© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 1619-1634 doi:10.1242/jcs.179036

RESEARCH ARTICLE Conditional mutation of Smc5 in mouse embryonic stem cells perturbs condensin localization and mitotic progression Marina V. Pryzhkova and Philip W. Jordan*

ABSTRACT Condensins are crucial for early embryonic development and Correct duplication of stem cell genetic material and its appropriate cerebral cortex formation in mice (Nishide and Hirano, 2014). segregation into daughter cells are requisites for tissue, organ and Misregulation of condensin II has been implicated in the organism homeostasis. Disruption of stem cell genomic integrity can neurodevelopmental disorder autosomal recessive primary lead to developmental abnormalities and cancer. Roles of the Smc5/6 microcephaly (MCPH) (Hirano, 2012; Trimborn et al., 2006). structural maintenance of complex in pluripotent stem Recently, hypomorphic mutations of a Smc5/6 component, cell genome maintenance have not been investigated, despite its NSMCE2, have been reported to cause primordial dwarfism and important roles in DNA synthesis, DNA repair and microcephaly in (Payne et al., 2014). Furthermore, SMC segregation as evaluated in other model systems. Using mouse mutations are associated with cancer (Leiserson et al., 2015; Jacome embryonic stem cells (mESCs) with a conditional knockout allele of et al., 2015). mutations have been identified in myeloid Smc5, we showed that Smc5 protein depletion resulted in leukemias (Leeke et al., 2014). The condensin component Smc2 destabilization of the Smc5/6 complex, accumulation of cells in G2 and the Smc5/6 complex component Smc6 are overexpressed in phase of the and apoptosis. Detailed assessment of mitotic cancers, such as colorectal, neuroblastoma and breast cancer mESCs revealed abnormal condensin distribution and perturbed (Davalos et al., 2012; Murakami-Tonami et al., 2014; Stevens chromosome segregation, accompanied by irregular spindle et al., 2011). Furthermore, mutation of Smc5 is linked to the morphology, lagging chromosomes and DNA bridges. Mutation of development of brain metastases (Saunus et al., 2015). Smc5 resulted in retention of and enrichment of Each SMC complex is composed of two SMC components condensin on chromosome arms. Furthermore, we observed reduced forming a V-shaped heterodimer, which is bridged by non-SMC levels of Polo-like kinase 1 at during . Our study subunits (Hirano, 2006, 2012). Cohesin comprises the Smc1 and α reveals crucial requirements of the Smc5/6 complex during cell cycle Smc3 heterodimer, bridged by the -kleisin subunit Rad21 and one progression and for stem cell genome maintenance. of two stromal antigen proteins, Stag1 or Stag2. The canonical function of the cohesin complex is to hold sister chromatids together KEY WORDS: Smc5, Smc6, Mouse embryonic stem cells, following DNA replication. Cohesin removal is required to ensure Pluripotency, Apoptosis, Genomic integrity, Chromosome chromosome segregation during cell division (Nasmyth and segregation Haering, 2009). There are two condensin complexes, condensin I and condensin II, both promote compaction and disentanglement of INTRODUCTION sister chromatids prior to chromosome segregation (Hirano, 2012). Pluripotent stem cells (PSCs) possess unlimited differentiation Condensin I and II share the core Smc2 and Smc4 heterodimer; potential and are considered to be a source of cells for regenerative however, they are made unique by their complex specific non-SMC medicine. PSCs also share multiple characteristics with cancer cells subunits. In mammals, the Smc5/6 complex contains a Smc5 and and are prone to acquisition of chromosomal abnormalities in cell Smc6 heterodimer and four non-SMC elements Nsmce1– Nsmce4 culture. Although the underlying mechanisms that initiate genomic (also known as Nse1–Nse4) (Hirano, 2006). In addition, two Smc5/ instability are yet to be elucidated, DNA replication stress and 6 complex localization factors (Slf1 and Slf2) have recently been defective chromosome condensation, decatenation and segregation discovered (Räschle et al., 2015). are likely contributing factors (Na et al., 2014; Lamm et al., 2015; Studies using budding and fission yeast mutants have shown that Damelin et al., 2005). the Smc5/6 complex is required for replication fork stability, It is expected that the structural maintenance of chromosomes facilitating the resolution of joint molecules and preventing the (SMC) complexes play crucial roles in PSC preservation, formation of aberrant joint molecules that can lead to mitotic proliferation and differentiation. The SMC complex family catastrophe (reviewed in Carter and Sjögren et al., 2012; Jeppsson includes cohesin, condensins and Smc5/6, and each complex et al., 2014; Langston and Weinert, 2015; Murray and Carr, 2008; contributes to maintaining eukaryotic genome integrity. Verver et al., 2016; Wu and Yu, 2012). The distinct roles of the Malfunction of SMC complexes is often associated with Smc5/6 complex in mammalian cells have yet to be defined. severe developmental disorders. Mutations and misexpression of However, localization and small interfering RNA (siRNA) cohesin lead to developmental disorders collectively known as knockdown studies in mammalian cells suggest that the complex cohesinopathies (Bose and Gerton, 2010; Skibbens et al., 2013). is required during DNA replication, DNA repair and chromosome segregation (Wu et al., 2012; Gallego-Paez et al., 2014; Gomez Department of Biochemistry and Molecular Biology, Johns Hopkins University et al., 2013). Bloomberg School of Public Health, Baltimore, MD 21205, USA. Faithful chromosome segregation depends on cooperative *Author for correspondence ([email protected]) functioning of the SMC complexes and multiple cell cycle kinases including polo-like kinases (Plks), cyclin-dependent

Received 13 August 2015; Accepted 22 February 2016 kinases (Cdks) and Aurora kinases. For instance, -mediated Journal of Cell Science

