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Developmental Cell
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RuvB-like ATPases Function in Chromatin Decondensation at the End of Mitosis

Open ArchivePublished:October 23, 2014DOI:https://doi.org/10.1016/j.devcel.2014.09.001

      Highlights

      • A cell-free assay reconstitutes mitotic chromatin decondensation
      • Chromatin decondensation requires ATP and GTP hydrolysis
      • RuvB-like ATPases and their ATPase activity function in chromatin decondensation
      • Both RuvBL1 and RuvBL2 can independently promote chromatin decondensation

      Summary

      Chromatin undergoes extensive structural changes during the cell cycle. Upon mitotic entry, metazoan chromatin undergoes tremendous condensation, creating mitotic chromosomes with 50-fold greater compaction relative to interphase chromosomes. At the end of mitosis, chromosomes reestablish functional interphase chromatin competent for replication and transcription through a decondensation process that is cytologically well described. However, the underlying molecular events and factors remain unidentified. We describe a cell-free system that recapitulates chromatin decondensation based on purified mitotic chromatin and Xenopus egg extracts. Using biochemical fractionation, we identify RuvB-like ATPases as chromatin decondensation factors and demonstrate that their ATPase activity is essential for decondensation. Our results show that decompaction of metaphase chromosomes is not merely an inactivation of known chromatin condensation factors but rather an active process requiring specific molecular machinery. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its coordination with concomitant processes.

      Graphical Abstract

      Introduction

      Cells have evolved highly elaborate mechanisms to transmit genetic information accurately to their offspring. These mechanisms often involve major cellular reorganization. In metazoa, the nucleus entirely disintegrates during each round of cell division (for a review, see
      • Kutay U.
      • Hetzer M.W.
      Reorganization of the nuclear envelope during open mitosis.
      ). At the beginning of mitosis, the nuclear envelope breaks down and the chromatin condenses to rod-shaped chromosomes, which are captured by the mitotic spindle and segregated to the emerging daughter cells. The two resulting cells and their nuclei must therefore reestablish the functional interphase state. This reestablishment during mitotic exit requires the complete reversal of events that occurred at the onset of mitosis. The chromosomes decondense, and the nuclear envelope and other nuclear structures reform.
      Whereas mitotic entry and the processes leading to successful spindle formation and chromatin segregation are comparatively well studied (
      • Walczak C.E.
      • Cai S.
      • Khodjakov A.
      Mechanisms of chromosome behaviour during mitosis.
      ,
      • Walczak C.E.
      • Heald R.
      Mechanisms of mitotic spindle assembly and function.
      ) much less is known about the important processes at the end of mitosis. In animal cells, mitotic exit is driven by the inactivation of mitotic kinases (
      • Peters J.M.
      The anaphase promoting complex/cyclosome: a machine designed to destroy.
      ), the extraction of ubiquitinylated Aurora B from chromosomes by the AAA+ (ATPases associated with diverse cellular activities) ATPase p97 (
      • Ramadan K.
      • Bruderer R.
      • Spiga F.M.
      • Popp O.
      • Baur T.
      • Gotta M.
      • Meyer H.H.
      Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin.
      ), and the activation of several protein phosphatases, most prominently, PP1 (
      • Landsverk H.B.
      • Kirkhus M.
      • Bollen M.
      • Küntziger T.
      • Collas P.
      PNUTS enhances in vitro chromosome decondensation in a PP1-dependent manner.
      ,
      • Steen R.L.
      • Martins S.B.
      • Taskén K.
      • Collas P.
      Recruitment of protein phosphatase 1 to the nuclear envelope by A-kinase anchoring protein AKAP149 is a prerequisite for nuclear lamina assembly.
      ,
      • Thompson L.J.
      • Bollen M.
      • Fields A.P.
      Identification of protein phosphatase 1 as a mitotic lamin phosphatase.
      ) and PP2A (
      • Schmitz M.H.
      • Held M.
      • Janssens V.
      • Hutchins J.R.
      • Hudecz O.
      • Ivanova E.
      • Goris J.
      • Trinkle-Mulcahy L.
      • Lamond A.I.
      • Poser I.
      • et al.
      Live-cell imaging RNAi screen identifies PP2A-B55alpha and importin-beta1 as key mitotic exit regulators in human cells.
      ). These events collectively result in the reversal of mitotic phosphorylation on a broad range of substrates (
      • Dephoure N.
      • Zhou C.
      • Villén J.
      • Beausoleil S.A.
      • Bakalarski C.E.
      • Elledge S.J.
      • Gygi S.P.
      A quantitative atlas of mitotic phosphorylation.
      ,
      • Olsen J.V.
      • Vermeulen M.
      • Santamaria A.
      • Kumar C.
      • Miller M.L.
      • Jensen L.J.
      • Gnad F.
      • Cox J.
      • Jensen T.S.
      • Nigg E.A.
      • et al.
      Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis.
      ), yet little is known about the actual machineries that mediate specific mitotic exit events (
      • Wurzenberger C.
      • Gerlich D.W.
      Phosphatases: providing safe passage through mitotic exit.
      ). This is especially evident for chromatin decondensation, a prerequisite for the formation of interphase nuclear structures. Metaphase chromosomes are highly condensed—DNA compaction is up to 50-fold higher than in interphase (
      • Belmont A.S.
      Mitotic chromosome structure and condensation.
      )—but how this condensation is achieved is still ill defined (for a review, see
      • Hansen J.C.
      Human mitotic chromosome structure: what happened to the 30-nm fibre?.
      ,
      • Ohta S.
      • Wood L.
      • Bukowski-Wills J.C.
      • Rappsilber J.
      • Earnshaw W.C.
      Building mitotic chromosomes.
      ). However, the process that reorganizes the genome into a structure competent for transcription and replication is largely unchartered territory. We are ignorant about the proteins that mediate chromatin decondensation, the distinct steps in this most likely multistep procedure, and its regulation.
      To date, chromatin decondensation has mainly been examined in the context of sperm chromatin remodeling after fertilization. Highly compacted sperm DNA undergoes reorganization due to the presence of nucleoplasmin (NPM2) stored in oocyte cytoplasm (
      • Philpott A.
      • Leno G.H.
      • Laskey R.A.
      Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin.
      ). This process has been intensively studied using Xenopus laevis egg extracts. Xenopus sperm chromatin consists of a complex mixture of sperm-specific basic proteins and histones H3 and H4. NPM2 replaces these basic proteins from the male pronucleus with histones H2A and H2B stored in the egg, relaxing the tightly wound sperm chromatin structure (
      • Philpott A.
      • Leno G.H.
      Nucleoplasmin remodels sperm chromatin in Xenopus egg extracts.
      ). However, as mitotic chromatin is already structured around H2A and H2B and does not contain these sperm-specific proteins, chromatin decondensation at the end of mitosis is likely to proceed by another yet-unknown mechanism.
      Here, we describe a cell-free assay that faithfully recapitulates decondensation of mitotic chromatin. Using this assay, we show that chromatin decondensation requires ATP and GTP hydrolysis and is, thus, an active process. We identify RuvB-like ATPases as crucial chromatin decondensation factors and show that their ATPase activity is essential for decondensation. Intriguingly, both metazoan RuvB-like proteins, RuvBL1 and RuvBL2 can function alone in chromatin decondensation in contrast to many other RuvBL1/RuvBL2-mediated processes, which require both components.

