO 25 50 10 (μM) etoposide ICRF-193

Figure S5. Inhibition of topoisomerase II leads to minor stretching of centromeres. HeLa cells were isolated by mitotic shake-off following a 2 hr treatment with (vehicle) DMSO or the Topo II inhibitors etoposide or ICRF-193, analyzed by 13/21 cen FISH, and quantified. Mitotic cells with 3 stretched centromeres were scored as having stretched 13/21 centromeres. (n=39-58 cells). A HeLa cell lines B TNKS1 shRNA cell line C CAP-D3 shRNA cell line shRNA: GFP TNKS1 Tx: Vec TNKS Topo D3 Tx: Vec D3 Topo TNKS -170 -170 -170 TNKS1- TNKS1- CAP-D3-

α-tubulin- α Topo IIα- -170 -55 Topo II - -170 -170 -170 CAP-D3- TNKS1-

α α-tubulin- -55 -tubulin- -55

Figure S6. Immunoblot analysis of transfected TNKS1 and CAP-D3 shRNA cell lines. (A) Immunoblot analysis of HeLaI.2.11 cell lines stably expressing GFP or TNKS1-13 shRNA. (B and C) Immunoblot analysis showing (B) TNKS1 and (C) CAP-D3 shRNA cell lines transfected with vector, TNKS1, CAP-D3, or topo II . BJ-hTERT cell lines

15 * GFP TNKS1 10

0 % deviation from modal copy number 15 cen 10 cen 6 cen 16 cen (acro) (submeta) (submeta) (meta)

Figure S7. Increased aneuploidy of acrocentric chromosome 15 compared to non-acrocentric chromosomes 10, 6, and 16 in TNKS1-depleted BJ-hTERT cells. BJ-hTERT interphase cells stably expressing GFP or TNKS1 shRNA were analyzed by 15 cen, 10 cen, 6 cen, or 16 cen FISH and quantified for deviation from the modal copy number. The analysis was performed in a blinded manner. Average of three independent experiments (n=91-121 cells each) ± SEM. *p 0.05, students unpaired t-test.