1619 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 1619-1634 doi:10.1242/jcs.179036 phosphorylation of cohesin stimulates removal of arm cohesin treatment, we kept a concentration of 0.05 µM 4-OH TAM in during (Giménez-Abián et al., 2004). Condensins are culture. Cells were evaluated for changes in gene and protein phosphorylated by Cdk1, Plk1 and Aurora B kinases to ensure expression, and cell cycle perturbations during the next three proficient chromosome condensation (Abe et al., 2011; Lipp et al., passages after initiating 4-OH TAM treatment (Fig. S2A). For 2007; Tada et al., 2011). In addition, condensins are required for simplicity, we refer to the wild-type Smc5 as ‘Smc5+’, floxed Smc5 appropriate localization of Aurora B and Plk1 kinases during the as ‘Smc5flox’ and deleted Smc5 as ‘Smc5−’. -to- transition and ensure accurate chromosome segregation (Abe et al., 2011; Kim et al., 2014; Green et al., 2012; mESCs and somatic cells display different phenotypes upon Kitagawa and Lee, 2015). Components of the Smc5/6 complex have Smc5 mutation been reported to be phosphorylated by Plk1 and Aurora B kinases Smc5 depletion in mESCs resulted in extensive cell death after 5– during mitosis (Hegemann et al., 2011). However, mechanistic links 8 days of 4-OH TAM treatment (Fig. 2A; Fig. S2D). However, this between Smc5/6 complex and cell cycle kinases have yet to be was not the case for immortalized mouse embryonic fibroblasts determined. (MEFs). Instead, the proliferation rate of fibroblasts diminished after To assess the requirements for the Smc5/6 complex in stem cell about 10 days of drug addition, but cell death was negligible genome maintenance, we aimed to use a knockout mouse approach. (Fig. S2E). mESCs proliferate very rapidly compared to MEFs. To Previous studies have reported that Smc5/6 components are exclude possible influence of slow cell expansion on the appearance essential for early embryonic development in mouse (Ju et al., of phenotypic changes, we assessed MEF proliferation during 2013; Jacome et al., 2015). Therefore, we created a Smc5 17 days. After 14 and 17 days of 4-OH TAM treatment, MEFs conditional knockout mouse, which we used to investigate increased in cell number by less than two-fold, whereas untreated functions of the Smc5/6 complex in mouse embryonic stem cells MEFs increased by six-fold (Fig. S2E). PCR analysis revealed that (mESCs). Cre-ERT2-mediated mutation of Smc5 impacted mitotic excision of Smc5 exon 4 was much less efficient in MEFs compared progression, leading to the formation of chromosomal bridges, to mESCs, requiring up to 10 days of 4-OH TAM treatment appearance of lagging chromosomes during anaphase and, (Fig. S2F). We speculated that a high expression level of Smc5/6 ultimately, to aneuploidy. mESCs accumulated in the G2 phase of components is essential for pluripotent mESCs, and lower levels of the cell cycle and activated apoptotic signaling. Microscopy studies Smc5/6 are required for cells at more advanced developmental revealed the irregular distribution of condensin, Plk1 and Aurora B stages. To support this hypothesis, we evaluated Smc5 and Smc6 in Smc5-depleted mitotic cells, which correlated with distorted expression during mESC differentiation in embryoid bodies. chromosome structure and abnormal spindle morphology. In Notably, our data showed a decrease in protein amount with summary, our data demonstrate that the absence of functional prolonged embryoid body culture (Fig. 2B). This result suggests that Smc5/6 complex in mESCs leads to rapid cell death as a result of higher levels of the Smc5/6 complex are required for the highly disrupted genomic integrity and mitotic failure. proliferative mESCs compared to differentiated cells.

RESULTS Smc5 deficiency leads to activation of the p53 pathway and Established mESC lines express pluripotency-associated Parp1 cleavage markers and form teratomas in vivo In contrast to somatic cells, PSCs are very sensitive to stress factors To investigate the role of the Smc5/6 complex in PSCs, we established and readily undergo differentiation or apoptosis under suboptimal two sets of mESC lines with experimental (Smc5flox/del, Cre-ERT2; conditions (Weissbein et al., 2014). We explored whether depletion called Smc5-13exp and Smc5-1exp) and control (Smc5wt/flox,Cre- of Smc5 would lead to changes in mESC profile ERT2;calledSmc5-3cont and Smc5-6cont) genotypes. Cell lines with (Fig. 2C; Fig. S2G). We did not observe a decrease in pluripotency both genotypes similarly displayed characteristic mESC colony markers Oct4 and Nanog. The mesendoderm marker brachyury (T ) morphology and expressed the pluripotency-associated markers Oct4 was expressed in all cell samples at a low level. No expression (also known as Pou5f1), Sox2, SSEA-1 (also known as Fut4) and a of the endoderm marker α-fetoprotein was detected. However, after high level of alkaline phosphatase activity (Fig. 1A; Fig. S1A). After 5 and 8 days (P2 and P3) of 4-OH TAM treatment to experimental injection into immuno-deficient mice, mESCs formed teratomas mESCs, we observed an upregulation of NeuroD1, which is an early representing derivatives of all three embryonic germ layers (Fig. 1B; marker of ectoderm. A slight increase in the expression of later Fig. S1B). Thus, using in vitro and in vivo assays, we confirmed ectoderm marker Pax6 was observed for the mESC Smc5-13exp pluripotency of established mESC lines. As an additional control, we (experimental; Smc5-3font/del, Cre-ERT12) cell line (Fig. S2G). We established a wild-type cell line with the same C57BL/6J genetic also observed upregulation of Myc in experimental mESCs after background (Fig. S1A). 5 and 8 days of 4-OH TAM treatment (Fig. S2G). Thus, the depletion of Smc5 protein in mESCs does not lead to Cre-ERT2 recombinase-induced mutation of Smc5 downregulation of pluripotency markers, but causes a shift in the To initiate Cre-ERT2 recombinase activity and excise the floxed pluripotent state by inducing the expression of differentiation Smc5 exon 4, two experimental and two control mESC lines were markers. treated with 0.2 µM 4-hydroxytamoxifen (4-OH TAM) (Fig. 1C; The rapid cell death we observed could be mediated by the stress Fig. S2A). This dose was sufficient to excise the targeted sequence response factor p53, which also impedes somatic cell within 3 days of treatment (Fig. 1D; Fig. S2B). The deletion of Smc5 reprogramming and promotes ESC differentiation (Weissbein exon 4 in experimental cell lines was accompanied by the loss of et al., 2014; Lin et al., 2012). The basal p53 level in ESCs is Smc5 protein expression and a dramatic decrease in the Smc6 level generally higher than in somatic cells, and regulation of p53 compared to control cell lines (Fig. 1E; Fig. S2C). signaling is different (Lin et al., 2012). ESCs activate p53 signaling Published studies and our observations have revealed that some in response to DNA damage; however, they do not arrest at the G1 cells can escape Cre recombinase activity and overtake cell cultures phase of the cell cycle as is the case for somatic cells (Weissbein

(Yoshida et al., 2010). Therefore, after 3 days of 0.2 µM 4-OH TAM et al., 2014). Previous studies on mouse and human ESCs have Journal of Cell Science

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Fig. 1. Characterization of mESC lines and conditional mutation of Smc5. (A) The mESC Smc5-13exp (experimental; Smc5flox/del, Cre-ERT2) line expressed the pluripotency markers Oct4, Sox2, alkaline phosphatase (AP) and SSEA1. mESC colonies are shown on MEF feeder layer. Scale bars: 100 µm. (B) Analysis of mESC Smc5-1exp (experimental; Smc5flox/del, Cre-ERT2) teratoma sections stained with hematoxylin and eosin revealed the presence of derivatives of all three embryonic germ layers: ectoderm, mesoderm and endoderm. Scale bars: 50 µm. (C) The scheme of the deletion of intron 4 in the floxed Smc5 allele using Cre-ERT2 recombinase. Genotyping primers are shown as arrows. Amplified DNA fragment sizes are depicted in the box on the right. (D) PCR analysis of experimental and control lines mESC Smc5-1exp and mESC Smc5-3cont (control; Smc5wt/flox, Cre-ERT2) showed efficient deletion of floxed DNA fragment. Shown are untreated cells (Unt) and mESCs after 1, 2 and 3 days of 4-OH TAM treatment. wt, wild type; del, deleted. (E) Western blot analysis revealed efficient depletion of Smc5 protein and a exp cont α substantial decrease in Smc6 level in the experimental line mESC Smc5-1 , but not in control mESC Smc5-3 cells. -tubulin was used as a loading control. Journal of Cell Science

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Fig. 2. See next page for legend. shown that acetylation of the C-terminus of p53 plays a crucial role did not change total p53 level. However, after 5 and 8 days (P2 and in its activation in response to genotoxic stress (Feng et al., 2005; P3) of 4-OH TAM treatment we observed p53 acetylation at Lys379