      Results

      A Cell-free Assay to Monitor Mitotic Chromatin Decondensation

      Chromatin decondensation at the end of mitosis is underinvestigated due to a lack of appropriate assays to monitor the process. To overcome this limitation, we have developed a cell-free assay that recapitulates chromatin decondensation in vitro. We incubated highly condensed chromosome clusters isolated from mitotic HeLa cells with cytosol and purified membranes derived from Xenopus egg extracts mimicking the postmitotic state. Using DAPI staining and confocal microscopy, we observed sequential morphological changes of chromatin structure (Figure 1A) that resembled chromosome decondensation in cells exiting mitosis (see Figure S1A available online). Highly compacted distinguishable metazoan chromosomes decondensed in a time-dependent manner. After 10–20 min, the individual chromosomes merged to an apparently single corpus, which became progressively spherical and finally adopted an interphasic nuclear appearance. Chromatin decondensation was not induced by the incubation of chromatin substrates with buffer alone, indicating the presence of an essential decondensation activity in egg extracts. Mitotic (cytostatic factor [CSF]-arrested) egg extracts did not support the decondensation of the chromatin substrate (Figure 1B). However, addition of 1 mM Ca2+ ions to mitotic extracts, which causes mitotic exit (
      • Murray A.W.
      Cell cycle extracts.
      ), did induce chromatin decondensation, indicating that postmitotic conditions are required for the process. An equal progressive decondensation was observed when, instead of HeLa cell chromatin, mitotic chromatin generated from Xenopus sperm DNA was used (Figure S1B) demonstrating the universality of the process.
      Figure thumbnail gr1
      Figure 1Reconstitution of Chromatin Decondensation in Xenopus Egg Extracts
      (A) Time course of the in vitro decondensation reaction. Mitotic chromatin clusters from HeLa cells were incubated with postmitotic Xenopus egg extracts for the indicated time. Samples were fixed with 4% PFA and 0.5% glutaraldehyde, stained with DAPI, and analyzed by confocal microscopy. For quantification of the decondensation reaction, the smoothness of the boundary of the chromatin (light gray) and the homogeneity of DAPI staining (dark gray) were analyzed. The means (± SEM) of three independent experiments are shown, each including at least ten chromatin substrates for each time point, ∗∗∗p < 0.001 by one-way ANOVA, Dunnett’s C post hoc test. rel, relative.
      (B) Mitotic chromatin clusters from HeLa cells were incubated for 120 min with CSF-arrested Xenopus egg extracts in the absence or presence of 1 mM CaCl2, which induces mitotic exit. Samples were fixed, and the decondensation reaction was quantified as in (A). The means (±SEM) of three independent experiments are shown, each including at least ten chromatin substrates, ∗∗∗p < 0.001 by Mann-Whitney test.
      Scale bars, 5 μm. See also .
      We quantified mitotic HeLa chromatin decondensation based on the homogeneity of DAPI staining and the smoothness of the chromatin boundary (Figure 1A; see Experimental Procedures for details). These features were chosen with the following rationale: when chromatin is completely decondensed, the nuclear shape is spherical and bulk chromatin appears to be distributed rather homogenously; when chromatin is condensed, the surface appears rough and bulk chromatin is clustered in distinct chromosomes. Both parameters increased over the time course of HeLa chromosome decondensation and reliably built up the process, indicating a highly reproducible progression of chromatin decondensation in our assay system.
      In addition to chromatin decondensation, our in vitro system recapitulates several other mitotic exit events. Histone H3 phosphorylation at serine 10, a marker of the mitotic state of chromatin (
      • Hendzel M.J.
      • Wei Y.
      • Mancini M.A.
      • Van Hooser A.
      • Ranalli T.
      • Brinkley B.R.
      • Bazett-Jones D.P.
      • Allis C.D.
      Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation.
      ), was rapidly diminished on incubation with postmitotic Xenopus egg extract (Figure 2A, upper panel). Dephosphorylation of this site also occurred when mitotic chromatin was incubated with buffer alone, indicating that the relevant phosphatase activity is present on mitotic chromatin. However, mitotic chromatin incubated with buffer remained condensed (Figures 1A and 2A), consistent with previous findings that this modification is not essential for the establishment or maintenance of condensed mitotic chromatin in yeast or vertebrates (
      • Hsu J.Y.
      • Sun Z.W.
      • Li X.
      • Reuben M.
      • Tatchell K.
      • Bishop D.K.
      • Grushcow J.M.
      • Brame C.J.
      • Caldwell J.A.
      • Hunt D.F.
      • et al.
      Mitotic phosphorylation of histone H3 is governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes.
      ,
      • MacCallum D.E.
      • Losada A.
      • Kobayashi R.
      • Hirano T.
      ISWI remodeling complexes in Xenopus egg extracts: identification as major chromosomal components that are regulated by INCENP-aurora B.
      ).
      Figure thumbnail gr2
      Figure 2Decondensing Chromatin Assembles into Functional Nuclei
      (A) Mitotic chromatin clusters from HeLa cells were incubated with Xenopus egg extracts for the indicated time and fixed with 4% PFA. Immunofluorescence shows histone H3 serine 10 phosphorylation (H3P, upper panel), nuclear pore complexes (NPC, middle panel), and chromatin (DAPI).
      (B) For visualization of nuclear envelope reformation, HeLa mitotic chromatin substrates and DiIC18 (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)-labeled membranes (upper panel) were added to the egg extracts or the buffer control. Samples were fixed at indicated time points with 4% PFA and 0.5% glutaraldehyde, stained with DAPI (lower panel), and analyzed by confocal microscopy.
      (C) Chromatin decondensation using HeLa mitotic chromatin was analyzed by transmission electron microscopy. Samples were fixed at indicated time points with 4% PFA and 2.5% glutaraldehyde, postfixed in 1% OsO4, and stained with 1% uranyl acetate. After embedding in Epon, ultrathin sections (50–70 nm) were stained with uranyl acetate and lead citrate and viewed with a Philips CM10 microscope.
      (D) HeLa mitotic chromatin was decondensed for 120 min. An enhanced green fluorescent protein (EGFP)-fused import substrate (left column) or a shuttling substrate containing a nuclear localization signal and a nuclear export signal (middle and right column) was added. Nuclear export was inhibited by the addition of 300 nM leptomycin B. Samples were stained with DAPI and analyzed by confocal microscopy. The weighted average percentage of two independent experiments, each including at least 100 randomly chosen chromatin substrates, is shown. Diamonds indicate data points of the individual experiments.
      Scale bars, 5 μm.
      The decondensing chromatin in our assay system was enclosed by membranes, which eventually formed a smooth nuclear envelope (Figures 2B and 2C). The nuclear envelope contained nuclear pore complexes, gatekeepers of the nucleus that mediate nuclear import and export. Nuclear pore complex formation was analyzed by immunofluorescence with mAB414 (
      • Davis L.I.
      • Blobel G.
      Identification and characterization of a nuclear pore complex protein.
      ), an antibody that recognizes four different nuclear pore complex proteins (Figure 2A, middle panel). Nuclear pore complex proteins labeled by this antibody were first detected approximately 20 min after initiation of decondensation. After a 60–120 min incubation in postmitotic Xenopus extracts, the nuclei were capable of nuclear import and export (Figure 2D). Taken together, these results show that our cell-free system recapitulates chromatin decondensation as well as nuclear envelope and pore reformation and is, thus, an invaluable tool for studying mitotic exit events. Notably, in the absence of added membranes, chromatin decondensation similarly occurred, although nuclear envelopes and pore complexes, as expected, did not reform (data not shown). This indicates that chromatin decondensation does not require a reforming nuclear envelope and functional pore complexes, but it is possible that this is a peculiarity of the cell-free assay.

