Distinct Functions of Condensin I and II in Mitotic Chromosome Assembly
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Research Article 6435 Distinct functions of condensin I and II in mitotic chromosome assembly Toru Hirota1,*, Daniel Gerlich2,*, Birgit Koch1, Jan Ellenberg2 and Jan-Michael Peters1,‡ 1Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, 1030 Vienna, Austria 2European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany *These authors contributed equally to this work ‡Author for correspondence (e-mail: [email protected]) Accepted 22 October 2004 Journal of Cell Science 117, 6435-6445 Published by The Company of Biologists 2004 doi:10.1242/jcs.01604 Summary Condensin is a protein complex associated with mitotic progression through prometaphase and metaphase, chromosomes that has been implicated in chromosome whereas normal condensin II levels are dispensable for condensation. In vertebrates, two types of condensin these processes. After depletion of both condensin complexes have recently been identified, called condensin I complexes, the onset of chromosome condensation is and II. Here, we show that in mammalian cells condensin delayed until the end of prophase, but is then initiated II associates with chromatin in prophase, in contrast to rapidly before nuclear envelope breakdown. These results condensin I which is cytoplasmic and can thus interact with reveal that condensin II and I associate with chromosomes chromosomes only after nuclear envelope breakdown. RNA sequentially and have distinct functions in mitotic interference experiments in conjunction with imaging of chromosome assembly. live and fixed cells revealed that condensin II is required for chromosome condensation in early prophase, whereas condensin I appears to be dispensable at this stage. By Supplementary material available online at contrast, condensin I is required for the complete http://jcs.biologists.org/cgi/content/full/117/26/6435/DC1 dissociation of cohesin from chromosome arms, for chromosome shortening and for normal timing of Key words: Chromatin, Condensation, Cohesin, Cohesion, Prophase Introduction and in small amounts on chromosome arms until metaphase During mitosis, chromatin is structurally reorganized into when its Kleisin subunit Scc1 is cleaved by the protease condensed chromosomes that become first visible as long separase (reviewed in Nasmyth and Schleiffer, 2004). threads in prophase. In prometaphase and metaphase, A different Smc-Kleisin complex, called condensin, chromosomes are shortened and the sister chromatids, which becomes specifically enriched in axial structures of sister each contain one copy of the replicated DNA, are partially chromatids during mitosis (Hirano and Mitchison, 1994; resolved from each other, resulting in the formation of Maeshima and Laemmli, 2003; Saitoh et al., 1994). This ‘chromosome arms’. By contrast, chromatids remain tightly observation and the finding that depletion of condensin from connected at centromeres, where kinetochores assemble and Xenopus leavis egg extracts prevents chromosome assembly are then captured by spindle microtubules. In anaphase, (Hirano et al., 1997) implies that condensin is required for cohesion between sister chromatids is lost, chromatids are chromosome condensation. Consistent with this hypothesis, it segregated towards opposite spindle poles and chromatin is has been observed that worm and chicken cells, which lack subsequently decondensed during telophase. functional condensin, have chromosome-condensation defects Both chromosome condensation and cohesion depend on during prophase (Hagstrom et al., 2002; Kaitna et al., 2002; large protein complexes that contain two ATPases of the Smc Hudson et al., 2003). However, condensin mutants largely family, a Kleisin that is thought to bridge the ATPase domains show normal degrees of chromosome condensation at later of the Smc proteins, and one or two additional subunits stages of mitosis, and the major defect observed in these (reviewed in Haering and Nasmyth, 2003; Hagstrom and mutants is abnormal anaphase (Saka et al., 1994; Bhat et al., Meyer, 2003; Hirano, 2002; Jessberger, 2002; Schleiffer et al., 1996; Giet and Glover, 2001; Steffensen et al., 2001; Hagstrom 2003). One type of complex, called cohesin, associates with et al., 2002; Kaitna et al., 2002; Hudson et al., 2003). chromatin in telophase, remains bound throughout interphase, Condensin is also essential for cell division in budding yeast, and is required for sister chromatid cohesion from S phase until where only a small degree of mitotic chromosome anaphase onset. Before anaphase, cohesion is dissolved by condensation is observed (Guacci et al., 1994; Lavoie et al., stepwise removal of cohesin from chromosomes. In prophase 2004), and where condensin function is required for the