Chung et al., 2014). Depletion of Smc5 in our experimental mESCs (Fig. 2D), indicating stimulation of p53 transcriptional activity. Journal of Cell Science

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Fig. 2. Smc5 depletion causes multiple changes in mESCs. (A) Compared components during cell cycle progression in mESCs with functional +/− −/− to control cells (Smc5 ), Smc5-deficient mESCs (Smc5 ) revealed Smc5. Smc6 and Nse1 proteins were enriched within the nucleus significant decrease in cell growth after 5 days of 4-OH TAM treatment. Cells during , and accumulated at the pericentromeric regions of were counted on the day of passaging. Bars represent mean±s.e.m. (n=4). Day 2 (P1), P=0.0757 (not statistically significant); day 5 (P2), P=0.0054; day 8 (P3), chromosomes during prometaphase, metaphase and anaphase P=0.0061 (based on paired, one tailed t-tests). (B) Western blot analysis of (Fig. 3A,B; Fig. S3A,B). At late stages of interphase, we also Smc5 and Smc6 expression in embryoid bodies on days 0, 6, 12 and 18. observed perinuclear granules of Nse1. These also persisted in the α-tubulin was used as a loading control. (C) Semi-quantitative RT-PCR analysis during anaphase (Fig. 3B). Similarly, antibodies against of pluripotency and differentiation marker expression during three passages. Smc5 detected protein enrichment near centromeres during exp Gapdh was used as internal control. mESC Smc5-1 (experimental; prometaphase, metaphase and anaphase (Fig. 3C). In telophase and Smc5flox/del, Cre-ERT2) line, mESC Smc5-3cont (control; Smc5wt/flox, Cre-ERT2) line, mESC-2wt, wild-type line. (D) Western blot analysis demonstrated that interphase, Smc5 immunocytochemistry showed nuclear and Smc5 and Smc6 protein depletion was coupled with caspase-mediated cytoplasmic staining (Fig. 3C). To support our data further, we cleavage of Parp1 and acetylation of p53 in 4-OH TAM-treated mESC performed immunostaining of chromosome spreads, which Smc5-1exp cells at P2 and P3. α-tubulin was used as a loading control. (E) FACS confirmed pericentromeric enrichment of Smc5/6 components on analysis of mESC cycle during three passages. Smc5-depleted mESCs − − mitotic chromosomes and localization along the condensed (Smc5 / ) demonstrated a decrease in S phase and accumulation in G2 phase −/flox +/flox +/− chromosome arms (Fig. 3D). at P2 and P3, whereas control samples (Smc5 , Smc5 and Smc5 ) We also observed Smc6 accumulation in distinct nuclear were not affected. Bars represent mean±s.e.m. (n=3). Day 2 (P1): *P=0.0615 (not statistically significant), **P=0.3358 (not statistically significant); day 5 (P2): foci during interphase and prominent spindle pole localization *P=0.0176, **P=0.0212; day 8 (P3), *P=0.0454, **P=0.0191 (based on paired, through mitosis and up to late anaphase (Fig. S3C,E). Smc6 one tailed t-tests). (F) FACS data corresponding to the graph in E showing localization correlated with centrosome positioning during mitosis accumulation of polyploid Smc5-depleted mESCs at P3 (arrow), but not in (Fig. S3C). Interestingly, Mad2 showed a similar localization 4-OH-TAM-treated control mESCs. (G) Geimsa stained mitotic chromosome pattern, including co-localization with Smc6 in interphase nuclei spreads demonstrating a normal karyotype (left) for Smc5-expressing mESCs foci (Fig. S3D,F). Nse1 was also detected at spindle poles in and an abnormal karyotype (right) for Smc5-depleted mESCs. [Cell viability assay of adherent cells in colonies revealed 85% and 78% viable Smc5- mitotic cells (Fig. S3G). Because of abundant cytoplasmic staining depleted cells at day 5 (P2) and day 8 (P3) of 4-OH TAM treatment versus 99% with anti-Smc5 antibodies, we did not obtain conclusive images for untreated.] P1, P2 and P3, passages 1, 2 and 3, respectively. with Smc5 spindle pole localization. However, we propose that this is also the case for Smc5, based on the decline in Smc6 levels upon Another common feature of apoptotic signaling involves Smc5 mutation, and the similar localization observed for Smc6 activation of caspases and subsequent cleavage of target proteins and Nse1. including Parp1 (Chaitanya et al., 2010; Luo and Kraus, 2012). Smc5 depletion in mESCs led to cleavage of Parp1 at P2 and P3 Smc5-deficient mESCs undergo mitotic catastrophe (Fig. 2D). This correlated with substantial cell death at these time Smc5-depleted mESCs demonstrated a dramatic increase in points (Fig. 2A). abnormal mitotic cells harboring lagging chromosomes, chromosome bridges or both (Fig. 4A,B). We also observed a Smc5 depletion causes mESC accumulation in G2 phase and prominent increase in mitotic cells with abnormal spindles an increase in polyploid cells demonstrating aberrant chromosome segregation (Fig. 4B). Given the important roles reported for the Smc5/6 complex in DNA Approximately 80% of Smc5-depleted mESCs could not replication, repair and chromosome segregation, we hypothesized complete accurate chromosome segregation. In contrast, 4-OH- that its deficiency would lead to changes in cell cycle dynamics and TAM-treated control cells did not demonstrate an increase in mitotic distribution. Indeed, the decrease in cell growth within two passages abnormalities (Fig. 4A). To evaluate the appearance of abnormal was associated with significant changes in cell cycle distribution mitotic cells in more detail, we also collected control and Smc5 (Fig. 2E). PSCs have a relatively short G1 phase, and prevalence of a mutant cells after nocodazole-induced mitotic block and release at cell population in S phase (Li et al., 2012). After 5 and 8 days (P2 time points corresponding to day 3, 4 and 5 of 4-OH TAM treatment and P3) of 4-OH TAM treatment, we observed a significant decrease (Fig. S4A). Numerous abnormal mitotic cells were clearly detected in the percentage of experimental cells in S phase (minus 10.19% after 4 and 5 days of 4-OH TAM treatment, but not after 3 days and 10.3% for P2 and P3, respectively, compared to plus 1% for P1) (Fig. S4A). and accumulation of cells in G2 phase (plus 10.87% and 10.7% for Our study of Smc5/6 complex localization during normal mESC P2 and P3, respectively, compared to plus 1.29% for P1) (Fig. 2E). mitosis showed that the complex accumulates at pericentromeric These changes in cell cycle distribution could be a result of DNA regions (Fig. 3). Depletion of Smc5 protein led to disappearance of damage during interphase and the inability of cells to segregate Smc5 from mitotic chromosomes (Fig. 4C). We also observed the chromosomes (Mankouri et al., 2013). We also detected an increase absence of Smc6 and Nse1 components from pericentromeric in the polyploid cell population (∼7%, Fig. 2F). Giemsa staining of regions of chromosomes in cells undergoing abnormal mitosis Smc5-depleted cells confirmed the presence of cells with an (Fig. 4D). These data underline the importance of Smc5/6 complex abnormal karyotype (Fig. 2G). We did not observe changes in cell in chromosome segregation. cycle distribution or polyploid cell population in either the untreated To further enhance our observations, we synchronized control (Smc5−/flox and Smc5+/flox) or control mESCs treated with 4-OH and Smc5-depleted mESCs in the G2 phase of the cell cycle and TAM (Smc5+/−) (Fig. 2E,F). analyzed cell progression by flow cytometry (Fig. 4E). In support of our previous data obtained using asynchronous cells, we observed a Smc5/6 complex is associated with pericentromeric mitotic delay and accumulation of Smc5-deficient mESCs in G2 and localizes at spindle poles during mitosis phase (Figs 2E and 4E). A previous study employing human To investigate the reasons for disrupted mitotic progression and death somatic RPE-1 cells has demonstrated the requirement of Smc5/6 of Smc5-depleted mESCs, we used whole-cell immunofluorescence complex for timely progression of DNA synthesis (Gallego-Paez microscopy. First, we characterized the localization of Smc5/6 et al., 2014). Given that DNA replication stress can further cause Journal of Cell Science