      Chromatin Decondensation Requires ATP and GTP Hydrolysis

      Having established the versatility of the assay, we first investigated the basic requirements of chromatin decondensation. The removal of endogenous nucleoside triphosphates from the extracts by hexokinase treatment blocked chromatin decondensation (Figure S2A), indicating that some energy-consuming step is required. Nonhydrolyzable ATP or GTP analogs inhibited chromatin decondensation, suggesting that both ATP- and GTP-dependent activities are involved in chromatin decondensation (Figure 3). ATP dependence might be explained by a requirement for the ATPase p97, which removes Aurora kinase B from chromatin during decondensation (
      • Ramadan K.
      • Bruderer R.
      • Spiga F.M.
      • Popp O.
      • Baur T.
      • Gotta M.
      • Meyer H.H.
      Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin.
      ). However, inhibition of Aurora kinase B by hesperadin, which bypasses the need for p97 in this process (
      • Ramadan K.
      • Bruderer R.
      • Spiga F.M.
      • Popp O.
      • Baur T.
      • Gotta M.
      • Meyer H.H.
      Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin.
      ), did not restore chromatin decondensation in the presence of nonhydrolyzable ATP analogs, suggesting that at least one other ATPase is involved (data not shown).
      Figure thumbnail gr3
      Figure 3Chromatin Decondensation Requires ATP and GTP Hydrolysis
      HeLa mitotic chromatin was decondensed in the presence of 10 mM ATPγS, 10 mM GTPγS, or buffer control (CTRL). Samples were fixed with 4% PFA and 0.5% glutaraldehyde at indicated time points, analyzed, and quantified. The means (±SEM) of three independent experiments are shown, each including at least ten chromatin substrates for each time point, ∗∗∗p < 0.001 by one-way ANOVA, Dunnett’s C post hoc test. rel, relative. Scale bar, 5 μm. See also .
      Although DNA transcription is thought to be absent in Xenopus egg extracts (
      • Newport J.
      • Kirschner M.
      A major developmental transition in early Xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage.
      ), we wanted to exclude that transcriptional activity is required for chromatin decondensation in our assay system. As expected, addition of the transcription inhibitors actinomycin D or 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole did not affect chromatin decondensation (Figure S2B).

      RuvBL1 and RuvBL2 Can Function Individually as ATPases in Chromatin Decondensation