and prometaphase the bulk of cohesin is removed from organization and segregation of ribosomal DNA and other chromosome arms, whereas cohesin persists at centromeres repetitive DNA sequences (D’Amours et al., 2004; Freeman et 6436 Journal of Cell Science 117 (26) al., 2000; Machin et al., 2004; Sullivan et al., 2004; Wang et expressing GFP-H2B were established as previously described, except al., 2004). that cells were selected with 2.0 µg/ml Blasticidin-S (Kanda et al., Condensin is composed of five proteins, Smc2, Smc4, CAP- 1998). D2/Eg7, CAP-G and Kleisin-γ/CAP-H (reviewed in Gassmann et al., 2004; Hagstrom and Meyer, 2003; Swedlow and Hirano, Cell extracts and fractionations 2003), but recent work has shown that Smc2 and Smc4 are also HeLa cell extracts were prepared as previously described subunits of a separate complex, called condensin II (Ono et al., (Waizenegger et al., 2000). Proteins were resolved by SDS-PAGE, and 2003; Schleiffer et al., 2003; Yeong et al., 2003). In addition transferred to Immobilon-P membrane (Millipore). Blocking and to Smc2 and Smc4, condensin II contains CAP-D3/hHCP-6, antibody-incubations were in 4% (w/v) low-fat milk in TBS-T CAP-G2 and Kleisin-β/CAP-H2, proteins that are distantly [150 mM NaCl, 20 mM Tris pH 8.0, 0.05% (v/v) Tween 20], and related to the non-Smc subunits of the originally identified washes were in TBS-T. Blots were developed by enhanced complex, now called condensin I. Condensin I and II are chemiluminescence (ECL). Crude chromatin-enriched fractions were enriched at different sites along chromatid axes, and their obtained by centrifugation of total cell extracts at 1300 g for 20 depletion causes different morphological changes when minutes using a table top centrifuge. For fractionation, 100,000 g supernatant fractions of cell extracts from NRK cells expressing chromosomes are treated with hypotonic buffers (Ono et al., γ 2003), but their relative contribution to chromosome structure EGFP–Flag–Kleisin- were prepared using IP buffer (Waizenegger et al., 2000) and centrifuged through a 5-20% (w/v) sucrose density and function is poorly understood. Here, we report that gradient for 18 hours at 150,000 g at 4°C, using a SW40 rotor condensin I and II differ in their intracellular location, the (Beckman). timing of their association with mitotic chromosomes, and that these complexes have distinct functions in chromosome condensation, dissociation of cohesin from chromosomes, Immunofluorescence microscopy and chromosome spreading chromosome shortening and mitotic progression. Cells grown on poly-L-lysine-coated coverglass were fixed with 4% (w/v) paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 20 minutes at room temperature, or with ice cold acetone-methanol (1:1) solution for 2 minutes. In the latter case, cells were washed for Materials and Methods 2 minutes at room temperature with PBS containing decreasing Antibodies amounts of methanol [95%, 90%, 80%, 70%, 50% (v/v)]. Fixed cells Polyclonal rabbit antibodies raised against the following peptides were permeabilized with 0.2% (v/v) Triton X-100–PBS, and were used: Smc2 (Pep639; CAKSKAKPPKGAHVEV), Smc4 incubated with 3% (w/v) BSA-PBS for 1 hour. Pre-extraction was (Pep638, KSVAVNPKEIASKGLC). Antibodies against CAP-D3 and carried out for 2 minutes in 0.1% Triton X-100–PBS and followed by Kleisin-γ were prepared as described (Yeong et al., 2003); CAP-D2 a 3-minute incubation in PBS before fixation. For Scc1-myc staining, antibodies were a generous gift from E. Watrin, UMR 6061-CNRS, mitotic cells were spun onto glass slides for 5 minutes at 1500 rpm Rennes. Monoclonal mouse antibodies specific for the following using a cytospin centrifuge (Shandon), pre-extracted and fixed with proteins were used: GFP (7.1 and 13.1, Roche), histone H3-phospho- 4% (w/v) paraformaldehyde. Cells were incubated with the primary Ser10 (6G3, Cell Signaling Technologies), myc (4A6, Upstate), and antibodies overnight at room temperature, followed by incubation topoisomerase IIα (Ki-S1, Boehringer Mannheim). Human CREST with secondary antibodies for 45 minutes. The secondary antibodies serum was kindly provided by A. Kromminga, IPM, Hamburg. used in this study were: goat anti-rabbit IgG Alexa fluor 488 and 568, goat anti-mouse Alexa fluor 488 and 568, and goat anti-human IgG Alexa fluor 568 (all Molecular Probes). For antibody dilutions, 0.01% RNA interference (v/v) Triton X-100 in PBS with 1% BSA (w/v) was used. After a 5- The sequences of the small interfering RNAs (siRNAs) were as minute incubation with 0.1 µg/ml 4′,6-diamidino-2-phenylindole follows: CAP-D2 5′-CCAUAUGCUCAGUGCUACATT-3′, CAP-D3 (DAPI), cells were mounted in Vectorshield mounting medium 5′-CAUGGAUCUAUGGAGAGUATT-3′, Smc2 5′-UGCUAUCACU- (Vector Laboratories). Images were captured on a Zeiss Axioplan 2 GGCUUAAAUTT-3′.