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Fig. 3. Evaluation of Smc5/6 complex component localization during mESC cycle. (A) Localization of Smc6 protein during mitosis. (B) Localization of Nse1 component during mitosis. (C) Localization of Smc5 protein during mitosis. (D) Chromosome spreads showing Smc5/6 component localization on mitotic chromosomes. DNA is stained with DAPI (blue). Scale bars: 5 µm. defects in stem cell chromosome condensation and abnormal Smc5 mutation in mESCs leads to abnormal distribution of segregation (Lamm et al., 2015), we synchronized mESCs in the G1 condensin along chromosomes phase of cell cycle and observed the progression through the S phase Based on previous siRNA-mediated knockdown studies of the (Fig. 4E). Owing to accumulation of Smc5-depleted mESCs in G2 Smc5/6 complex in mammalian cell lines, we hypothesized that the phase, we could not obtain a G1-enriched mESC population similar chromosome bridges and lagging chromosomes we observed after to controls. However, the majority of cells entered S phase and did Smc5 depletion could be attributed to defects in chromosome not demonstrate a delay in DNA replication. There was a minor cell cohesion, condensation and/or topoisomerase IIα (Topo IIα) population that remained in G1 phase. Given that mESCs do not function (Wu et al., 2012; Gallego-Paez et al., 2014; Gomez demonstrate G1 cell cycle arrest, accumulation of cells in G1 phase et al., 2013). could be explained by acquisition of chromosomal damage during Timely removal of pericentromeric cohesin is a prerequisite for mitosis (Fig. 4E). accurate sister chromatid segregation during the metaphase-to- Journal of Cell Science

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− − Fig. 4. Smc5 deficiency causes mitotic abnormalities. (A) Smc5 depletion (Smc5 / ) leads to an increase in abnormal mitotic mESCs. Bars represent mean±s.e.m. (three independent experiments, n>150 cells for each group). *P=0.01, **P=0.0112 (based on paired, one tailed t-tests). (B) DAPI-stained control anaphase and Smc5-depleted mitotic cells with lagging chromosomes, DNA bridges and abnormal mitotic spindles with mis-segregated chromosomes. (C) mESC chromosome spreads showing Smc5 depletion. (D) Loss of Smc6 and Nse1 proteins at pericentromeric regions of chromosomes was observed in Smc5-depleted mitotically abnormal mESCs. (E) FACS analysis of mESC cycle progression after synchronization in G1 and G2 phases. After release from mitotic − − block, Smc5-deficient cells (Smc5 / ) demonstrated obstructed mitotic progression. However, there was no delay in proceeding through S phase after the release from G1 phase arrest. This experiment was repeated twice, and consistent results were obtained. All cells in this figure were analyzed on day 5 of 4-OH TAM treatment. Scale bars: 5 µm. Journal of Cell Science

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Fig. 5. Immunofluorescence microscopy analyses of proteins involved in chromosome cohesion and resolution of DNA joint molecules. (A,B) The cohesin component Rad21 persisted at pericentromeric regions of chromosomes in prometaphase cells (top panels) and dissociated from chromosomes in − − − anaphase (bottom panel) in both untreated (Smc5 /flox) (A) and 4-OH TAM-treated (Smc5 / ). (B) experimental mESCs. (C) Chromosome spreads showing no changes in Rad21 localization on mitotic chromosomes after depletion of Smc5. (D) Chromosome spreads showing no changes in Topo IIα localization on mitotic chromosomes after depletion of Smc5. (E) Topo IIα showed pericentromeric localization in mitotic untreated experimental mESCs and was not affected after Smc5 depletion (F). All mESCs were assessed after 5 days of 4-OH TAM treatment. Scale bars: 5 µm. anaphase transition (Giménez-Abián et al., 2004). Thus, we tested mitotic chromosome spreads did not reveal mislocalization of Topo the localization of Rad21, a cohesin subunit, in mitotic mESCs IIα (Fig. 5D). (Fig. 5A–C). In cells with functional Smc5/6, cohesin was enriched Condensins are required for structural organization of mitotic at pericentromeric regions in prometaphase and dissociated from chromosomes ensuring accurate segregation during anaphase chromosomes after anaphase onset (Fig. 5A,C). We observed (Hirano, 2012; Thadani et al., 2012). Previous studies have shown similar localization pattern for cohesin in Smc5-depleted cells, aberrant distribution of condensin in Smc5/6-depleted human suggesting that aberrant cohesin removal was not the reason for RPE-1 cells (Gallego-Paez et al., 2014). Thus, we investigated lagging chromosomes and DNA bridges (Fig. 5B,C). whether the localization of condensin subunit Smc4 was affected. In Incomplete decatenation of centromeric DNA and the presence of mESCs with a functional Smc5/6 complex, we observed enrichment unresolved joint molecules can lead to chromatin bridge formation of condensin at pericentromeric regions during prometaphase in mitotic cells (Mankouri et al., 2013). The separation of sister (Fig. 6A). In contrast, most Smc5-depleted prometaphase cells centromeres requires the activity of Topo IIα (Liu et al., 2014). demonstrated decreased condensin accumulation at pericentromeric Previous studies on human Smc5/6-depleted RPE-1 cells have regions, and the condensin signal was increased along chromosome reported mislocalization of Topo IIα from pericentromeric arms (Fig. 6B). Quantification of the condensin signal along regions to arms and distal ends of chromosomes (Gallego-Paez chromosome arms and pericentromeric regions supported our et al., 2014). However, we did not observe a defect in Topo IIα observations (Fig. 6C–E). We also evaluated condensin distribution localization. In Smc5-depleted mESCs, that were undergoing on mitotic chromosome spreads from control and Smc5 mutant chromosome missegregation, Topo IIα remained enriched at the mESCs, which confirmed the data obtained with whole-cell pericentromeric regions (compare Fig. 5E with Fig. 5F). Analysis of immunocytochemistry (Fig. 6F,G). In summary, our results Journal of Cell Science