      To identify essential chromatin decondensation factors, we fractionated the cytosol derived from postmitotic Xenopus egg extracts and assayed for chromatin decondensation activity. Differential ammonium sulfate precipitation yielded two fractions that individually had severely reduced decondensation activity but were highly active when combined (Figure 4A; Figure S3A). We further purified the first of these ammonium sulfate fractions by ion exchange and size exclusion chromatography (see Experimental Procedures for detailed information) and assayed the activities of the obtained fractions in combination with the second ammonium sulfate fraction. By mass spectrometry analysis of the gel filtration fractions with highest decondensation activity (G13–G15), we identified several candidate chromatin decondensation factors, including the ATPase RuvBL2. RuvBL2 is known to form a double hexameric ring complex with a second ATPase, RuvBL1 (
      • Jha S.
      • Dutta A.
      RVB1/RVB2: running rings around molecular biology.
      ,
      • Puri T.
      • Wendler P.
      • Sigala B.
      • Saibil H.
      • Tsaneva I.R.
      Dodecameric structure and ATPase activity of the human TIP48/TIP49 complex.
      ). Indeed, western blot analysis confirmed the presence of RuvBL1 and RuvBL2 in the active fractions throughout the purification procedure and enrichment in the most active gel filtration fractions (Figure 4A), which makes these proteins possible candidates for the decondensation activity. RuvBL1/RuvBL2 (also known as RVB1/RVB2, pontin/reptin, and TIP49/TIP48) are two highly conserved members of the AAA+ superfamily. They associate with diverse chromatin remodeling complexes, which are implicated in a variety of nuclear processes, including transcriptional regulation, DNA damage response, and small nuclear ribonucleoprotein particle (snoRNP) assembly (for a review, see
      • Jha S.
      • Dutta A.
      RVB1/RVB2: running rings around molecular biology.
      ,
      • Nano N.
      • Houry W.A.
      Chaperone-like activity of the AAA+ proteins Rvb1 and Rvb2 in the assembly of various complexes.
      ,
      • Tosi A.
      • Haas C.
      • Herzog F.
      • Gilmozzi A.
      • Berninghausen O.
      • Ungewickell C.
      • Gerhold C.B.
      • Lakomek K.
      • Aebersold R.
      • Beckmann R.
      • Hopfner K.P.
      Structure and subunit topology of the INO80 chromatin remodeler and its nucleosome complex.
      ).
      Figure thumbnail gr4
      Figure 4Chromatin Decondensation Requires RuvB-like ATPases
      (A) Xenopus egg extracts were fractionated by differential ammonium sulfate precipitation, ion exchange, and size exclusion chromatography (see fractionation scheme on the left with the fractions showing decondensation activity in black) and were tested for the state of chromatin decondensation on HeLa mitotic chromatin after 120 min. For ion exchange and size exclusion fractions, reactions were performed in the presence of fraction B of the ammonium sulfate precipitation. The lower panels show the distribution of RuvBL1 and RuvBL2 in fractions analyzed by western blotting. Representative quantification and western blot analysis of one fractionation experiment is shown. FT, flowthrough. rel, relative.
      (B) Chromatin decondensation on HeLa mitotic chromatin was performed for 120 min in the presence of 4 mg/ml affinity-purified IgG against RuvBL1, RuvBL2, or control IgGs.
      (C) Western blot of untreated (UNTR), mock, and RuvBL1/2-depleted extracts, the latter two generated by two passages over control IgG- or anti-RuvBL1 or anti-RuvBL2 IgG-bound beads, respectively. NPM2 serves as a control protein unaffected by this treatment.
      (D) Mock or RuvBL1/2-depleted extracts supplemented with buffer or purified recombinant RuvBL1-RuvBL2 complex (0.04 μg/μl to match the endogenous concentration) were tested for chromatin decondensation on HeLa mitotic chromatin (120 min time point).
      In (B) and (D), the means (±SEM) of three independent experiments are shown, each including at least 20 chromatin substrates. ∗∗∗p < 0.001 by one-way ANOVA, Dunnett’s C post hoc test. Scale bars, 5 μm. See also .
      To assess the relevance of RuvBL1 and RuvBL2 for chromatin decondensation, we performed antibody inhibition experiments in the decondensation assay. The addition of purified anti-RuvBL1 or anti-RuvBL2 immunoglobulin G (IgG) to the reactions significantly impaired chromatin decondensation compared to the addition of control IgG (Figures 4B and S3B). Immunodepletion using antibodies against either RuvBL1 or RuvBL2, respectively, removed both proteins efficiently from the extracts (Figure 4C), indicating that, in Xenopus egg extracts, RuvBL1 and RuvBL2 occur mostly together in heteromeric complexes. Both immunodepletion procedures rendered egg extracts incompetent for chromatin decondensation in contrast to control depletions (Figure 4D). The addition of purified recombinant RuvBL1-RuvBL2 complexes to a final concentration of 0.04 μg/μl, which matches the endogenous concentration (Figure S3C), was sufficient to rescue the depletion phenotype (Figure 4D), indicating on-target specificity of the immunodepletion. These experiments demonstrate that RuvBL1/2 indeed function in chromatin decondensation and are crucial for this process.

      RuvBL1 and RuvBL2 Can Function Individually as ATPases in Chromatin Decondensation

