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 37C for 2 min, and dehydrated in an 3 ethanol series of 70%, 80%, and 95% for 2 min each. Cells were denatured at 75C 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 72C 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 (0.1g/ml) (Bethyl, A300-604A); rabbit anti-CAP-D2 serum (1:2000) (Upstate, 07-701); rabbit anti-CAP-H (0.1g/ml) (Bethyl, A300-603A); anti-CAP-H2 (0.8g/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 4C 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.