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Fig. 6. Immunofluorescence microscopy analysis of condensin protein Smc4. (A) The core condensin component Smc4 was enriched at pericentromeric − regions in untreated experimental mESCs (Smc5 /flox) at prometaphase (top panel) and decreased after anaphase onset (bottom panel). (B) Smc5-depleted cells − − (Smc5 / ) showed decreased pericentromeric Smc4 and increased condensin distribution along chromosome arms before (top panel) and after (bottom panel) anaphase onset. (C–E) Quantification data showing aberrant distribution of condensin in Smc5-depleted cells in comparison with control cells. Data demonstrated an increase of condensin on chromosome arms (C), a decrease of condensin at pericentromeric regions (D), and a decrease in the ratio of centromere/arm condensin signal (E). Bars represent mean±s.d., and P values are given for indicated comparisons (Mann–Whitney, two-tailed, n>30 for each genotype). (F,G) Chromosome spreads showing an increase of condensin on chromosome arms, a decrease at pericentromeric regions and less-defined distribution along chromosome arms after Smc5 depletion (G). All mESCs were assessed after 5 days of 4-OH TAM treatment. Scale bars: 5 µm. demonstrate that the Smc5/6 complex is essential for normal In mESCs, Plk1 was enriched at pericentromeric regions of chromosome condensation in mESCs. chromosomes from prometaphase through mitosis and also redistributed to spindle poles and microtubules during metaphase Smc5 mutation in mESCs causes aberrant localization of and anaphase (Fig. 7A). Smc5-depleted mitotic cells displayed Plk1 and Aurora B diminished Plk1 enrichment at pericentromeric regions (Fig. 7B). Dynamic binding to chromatin and DNA supercoiling activity of Aurora B is required for localization of spindle condensin is regulated by mitotic kinases, including Aurora B assembly checkpoint (SAC) proteins, including Mad2 (also known and Polo-like kinase 1 (Plk1) (Hirano, 2012; Thadani et al., 2012). as MAD2L1), which are collectively required to ensure amphitelic Journal of Cell Science

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Fig. 7. Effects of Smc5 depletion on localization of mitotic kinase Plk1 and SAC protein Mad2. (A) Pericentromeric and spindle pole localization of Plk1 in − experimental mESCs with functional Smc5/6 complex (Smc5 /flox) at prometaphase (top panel), metaphase (middle) and anaphase (bottom panel), including − − relocalization of Plk1 on spindle microtubules at cleavage furrow in anaphase cells (bottom panel). (B) In Smc5-depleted cells (Smc5 / ) undergoing abnormal mitosis, Plk1 still persisted at spindle poles, but was diminished at pericentromeric regions of chromosomes (all three panels). (C) In mESCs with functional Smc5/ 6 complex, Mad2 is located at pericentromeric regions and centrosomes at prometaphase (top panel), at spindle poles during metaphase (middle panel) and dissociates from mitotic apparatus after anaphase onset (bottom panel). (D) Smc5-depleted cells show persistence of Mad2 protein at pericentromeric regions in prometaphase mESCs (top panel). However, the Mad2 signal was generally absent in cells undergoing mitosis (middle and bottom panels). (E,F) Chromosome spreads showing no changes in Mad2 localization on mitotic chromosomes after depletion of Smc5. All mESCs were assessed after 5 days of 4-OH TAM treatment. Scale bars: 5 µm. kinetochore attachment to microtubules prior to the metaphase-to- (Schuyler et al., 2012). The decrease in Mad2 level in Smc5- anaphase transition (Maresca, 2011; Schuyler et al., 2012). depleted cells suggests that SAC was either satisfied prior to Although we observed persistence of Mad2 protein at chromosome segregation or SAC function was disrupted (Schuyler pericentromeric regions in prometaphase mESCs and on et al., 2012). chromosome spreads, the Mad2 signal was diminished after Smc5 In addition to its canonical role in regulating the SAC, Aurora B depletion in mitotic whole cells (Fig. 7C–F). Mad2 was generally kinase is required for appropriate chromosome condensation absent in anaphase cells, consistent with its role in SAC (Fig. 7C,D) (Kitagawa and Lee, 2015). In mESCs, we detected Aurora B Journal of Cell Science

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− Fig. 8. Effect of Smc5 depletion on localization of mitotic kinase Aurora B. (A) In mESCs with functional Smc5/6 complex (Smc5 /flox), Aurora B localized along chromosome arms and at centromeres during prophase (top panel), concentrating at centromeric regions in prometaphase and metaphase (2nd and 3rd row) and relocating on spindle microtubules at the cleavage furrow in anaphase cells (two bottom panels). (B) In Smc5-depleted mESCs Aurora B dissociation − from chromosome arms was obstructed: (top panel) unaffected control cells (Smc5 /flox) showing pericentromeric Aurora B localization, and (middle and − − bottom panels) Smc5-depleted mESCs (Smc5 / ) showing persistence of Aurora B on chromosome arms during mitosis. (C) Chromosome spreads showing an increase of Aurora B concentration along chromosome arms after Smc5 depletion. (D) Quantification of Aurora B signal showing increased intensity on chromosome arms in Smc5-depleted cells in comparison with control cells. (E) No significant difference in Aurora B intensity observed at pericentromeric regions of control and Smc5-deficient mESCs. Bars represent mean±s.d., and the P value is given for the indicated comparison (Mann–Whitney, two-tailed, n>30 for each genotype). All mESCs were assessed after 5 days of 4-OH TAM treatment. Scale bars: 5 µm. along chromosome arms and near centromeres during prophase, Excessive Aurora B activity during mitosis induces defective near centromeres during prometaphase and metaphase, and on chromosome congression and causes chromosome missegregation spindle microtubules at the cleavage furrow during anaphase (Dobrynin et al., 2011). Thus, we tested Aurora B activity by (Fig. 8A). Smc5-depleted cells displayed aberrant distribution of assessing H3 phosphorylation at the Ser10 residue (H3S10). Aurora B in mitotic cells. Aurora B was enriched at centromeric In mESCs phosphorylation of H3S10 is detectable in the S phase regions, but in contrast to control cells, persisted along chromosome of cell cycle and reaches the maximum during mitosis (Mallm arms (Fig. 8B–E). and Rippe, 2015). Our analysis did not reveal aberrancies in the Journal of Cell Science