      In many cellular processes, RuvBL1 and RuvBL2 operate together by forming heteromeric complexes (
      • Jha S.
      • Dutta A.
      RVB1/RVB2: running rings around molecular biology.
      ,
      • Nano N.
      • Houry W.A.
      Chaperone-like activity of the AAA+ proteins Rvb1 and Rvb2 in the assembly of various complexes.
      ,
      • Nguyen V.Q.
      • Ranjan A.
      • Stengel F.
      • Wei D.
      • Aebersold R.
      • Wu C.
      • Leschziner A.E.
      Molecular architecture of the ATP-dependent chromatin-remodeling complex SWR1.
      ,
      • Tosi A.
      • Haas C.
      • Herzog F.
      • Gilmozzi A.
      • Berninghausen O.
      • Ungewickell C.
      • Gerhold C.B.
      • Lakomek K.
      • Aebersold R.
      • Beckmann R.
      • Hopfner K.P.
      Structure and subunit topology of the INO80 chromatin remodeler and its nucleosome complex.
      ,
      • Venteicher A.S.
      • Meng Z.
      • Mason P.J.
      • Veenstra T.D.
      • Artandi S.E.
      Identification of ATPases pontin and reptin as telomerase components essential for holoenzyme assembly.
      ); however, in some instances, these proteins act antagonistically (
      • Bauer A.
      • Chauvet S.
      • Huber O.
      • Usseglio F.
      • Rothbächer U.
      • Aragnol D.
      • Kemler R.
      • Pradel J.
      Pontin52 and reptin52 function as antagonistic regulators of beta-catenin signalling activity.
      ,
      • Rottbauer W.
      • Saurin A.J.
      • Lickert H.
      • Shen X.
      • Burns C.G.
      • Wo Z.G.
      • Kemler R.
      • Kingston R.
      • Wu C.
      • Fishman M.
      Reptin and pontin antagonistically regulate heart growth in zebrafish embryos.
      ). Surprisingly, the addition of either purified homohexameric RuvBL1 or RuvBL2 complexes to depleted extracts restored decondensation activity as efficiently as the addition of the heteromeric RuvBL1-RuvBL2 complex (Figure 5A). This indicates that both proteins can function redundantly and independently of each other in this process.
      Figure thumbnail gr5
      Figure 5RuvBL1 or RuvBL2 Alone Is Sufficient to Support Chromatin Decondensation and Require ATPase Activity
      (A) RuvBL1/2-depleted extracts (generated by two passages over anti-RuvBL1 or anti-RuvBL2 IgG-bound beads, respectively) were supplemented with purified recombinant RuvBL1 or RuvBL2 (0.02 μg/μl to match the endogenous concentration) and tested for chromatin decondensation on HeLa mitotic chromatin. rel, relative.
      (B) Chromatin decondensation was analyzed in RuvBL1/2-depleted extracts (generated by consecutive passage over anti-RuvBL1 and anti-RuvBL2 IgG-bound beads) supplemented with ATPase-deficient mutant versions of the RuvBL1, RuvBL2, or the RuvBL1-RuvBL2 complex (RuvBL1 D302N and RuvBL2 D298N) matching the endogenous concentration. WT, wild-type.
      (C) Chromatin decondensation in the presence of 40-fold excess compared to endogenous concentrations of recombinant wild-type RuvBL1, RuvBL2, or the RuvBL1-RuvBL2 complex or ATPase-deficient mutants of the respective proteins.
      Samples were analyzed after 120 min. The means (±SEM) of three independent experiments are shown, each including at least 20 chromatin substrates. ∗∗∗p < 0.001 by two-way ANOVA, Sidlak post hoc test for (A) and (B) and by one-way ANOVA, Dunnett’s C post hoc test for (C). WT, wild-type. Scale bars, 5 μm. See also .
      The addition of recombinant ATPase-deficient RuvBL1/2 mutants, either individually or in a heteromeric complex (RuvBL1 D302N/RuvBL2 D298N) (
      • Matias P.M.
      • Gorynia S.
      • Donner P.
      • Carrondo M.A.
      Crystal structure of the human AAA+ protein RuvBL1.
      ,
      • Mézard C.
      • Davies A.A.
      • Stasiak A.
      • West S.C.
      Biochemical properties of RuvBD113N: a mutation in helicase motif II of the RuvB hexamer affects DNA binding and ATPase activities.
      ) (Figure S4C), did not rescue the depletion phenotype, indicating that the ATPase function of either proteins is required for its role in chromatin decondensation (Figure 5B). The addition of excess RuvBL1 D302N, RuvBL2 D298N, or the RuvBL1 D302N/RuvBL2 D298N complex to untreated extracts inhibited chromatin decondensation, while the wild-type proteins and complexes had no effect (Figures 5C and S4A). RuvB-like ATPases perform their different cellular functions in conjunction with a variety of cofactors (for a review, see
      • Jha S.
      • Dutta A.
      RVB1/RVB2: running rings around molecular biology.
      ,
      • Nano N.
      • Houry W.A.
      Chaperone-like activity of the AAA+ proteins Rvb1 and Rvb2 in the assembly of various complexes.
      ), and this is most likely also the case for chromatin decondensation (see Discussion). Thus, the dominant-negative effect of ATPase-deficient RuvBL1/2 mutants is likely to be caused by a sequestration of these cofactors. Together, these experiments using ATPase-deficient RuvBL1/2 versions demonstrate that chromatin decondensation depends on ATPase-proficient RuvB-like proteins.
      Although RuvB-like proteins are required for chromatin decondensation, they are not sufficient. When purified recombinant RuvBL1, RuvBL2, or the heteromeric RuvBL1/2 complex were added to HeLa mitotic chromatin in buffer in the presence of ATP, no chromatin decondensation was detected (Figure S4B), indicating that other factors are also crucially required (see Discussion).

      RuvBL1 and RuvBL2 Localize on the Decondensing Chromatin

      We next analyzed the localization of RuvBL1 and RuvBL2 during mitotic exit. Consistent with their role in chromatin decondensation, RuvBL1 and RuvBL2 localize and enrich on postmitotic decondensing chromatin, both in the in vitro assay (Figure 6A) and in HeLa cells (Figure S5A). Both RuvB-like proteins are excluded from chromatin during earlier stages of mitosis, including metaphase in agreement with previous reports (
      • Gartner W.
      • Rossbacher J.
      • Zierhut B.
      • Daneva T.
      • Base W.
      • Weissel M.
      • Waldhäusl W.
      • Pasternack M.S.
      • Wagner L.
      The ATP-dependent helicase RUVBL1/TIP49a associates with tubulin during mitosis.
      ,
      • Sigala B.
      • Edwards M.
      • Puri T.
      • Tsaneva I.R.
      Relocalization of human chromatin remodeling cofactor TIP48 in mitosis.
      ).
      Figure thumbnail gr6
      Figure 6RuvBL1 and RuvBL2 Localize to the Decondensing Chromatin
      (A) HeLa mitotic chromatin was incubated with extracts for the indicated time. RuvBL1/2-depleted extracts (generated by consecutive passage over anti-RuvBL1 and anti-RuvBL2 IgG-bound beads) were supplemented with buffer, recombinant RuvBL1-RuvBL2 complex, RuvBL1, or RuvBL2 or ATPase-deficient versions of the proteins (matching the endogenous concentrations) and used in the decondensation reaction for 120 min. Samples were fixed with 4% PFA and processed for immunofluorescence, or chromatin was reisolated and analyzed by western blot (histone H2B shows equal chromatin loading). Scale bars, 5 μm.
      (B) HeLa mitotic chromatin incubated as in (A) was reisolated and probed for the presence of Mel28/ELYS, the condensing I and II complex (CAP-G or CAP-D3 antibodies, respectively), topoisomerase IIα, the chromokinesin KIF4A, and Repo-MAN. Please note that during the reisolation procedure, rapid rebinding of Xenopus proteins to chromatin and/or their exchange with the HeLa proteins occurs so that they can be detected already at t = 0.
      See also .
      When depleting the endogenous RuvBL1/2 complex, both recombinant RuvBL1 and RuvBL2 could be detected on the chromatin template (Figure 6A), indicating that both proteins can independently localize to chromatin. This observation is consistent with the finding that either homomeric complex can substitute the heteromeric complex to support chromatin decondensation (Figure 5A). The ATPase-deficient mutants similarly localized to chromatin, indicating that the ATPase function is not required for chromatin localization.
      Having identified RuvBL1/2 as chromatin decondensation factors, we analyzed the fate of known chromatin condensation factors on the chromatin on depletion of RuvB-like proteins. Topoisomerase II, KIF4A, and the condensin II complex were detected on the chromatin at all stages of the decondensation reaction (Figures 6B and S5B), as expected (
      • Gerlich D.
      • Hirota T.
      • Koch B.
      • Peters J.M.
      • Ellenberg J.
      Condensin I stabilizes chromosomes mechanically through a dynamic interaction in live cells.
      ,
      • Mazumdar M.
      • Sundareshan S.
      • Misteli T.
      Human chromokinesin KIF4A functions in chromosome condensation and segregation.
      ,
      • Tavormina P.A.
      • Côme M.G.
      • Hudson J.R.
      • Mo Y.Y.
      • Beck W.T.
      • Gorbsky G.J.
      Rapid exchange of mammalian topoisomerase II alpha at kinetochores and chromosome arms in mitosis.
      ). A similar pattern was observed for Repo-Man, also known as CDCA2, which recruits the protein phosphatase PP1 to chromatin during mitotic exit and was shown to coordinate chromatin decondensation and nuclear envelope reformation (
      • Vagnarelli P.
      • Ribeiro S.
      • Sennels L.
      • Sanchez-Pulido L.
      • de Lima Alves F.
      • Verheyen T.
      • Kelly D.A.
      • Ponting C.P.
      • Rappsilber J.
      • Earnshaw W.C.
      Repo-Man coordinates chromosomal reorganization with nuclear envelope reassembly during mitotic exit.
      ); and for Mel28 (also referred to as ELYS), a chromatin-binding protein that acts as a seeding point for nuclear pore complex formation (
      • Franz C.
      • Walczak R.
      • Yavuz S.
      • Santarella R.
      • Gentzel M.
      • Askjaer P.
      • Galy V.
      • Hetzer M.
      • Mattaj I.W.
      • Antonin W.
      MEL-28/ELYS is required for the recruitment of nucleoporins to chromatin and postmitotic nuclear pore complex assembly.
      ). The condensin I complex is lost from the chromatin in the course of decondensation (
      • Gerlich D.
      • Hirota T.
      • Koch B.
      • Peters J.M.
      • Ellenberg J.
      Condensin I stabilizes chromosomes mechanically through a dynamic interaction in live cells.
      ). In all instances, depletion of RuvBL1/2 did not affect the spatiotemporal localization of these proteins on decondensing chromatin, indicating that RuvB-like ATPases act independently of these factors during decondensation.