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H3S10 phosphorylation pattern (Fig. S4B). Interestingly, H3S10 Our observations also complement a recent report using budding phosphorylation signal revealed chromatin fibers that appeared to yeast, which demonstrated that the absence of Smc5/6 during connect chromosomes during mitosis (Fig. S4B). S-phase does not impact upon DNA replication efficiency and cell Finally, intrigued by the increased amount of abnormal mitotic proliferation, whereas the absence of Smc5/6 during the G2/M cells after Smc5 depletion, we used H3S10 phosphorylation to transition causes cell cycle arrest and lethality (Menolfi et al., 2015). calculate the percentage of cells at different stages of mitosis, as well That study specified that Smc5/6 is required to resolve as cells in prophase. We recorded an increase in the population of recombination structures formed from endogenous replication cells in late prophase (Fig. S4C). This finding suggests that defects stress and late replication pausing sites. Human PSCs are prone to observed in Smc5-depleted mESCs during mitosis are due to genomic instability due to replicative stress and impaired aberrancies that occur during the prophase-to-prometaphase chromosome condensation (Lamm et al., 2015). It is likely that transition. Smc5/6 plays a crucial role in avoiding these sources of genomic instability in PSCs. We speculate that perturbation of Smc5/6 DISCUSSION function could result in PSC transformation and tumor formation PSCs are unique cells capable of differentiation into numerous from other cell types with impaired intra-S or decatenation tissue-specific cell types and share some characteristics with cancer checkpoints. cells (Evans and Kaufman, 1981; Martin, 1981; Thomson et al., Observing cells with abnormal mitotic spindles and 1998). Studying PSCs might help elucidate processes supporting missegregating chromosomes prompted us to investigate mitotic genetic stability of stem cells, reveal factors leading to malignant regulators involved in chromosome congression and segregation. transformation and possibly find therapeutic targets to cure cancers. We showed that Smc5 depletion in mitotic mESCs resulted in a Functions of the Smc5/6 complex have been investigated in decrease of condensin signal at the pericentromeric regions and different model systems and cell types from budding yeast to persistence of condensin along chromosome arms. Abnormal human cell lines (reviewed in Carter and Sjögren et al., 2012; condensin distribution on mitotic chromosomes correlated with Jeppsson et al., 2014; Langston and Weinert, 2015; Murray and diminished Plk1 enrichment at pericentromeric regions. A previous Carr, 2008; Verver et al., 2016; Wu and Yu, 2012). These reports study on human cells showed that stable localization of Plk1 to the have provided information about the crucial roles of the Smc5/6 kinetochores requires an interaction with the condensin II subunit complex in preserving genomic integrity. However prior to our Ncapg2 (Kim et al., 2014). Plk1 is required for stabilizing research, mammalian PSCs had not been assessed. Furthermore, kinetochore–microtubule attachments by counteracting the spindle previous reports studying the function of Smc5/6 using human cells assembly checkpoint function of Aurora B (Suijkerbuijk et al., have been hindered by siRNA off-target effects (Wu et al., 2012). 2012). As we observed reduced Plk1 localization, but no effect on Our conditional knockout strategy using Cre-ERT2 recombinase- the enrichment of Aurora B at centromeric regions, it is likely that induced mutation of Smc5 avoided off-target effects, and resulted in Aurora B is overactive during mitosis. Studies using mammalian cell efficient depletion of Smc5 as well as Smc6. This suggests that lines have shown that Plk1 and Aurora B are also required to Smc5 and Smc6 are stabilized within the context of the Smc5/6 coordinate chromosome condensation. Plk1 phosphorylates the complex. condensin II subunit Cap-D3 (also known as Ncapd3), which leads We revealed varying effects of Smc5 depletion in embryonic to correct chromosome assembly prior to segregation (Abe et al., versus somatic cells. mESCs begin to undergo apoptosis shortly 2011). Association of condensin I to the chromatin is regulated by after Smc5 depletion, whereas MEFs display diminished Aurora-B-mediated phosphorylation (Lipp et al., 2007; Tada et al., proliferative capacity. Observed outcomes highlight different 2011). As we observe increased enrichment of condensin and needs for the Smc5/6 complex or distinct regulation of cellular Aurora B along chromosome arms, Smc5/6 might be essential for processes in these cell types. Similar observations have been coordinating the release of Aurora B from chromosome arms after reported when condensin I and II are both depleted in mESCs condensin I deposition. A systematic phosphorylation screen of compared to MEFs (Fazzio and Panning, 2010). Species- and cell- human mitotic protein complexes demonstrated that Smc5, Smc6 type-specific differences have been also reported by others. For and NSMCE4 are phosphorylated, and these phosphorylation example, after depletion of the core condensin subunit Smc2, mouse events are sensitive to chemical inhibition of either Aurora B neural stem cells (NSCs) progressed through mitosis, underwent or Plk1 (Hegemann et al., 2011). Therefore, determining the defective chromosome segregation and DNA damage-induced importance of these modifications during mitosis might provide apoptosis. However, the depletion of Smc2 in human RPE-1 cells insight into the mechanistic link between the Smc5/6 complex and causes senescent-like phenotype and cell cycle arrest in G1 phase these cell cycle kinases. (Nishide and Hirano, 2014). Additionally, Smc5 knockdown in Depletion of the protein degradation chaperone complex Cdc48– chicken DT40 lymphoma cells and human RPE-1 cells does not Ufd1–Npl4 (Cdc48 is also known as p97 and VCP; Npl4 is also affect cell viability, although cells showed lower proliferation rates known as NPLOC4), in mitotic HeLa cells has been reported to (Stephan et al., 2011; Gallego-Paez et al., 2014). cause abnormal distribution of Aurora B along chromosome arms Contrary to somatic cells, ESCs do not have effective G1/S phase (Dobrynin et al., 2011). Furthermore, the persistence of Aurora B cell cycle checkpoints. Instead, ESCs with acquired DNA damage resulted in defects in chromosome congression and segregation, proceed to S phase and accumulate in G2 phase (Weissbein et al., similar to what we observe in Smc5 mutant mESCs. The Ufd1 2014; van der Laan et al., 2013; Desmarais et al., 2012). We subunit of this chaperone complex has been shown to bind ubiquitin observed an increase in the G2 population of Smc5-depleted and small ubiquitin-like modifications (SUMO) (Nie et al., 2012). mESCs. We also observed a minor fraction of polyploid cells, which SUMO modification of Aurora B stimulates its removal from suggests that some cells did not complete mitosis and underwent chromosome arms during prometaphase (Fernandez-Miranda et al., another round of DNA replication. Our results echo the outcomes of 2010). The Smc5/6 complex components Nse1 and Nse2 are E3 condensin knockdown studies in mESCs (Fazzio and Panning, ubiquitin and SUMO ligases, respectively (reviewed in Verver et al.,

2010). 2016). Our study allows us to speculate that the Smc5/6 complex Journal of Cell Science

1630 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 1619-1634 doi:10.1242/jcs.179036 might be required for the ubiquitin or SUMO modification of mitotic kinases Plk1 and Aurora B. We evaluated condensin Aurora B, which ensures timely removal of Aurora B from localization by visualizing Smc4, a common subunit of condensin I chromosome arms. Interestingly, in budding yeast, it has been and II, which prompts future investigation of each condensin shown that Nse2 is required for normal SUMOylation levels of Bir1 complex. Our study provides the platform for further detailed (also known as survivin and Birc5), which is a direct interaction investigation of Smc5/6 functions in stem cell maintenance, DNA partner of Aurora B (Yong-Gonzales et al., 2012). However, direct replication and mitosis. Collectively, this will lead to a better SUMO modification by Nse2 might not be essential, as mice understanding of stem cell genomic instability and the expressing an Nse2 mutant allele deficient for SUMOylation developmental abnormalities caused by Smc5/6 perturbation. activity did not show any phenotypic aberration (Jacome et al., 2015). MATERIALS AND METHODS During DNA replication, sister chromatids become topologically Animal use and care intertwined and these catenane structures must be resolved by Topo Mice were bred by the investigators at The Jackson Laboratory (JAX, Bar IIα to permit chromosome segregation (Liu et al., 2014). Mutation Harbor, ME) and Johns Hopkins University (JHU, Baltimore, MD) in of Smc5 in mESCs resulted in chromosome segregation defects accordance with criteria of the NIH and USDA. All animal procedures were reminiscent of a defect in Topo IIα function. Similar to our conducted with approval from the IACUC of JAX and JHU. observations, Smc5/6 depletion in human RPE-1 cells results in the presence of DNA bridges and lagging chromosomes during mitosis Mice (Gallego-Paez et al., 2014). This phenotype was attributed to mESC clone EPD0395_1_F05 (C57BL/6N-A/a genetic background) bearing a ‘knock-out first’ allele of Smc5 (Smc5tm1a(KOMP)Wtsi) were impaired localization of Topo IIα and condensin to pericentromeric α acquired from the Knockout Mouse Project (http://www.mousephenotype. regions. Although we did not observe mislocalization of Topo II in org/data/alleles/MGI:2385088/tm1a%28KOMP%29Wtsi). our Smc5 mutant mESCs, we did so for condensin, and this might be Chimeras were obtained by microinjection of EPD0395_1_F05 mESCs the direct cause of chromosome missegregation. In budding yeast, it into C57BL/6J blastocyst-stage mouse embryos and were assessed for has been shown that condensin is required to facilitate Topo-IIα- germline transmission. Heterozygous progeny were bred with a C57BL/6J mediated resolution of catenanes prior to the metaphase-to- Flp recombinase deleter strain (B6.129S4-Gt(ROSA)26Sortm1(FLP1)Dym/ anaphase transition (Charbin et al., 2014). Our results suggest that RainJ, JAX) to remove the SA-LacZ and Neo selection cassette and flox the Smc5/6 complex is required for stable localization of condensin produce the floxed exon 4 (designated Smc5 ). To produce offspring heterozygous for the deleted exon 4 (designated to the pericentromeric regions, and in its absence it is conceivable del flox that Topo IIα cannot efficiently stimulate sister chromatid Smc5 ), heterozygous Smc5 males were mated to Sox2-Cre C57BL/6J (B6.Cg-Tg(Sox2-cre)1Amc/J, JAX) mice. Then, heterozygous Smc5del decatenation. Alternatively, from work using DT40 cells and T2 α mice were bred to mice harboring the conditional Cre-ER (B6.129- budding yeast, it has been reported that condensin and Topo II Gt(ROSA)26Sortm1(cre/ERT2)Tyj/J, JAX), which resulted in progeny demonstrate opposing activities, where condensin promotes DNA heterozygous for the Smc5del allele and hemizygous for the Cre-ERT2 compaction and Topo IIα promotes DNA relaxation (Samejima genotype. These mice were bred to homozygous Smc5flox mice to derive et al., 2012; Leonard et al., 2015). Smc5/6 might be required to experimental (Smc5flox/del, Cre-ERT2) and control (Smc5wt/flox, Cre-ERT2) mediate the balance between condensin and Topo IIα during genotypes. mitosis, which is essential for the efficient resolution of intertwined sister chromatids. Establishment of mESC lines Smc5/6 components primarily localize to the pericentromeric For mESC derivation and culture we used 2i medium described by Ying , but also along chromosome arms in mESCs. This et al. (2008) and Hanna et al. (2010). Briefly, mESC culture medium is consistent with what was previously published using MEFs included 1:1 mixture of DMEM/F12 and neurobasal medium with 1% N2 and 2% B27 supplements, 1 mM L-Glutamine, 1% MEM NEAA (Gomez et al., 2013). These observations are in contrast to what has β been reported using RPE-1 cells, where Smc5 and Smc6 are (Invitrogen), 50 µM -mercaptoethanol (Sigma), 50 µg/ml BSA (Sigma), 10 ng/ml human leukemia inhibitory factor (Cell Signaling), 1 µM MEK enriched within the nucleus during interphase, but then greatly inhibitor PD 0325901 and 3 µM GSK-3 inhibitor CHIR 99021 (Tocris). diminished from the chromatin during mitosis (Gallego-Paez et al., mESC lines were established from whole embryos (Behringer et al., 2014). 2014). However, some Smc5 and Smc6 is still present in the After 5–6 passages on primary MEF feeder, mESC lines were switched chromatin fraction during mitosis in RPE-1 cells, and the to a feeder-free culture on 0.2% gelatin and passaged every 3 days with immunocytochemistry with anti-GFP antibodies demonstrates a seeding density 5×103/cm2. mESCs were analyzed within 20 passages of strong chromatin signal in mitotic HeLa cells expressing EGFP– culture. Smc5 used in the same study. Therefore, the differences might be easily reconciled with further detailed assessments. MEF derivation Budding yeast Smc5 binds directly to microtubules and mutation Primary MEFs were derived from F1 FVBxC57BL/6J embryos at 13.5 days of the microtubule-binding region of Smc5 results in compromised post coitum (dpc) (Behringer et al., 2014). MEF culture medium consisted microtubule stability (Laflamme et al., 2014). We report that Smc5/ of 89% DMEM (Invitrogen), 10% fetal bovien serum (FBS; Hyclone), 1% γ 6 components localize to the spindle poles, and depletion of Smc5 penicillin and streptomycin (100 U/100 µg). For feeder preparation, - results in the presence of abnormal mitotic spindles. It is possible irradiated MEFs (20 Gy) were plated into six-well plates precoated with 0.1% gelatin at density 2×104/cm2. Feeder was used for mESC culture on that the Smc5/6 complex is involved in the regulation of centrosome the next day after cell plating. The standard NIH 3T3 protocol was used for function, and required for stabilizing microtubules that promote establishing immortal MEF cell lines. normal mitotic spindle formation. Our study has demonstrated that the Smc5/6 complex is crucial Embryoid body formation for mESC maintenance and cell cycle progression. Destabilization For embryoid body formation, mESC aggregates were grown in a mixture of of the Smc5/6 complex in mESCs affects multiple aspects of the cell differentiation medium [15% FBS (HyClone) and 85% DMEM/F12 cycle and leads to mitotic catastrophe. We have shown that Smc5/6 (Invitrogen)] and mESC culture medium at a ratio of 2:1. After 3 days, is required for regular distribution and functioning of condensin and medium was changed to differentiation medium only. Medium was changed Journal of Cell Science