      RuvB-like ATPases Are Not Required for Nuclear Envelope and Pore Complex Formation

      Our data show that the RuvB-like ATPases function as key decondensation factors of mitotic chromatin. In organisms undergoing open mitosis, the nuclear envelope and nuclear pore complexes break down at the beginning of mitosis and reform on the decondensing chromatin in telophase (for a review, see (
      • Kutay U.
      • Hetzer M.W.
      Reorganization of the nuclear envelope during open mitosis.
      ,
      • Schooley A.
      • Vollmer B.
      • Antonin W.
      Building a nuclear envelope at the end of mitosis: coordinating membrane reorganization, nuclear pore complex assembly, and chromatin de-condensation.
      ). On depletion of RuvBL1/2 in the decondensation assay, we did not observe formation of a closed nuclear envelope and nuclear pore complex reassembly (data not shown). This could indicate that RuvB-like ATPases are also involved in these processes. Alternatively, chromatin decondensation might be a prerequisite for nuclear envelope and pore complex assembly. To distinguish these two possibilities, we sought to bypass the need of RuvBL1/2 for chromatin decondensation by using an already decompacted chromatin template. For this, Xenopus sperm heads were incubated in postmitotic egg extracts. In this assay, which recapitulates the processes naturally occurring after entry of sperm DNA into an egg, pronuclei with intact nuclear envelopes and pore complexes are formed, and this system has been widely used to study these assembly processes (
      • Gant T.M.
      • Wilson K.L.
      Nuclear assembly.
      ). Notably, sperm DNA is, in this experimental setup, decompacted by the NPM2-mediated exchange of protamines to histones H2A and H2B (
      • Philpott A.
      • Leno G.H.
      Nucleoplasmin remodels sperm chromatin in Xenopus egg extracts.
      ). When sperm heads were incubated with control or RuvBL1/2-depleted postmitotic extracts, pronuclei with closed nuclear envelopes and intact nuclear pore complexes were formed (Figure 7). These experiments demonstrate that, as expected, RuvB-like proteins are not required for sperm DNA decompaction. Notably, they are also not crucial for nuclear envelope and pore complex formation. In this experimental system, the pronuclei undergo nuclear expansion after initial NPM2-dependent sperm DNA decompaction. This process, which is also referred to as nuclear swelling/expansion or secondary decondensation, requires nuclear import and, thus, a functional nuclear envelope including pore complexes (
      • Philpott A.
      • Leno G.H.
      • Laskey R.A.
      Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin.
      ,
      • Wright S.J.
      Sperm nuclear activation during fertilization.
      ). The DAPI staining of the pronuclei assembled in the absence of RuvBL1/2 indicates that this nuclear swelling does not require RuvB-like ATPases. These data also show that distinct mitotic exit events such as chromatin decondensation and nuclear envelope/pore complex reformation can be uncoupled in vitro.
      Figure thumbnail gr7
      Figure 7RuvB-like ATPases Are Specifically Required for Chromatin Decondensation during Mitotic Exit
      Pronuclei were assembled on Xenopus sperm chromatin in mock-treated or RuvBL1/2-depleted extracts (using anti-RuvBL1 or anti-RuvBL2 antibodies). After 120 min, samples were fixed with 4% PFA and 0.5% glutaraldehyde and analyzed for membrane staining (DiIC18, upper panel) or for nuclear pore complexes (NPC, lower panel) by immunofluorescence with the antibody mAB414. Chromatin was stained with DAPI. Right panel shows the quantitation of chromatin substrates with closed nuclear envelopes as weighted average percentage of two independent experiments, each including at least 100 chromatin substrates. Diamonds indicate data points of the individual experiments. Scale bar, 5 μm. See also .
      Mitotic chromatin decondensation does not require NPM2, which, in turn, is needed for sperm DNA decompaction (Figure S6). This supports the view that sperm DNA and mitotic chromatin decondensation are mechanistically fundamentally different.