1631 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 1619-1634 doi:10.1242/jcs.179036 every 3 days. Embryoid bodies were collected for western blot analysis on following nocodazole treatment, thymidine was used at a concentration day 0, 6, 12 and 18 of differentiation. 2 mM for the next 4 h. Cells were collected for cell cycle analysis at indicated time points. Teratoma formation assay mESCs were mechanically collected from tissue culture plastic, washed once Cell cycle analysis in PBS and resuspended in 80% of DMEM/F12 and 20% Geltrex (Invitrogen). Approximately 1×106–2×106 mESCs were collected, washed in PBS mESCs (2×106–4×106) were injected subcutaneously into right hind limb of and resuspended in 1 ml of PBS. Cells were fixed by adding 3 ml cold the NOD/SCID mouse (The Jackson Laboratory). Teratomas were collected (−20°C) absolute ethanol to cell suspension while vortexing and were kept after 4 weeks with a size of 1–2cm3. Isolated teratomas were fixed in 10% overnight at 4°C. For cell cycle analysis, cells washed in PBS were formalin, embedded in paraffin, sectioned onto glass microscope slides with a resuspended in 1 ml of staining solution (0.1% Triton X-100 in PBS, 5-µm thickness and stained with hematoxylin and eosin. 0.2 mg/ml RNase A and 20 μg/ml propidium iodide) and kept overnight at 4°C in the dark. DNA content was determined by flow cytometry using BD Cre recombinase-induced excision of Smc5 intron 4 FACS Calibur and Cell Quest software. Total populations were gated to Cre recombinase activity was induced by 4-hydroxytamoxifen (4-OH remove cell debris and doublets. Data were analyzed using FlowJo software. TAM) (Sigma) at a dose of 0.2 µM, which was added into cell culture medium for 3 days (mESCs) or 10 days (MEFs). After 3 or 10 days of Semi-quantitative RT-PCR treatment the dose of 4-OH TAM was decreased to 0.05 µM and kept at Total RNA was isolated using PureLink RNA kit (Ambion) followed by this level until the end of experiments. Fresh 4-OH TAM was added daily DNase I (RNase-free, Ambion) treatment. First strand cDNA synthesis was into mESC culture and every 2 days for MEFs. Excision of intron 4 was carried out using the Enhanced Avian HS RT-PCR kit (Sigma). PCR confirmed by PCR analysis on days 1–3 of treatment in mESCs, and on amplification of cDNA samples was set with HotMaster Taq DNA day 5 and day 10 of treatment for MEFs. For further information see the polymerase (5 Prime). Primers are listed in Table S2. PCR reaction Results section and Fig. S2A. conditions were: initial denaturation at 94°C for 2 min, (denaturation at 94°C for 20 s, annealing at 55°C for 10 s, amplification at 70°C for 30 s)×28 Cell collection and viability cycles, and final extension at 70°C for 10 min. Cells were expanded in large quantity and simultaneously collected for DNA, RNA, western blotting and cell cycle analyses. Cells were washed in Immunocytochemistry PBS, counted and snap-frozen in aliquots for further evaluation or were For pluripotency marker analysis, mESCs were plated on glass coverslips processed immediately. Cell viability was determined based on Trypan Blue (Electron Microscopy Sciences) precoated with 0.1% gelatin and containing die incorporation using a Bio-Rad TC20 cell counter. MEF feeder. mESCs were fixed with 10% formalin for 20 min, washed in PBS and used for immunostaining. For single-cell analysis, mESCs were fixed in ∼ – Genotyping suspension and 50,000 100,000 cells were spun onto glass micro slides DNA was extracted using the GeneJet Genomic DNA purification kit (Cardinal Health) in Shandon Cytospin 4 centrifuge (200 g, 3 min). For (Thermo Scientific) and 20 ng was used for each PCR. PCRs were set with chromosome spread preparation, mESCs were treated with 0.1 µg/ml HotMaster Taq DNA polymerase (5 Prime). The floxed allele (563 bp) KaryoMax colcemid solution (Invitrogen) for 1.5 h. Cells were collected, and the wild-type allele (410 bp) were amplified with primers 1 (forward placed into hypotonic solution (PBS:water, 4:6 mix) for 5 min at room 5′-ACTCAGTCTCACACGGCAAG-3′) and 2 (reverse 5′-ATCCTTCCC- temperature and dropped onto ice-cold glass slides. Chromosome spreads were ACCTTGGAAAC-3′), the floxed allele (644 bp) was amplified with fixed and permeabilized in 10% formalin with 0.5% Triton X-100 for 15 min. primers 3 (forward 5′-AGAAAGACATCAAACTAACCGCTGGC-3′) and Immunocytochemistry was performed as described previously (Pryzhkova 4 (reverse 5′-GAGATGGCGCAACGCAATTAAT-3′). The deletion allele et al., 2014). Primary antibodies used are listed in Table S1. Secondary was amplified as a 763-bp product with primers 1 and 4. PCR reaction Alexa-Fluor-conjugated (488, 568 or 633) antibodies were goat anti-human- conditions were: initial denaturation at 94°C for 2 min, (denaturation at 94°C IgG, anti-mouse-IgG and anti-rabbit-IgG antibodies (Invitrogen). To for 20 s, annealing at 58°C for 30 s, amplification at 72°C for 1 min)×30 detect cell DNA, we used VectaShield mounting medium with DAPI cycles, and final extension at 72°C for 10 min. (Vector Laboratories). The detection of alkaline phosphatase activity was carried out using Vector Red Alkaline Phosphatase Substrate (Vector Laboratories). Karyotyping mESCs (untreated and treated with 4-OH TAM for 5 days) were mitotically Microscopy arrested by incubating with 0.1 µg/ml KaryoMax colcemid solution Samples were analyzed using either a Zeiss AxioImager A2 or (Invitrogen) for 2 h. Cells were collected, placed into hypotonic solution Cell Observer Z1 fluorescent microscopes and captured using AxioCam (0.075 M KCl) at 37°C for 20 min and fixed in methanol:acetic acid (3:1). ERc 5 s (Zeiss) or a ORCA-Flash 4.0 CMOS camera (Hamamatsu), Fixed cells were dropped onto wet, ice-cold glass micro slides (Cardinal respectively. Images were analyzed and processed using ZEN 2012 blue Health) and stained with KaryoMax Giemsa stain solution following the edition imaging software (Zeiss). Photoshop (Adobe) was used to prepare manufacturer’s instructions (Invitrogen). figure images.

Western blot analysis Acknowledgements Cell lysates were prepared in RIPA buffer (Santa Cruz Biotechnology) We thank Mike Matunis for critical comments on the manuscript. We thank Laura supplemented with protease inhibitor cocktail (Roche). Equal amounts of Morsberger and Raluca Yonescu from the JHU Cytogenetics Core Facility for proteins were fractionated by SDS-PAGE and transferred to PVDF guidance with karyotype analysis. membrane (Bio-Rad). Primary antibodies are provided in Table S1. We used horseradish peroxidase (HRP)-conjugated goat anti-mouse-IgG and Competing interests anti-rabbit-IgG secondary antibodies (Invitrogen). Signal was detected The authors declare no competing or financial interests. using Clarity Western ECL Substrate (Bio-Rad) and imaged using Syngene XR5 system. Author contributions M.V.P. and P.W.J. conceived, designed and performed experiments, analyzed data and wrote the paper. Cell synchronization To synchronize mESCs (untreated and 4-OH TAM-treated for 5 days) in the Funding G2/M phase of cell cycle, nocodazole at a concentration of 50 ng/ml was This work was supported by the National Institute of Child Health and Human added to cell culture medium for 6 h. For cell synchronization in G1 phase, Development [grant number R00HD069458 to P.W.J.]; and National Institute of Journal of Cell Science

1632 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 1619-1634 doi:10.1242/jcs.179036

General Medical Sciences [grant number R01GM117155 to P.W.J.] of the National Hirano, T. (2012). Condensins: universal organizers of chromosomes with diverse Institutes of Health. Deposited in PMC for release after 12 months. functions. Genes Dev. 26, 1659-1678. Jacome, A., Gutierrez-Martinez, P., Schiavoni, F., Tenaglia, E., Martinez, P., ı́ ́ Supplementary information Rodr guez-Acebes, S., Lecona, E., Murga, M., Mendez, J., Blasco, M. A. et al. Supplementary information available online at (2015). NSMCE2 suppresses cancer and aging in mice independently of its SUMO ligase activity. EMBO J. 34, 2604-2619. http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.179036/-/DC1 Jeppsson, K., Kanno, T., Shirahige, K. and Sjögren, C. (2014). The maintenance of chromosome structure: positioning and functioning of SMC complexes. Nat. References Rev. Mol. Cell Biol. 15, 601-614. 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