      Discussion

      Here, we show that chromatin decondensation can be faithfully reconstituted in a cell-free assay. Using this system, we demonstrate that the process requires ATP and GTP hydrolysis. It is not merely an inactivation of known chromatin condensation factors but an active process involving specific molecular machinery. We identify a defined requirement for the RuvB-like ATPases in chromatin decondensation, but not for nuclear envelope and pore complex formation. Our assay system is, therefore, a valuable tool for the dissection of the cellular processes that lead to the assembly of functional interphase chromatin after mitosis.
      Cell-free extracts derived from frog eggs, especially from Xenopus laevis, have been widely used to study cell cycle regulation as well as many mitotic and nuclear processes since their development and first use 30 years ago (
      • Lohka M.J.
      • Masui Y.
      Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components.
      ). These extracts recapitulate complex cellular reactions such as chromatin condensation, spindle assembly, and nuclear envelope breakdown (
      • Galy V.
      • Antonin W.
      • Jaedicke A.
      • Sachse M.
      • Santarella R.
      • Haselmann U.
      • Mattaj I.
      A role for gp210 in mitotic nuclear-envelope breakdown.
      ,
      • Maresca T.J.
      • Heald R.
      Methods for studying spindle assembly and chromosome condensation in Xenopus egg extracts.
      ). Nuclear envelope and pore complex formation has been intensively studied in pronucleus formation using sperm DNA as a chromatin template (for a review, see
      • Gant T.M.
      • Wilson K.L.
      Nuclear assembly.
      ). Here, we use mitotic chromatin to study chromatin decondensation and nuclear reformation during mitotic exit. We show that the nuclei formed on the decondensing chromatin contain a closed nuclear envelope with two membranes and nuclear pore complexes (Figure 2). These nuclei are competent for nuclear import and export and DNA replication (Figure 2D; A.M. and W.A., unpublished data), showing that they represent a functional interphasic status.
      So far, chromatin decondensation has been mainly investigated in the context of male pronucleus formation around sperm DNA. However, it is unlikely that this involves the same machinery as chromatin decondensation at the end of mitosis. Indeed, our data show that sperm DNA decompacts in the absence of the RuvB-like ATPases (Figure 7), which are required for mitotic chromatin decondensation. In contrast, sperm DNA decondensation depends on the histone chaperone NPM2 (
      • Philpott A.
      • Leno G.H.
      Nucleoplasmin remodels sperm chromatin in Xenopus egg extracts.
      ,
      • Philpott A.
      • Leno G.H.
      • Laskey R.A.
      Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin.
      ), which conversely is not necessary for mitotic chromatin decondensation (Figure S6), consistent with the fact that NPM2 is absent in somatic cells (
      • Burns K.H.
      • Viveiros M.M.
      • Ren Y.
      • Wang P.
      • DeMayo F.J.
      • Frail D.E.
      • Eppig J.J.
      • Matzuk M.M.
      Roles of NPM2 in chromatin and nucleolar organization in oocytes and embryos.
      ).
      In contrast to sperm DNA decompaction, which is an energy-independent process (
      • Philpott A.
      • Leno G.H.
      • Laskey R.A.
      Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin.
      ), mitotic chromatin decondensation requires cellular energy (Figure S2). The inhibition of mitotic chromatin decondensation observed in the presence of nonhydrolyzable ATP (Figure 3) suggests that ATPases are involved in the process. Indeed, we show that RuvB-like ATPases and, specifically, their ATPase functions are compulsory in addition to p97, the only protein previously implicated in the postmitotic decondensation of chromatin (
      • Ramadan K.
      • Bruderer R.
      • Spiga F.M.
      • Popp O.
      • Baur T.
      • Gotta M.
      • Meyer H.H.
      Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin.
      ).
      We envision chromatin decondensation as a multistep procedure involving several activities. Indeed, each fraction of our ammonium sulfate fractionation is largely inactive on its own, and only when they are recombined is decondensation activity restored (Figures 4A and S3A). Consistent with the notion of multiple necessary decondensation factors, RuvB-like ATPases are not sufficient to promote chromatin decondensation if added alone to the mitotic chromatin template (Figure S4B). Most likely, yet-unidentified RuvBL1/2 interacting factors are crucially required for the RuvBL1/2-mediated step in chromatin decondensation as in other processes mediated by these ATPases (for a review, see
      • Jha S.
      • Dutta A.
      RVB1/RVB2: running rings around molecular biology.
      ,
      • Nano N.
      • Houry W.A.
      Chaperone-like activity of the AAA+ proteins Rvb1 and Rvb2 in the assembly of various complexes.
      ). In addition, chromatin decondensation most likely involves other RuvBL1/2-independent steps. The inhibition by nonhydrolyzable GTP (Figure 3) suggests that at least one GTPase is involved. The nature of the GTPases is currently unknown but an interesting avenue for future research.
      RuvB-like proteins are highly conserved and essential eukaryotic AAA+ ATPases involved in a wide range of cellular reactions as components of large protein complexes (for a review, see
      • Jha S.
      • Dutta A.
      RVB1/RVB2: running rings around molecular biology.
      ,
      • Nano N.
      • Houry W.A.
      Chaperone-like activity of the AAA+ proteins Rvb1 and Rvb2 in the assembly of various complexes.
      ). These include many chromatin-related, but also other, processes such as chromatin remodeling, transcriptional regulation, and DNA damage response, as well as snoRNP, telomere, and spindle assembly (
      • Ducat D.
      • Kawaguchi S.
      • Liu H.
      • Yates 3rd, J.R.
      • Zheng Y.
      Regulation of microtubule assembly and organization in mitosis by the AAA+ ATPase Pontin.
      ,
      • Ikura T.
      • Ogryzko V.V.
      • Grigoriev M.
      • Groisman R.
      • Wang J.
      • Horikoshi M.
      • Scully R.
      • Qin J.
      • Nakatani Y.
      Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis.
      ,
      • Jónsson Z.O.
      • Dhar S.K.
      • Narlikar G.J.
      • Auty R.
      • Wagle N.
      • Pellman D.
      • Pratt R.E.
      • Kingston R.
      • Dutta A.
      Rvb1p and Rvb2p are essential components of a chromatin remodeling complex that regulates transcription of over 5% of yeast genes.
      ,
      • Krogan N.J.
      • Keogh M.C.
      • Datta N.
      • Sawa C.
      • Ryan O.W.
      • Ding H.
      • Haw R.A.
      • Pootoolal J.
      • Tong A.
      • Canadien V.
      • et al.
      A Snf2 family ATPase complex required for recruitment of the histone H2A variant Htz1.
      ,
      • Lim C.R.
      • Kimata Y.
      • Ohdate H.
      • Kokubo T.
      • Kikuchi N.
      • Horigome T.
      • Kohno K.
      The Saccharomyces cerevisiae RuvB-like protein, Tih2p, is required for cell cycle progression and RNA polymerase II-directed transcription.
      ,
      • Newman D.R.
      • Kuhn J.F.
      • Shanab G.M.
      • Maxwell E.S.
      Box C/D snoRNA-associated proteins: two pairs of evolutionarily ancient proteins and possible links to replication and transcription.
      ,
      • Shen X.
      • Mizuguchi G.
      • Hamiche A.
      • Wu C.
      A chromatin remodelling complex involved in transcription and DNA processing.
      ,
      • Venteicher A.S.
      • Meng Z.
      • Mason P.J.
      • Veenstra T.D.
      • Artandi S.E.
      Identification of ATPases pontin and reptin as telomerase components essential for holoenzyme assembly.
      ,
      • Wood M.A.
      • McMahon S.B.
      • Cole M.D.
      An ATPase/helicase complex is an essential cofactor for oncogenic transformation by c-Myc.
      ,
      • Zhao R.
      • Davey M.
      • Hsu Y.C.
      • Kaplanek P.
      • Tong A.
      • Parsons A.B.
      • Krogan N.
      • Cagney G.
      • Mai D.
      • Greenblatt J.
      • et al.
      Navigating the chaperone network: an integrative map of physical and genetic interactions mediated by the hsp90 chaperone.
      ). RuvB-like ATPases show similarity to prokaryotic RuvB proteins but, because of an insertion into the ATPase domain, lack the helicase activity found in the bacterial proteins (
      • Ikura T.
      • Ogryzko V.V.
      • Grigoriev M.
      • Groisman R.
      • Wang J.
      • Horikoshi M.
      • Scully R.
      • Qin J.
      • Nakatani Y.
      Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis.
      ,
      • Matias P.M.
      • Gorynia S.
      • Donner P.
      • Carrondo M.A.
      Crystal structure of the human AAA+ protein RuvBL1.
      ). Currently, the precise function of RuvB-like ATPases in the different chromatin remodeling and other complexes is unclear (
      • Jha S.
      • Dutta A.
      RVB1/RVB2: running rings around molecular biology.
      ,
      • Rosenbaum J.
      • Baek S.H.
      • Dutta A.
      • Houry W.A.
      • Huber O.
      • Hupp T.R.
      • Matias P.M.
      The emergence of the conserved AAA+ ATPases Pontin and Reptin on the signaling landscape.
      ). Here, we add chromatin decondensation, a yet-ill-defined but nevertheless essential process during mitosis, to the list of RuvBL1/2-dependent processes. We show that the ATPase activity of RuvBL1/2 is mandatory for chromatin decondensation (Figure 5), in contrast to other RuvBL1/2-dependent processes such as transcriptional regulation (
      • Jónsson Z.O.
      • Dhar S.K.
      • Narlikar G.J.
      • Auty R.
      • Wagle N.
      • Pellman D.
      • Pratt R.E.
      • Kingston R.
      • Dutta A.
      Rvb1p and Rvb2p are essential components of a chromatin remodeling complex that regulates transcription of over 5% of yeast genes.
      ). Because RuvB-like ATPases are part of several chromatin remodeling complexes (for a review, see
      • Jha S.
      • Dutta A.
      RVB1/RVB2: running rings around molecular biology.
      ,
      • Rosenbaum J.
      • Baek S.H.
      • Dutta A.
      • Houry W.A.
      • Huber O.
      • Hupp T.R.
      • Matias P.M.
      The emergence of the conserved AAA+ ATPases Pontin and Reptin on the signaling landscape.
      ), it is tempting to speculate that chromatin decondensation at the end of mitosis functionally requires histone rearrangements, a hypothesis that needs to be addressed in the future.
      Many RuvBL1/2-dependent processes rely on a heterododecameric complex formed by both proteins (
      • Nguyen V.Q.
      • Ranjan A.
      • Stengel F.
      • Wei D.
      • Aebersold R.
      • Wu C.
      • Leschziner A.E.
      Molecular architecture of the ATP-dependent chromatin-remodeling complex SWR1.
      ,
      • Tosi A.
      • Haas C.
      • Herzog F.
      • Gilmozzi A.
      • Berninghausen O.
      • Ungewickell C.
      • Gerhold C.B.
      • Lakomek K.
      • Aebersold R.
      • Beckmann R.
      • Hopfner K.P.
      Structure and subunit topology of the INO80 chromatin remodeler and its nucleosome complex.
      ,
      • Venteicher A.S.
      • Meng Z.
      • Mason P.J.
      • Veenstra T.D.
      • Artandi S.E.
      Identification of ATPases pontin and reptin as telomerase components essential for holoenzyme assembly.
      ,
      • Zhao R.
      • Davey M.
      • Hsu Y.C.
      • Kaplanek P.
      • Tong A.
      • Parsons A.B.
      • Krogan N.
      • Cagney G.
      • Mai D.
      • Greenblatt J.
      • et al.
      Navigating the chaperone network: an integrative map of physical and genetic interactions mediated by the hsp90 chaperone.
      ), and our results confirm that, also in Xenopus eggs, these proteins are found to a large extent in heteromeric complexes. In other processes, such as Polycomb or NF-κB-mediated gene repression and β-catenin signaling, RuvBL1 and RuvBL2 act antagonistically (
      • Baek S.H.
      • Ohgi K.A.
      • Rose D.W.
      • Koo E.H.
      • Glass C.K.
      • Rosenfeld M.G.
      Exchange of N-CoR corepressor and Tip60 coactivator complexes links gene expression by NF-kappaB and beta-amyloid precursor protein.
      ,
      • Bauer A.
      • Chauvet S.
      • Huber O.
      • Usseglio F.
      • Rothbächer U.
      • Aragnol D.
      • Kemler R.
      • Pradel J.
      Pontin52 and reptin52 function as antagonistic regulators of beta-catenin signalling activity.
      ,
      • Diop S.B.
      • Bertaux K.
      • Vasanthi D.
      • Sarkeshik A.
      • Goirand B.
      • Aragnol D.
      • Tolwinski N.S.
      • Cole M.D.
      • Pradel J.
      • Yates 3rd, J.R.
      • et al.
      Reptin and Pontin function antagonistically with PcG and TrxG complexes to mediate Hox gene control.
      ,
      • Kim J.H.
      • Kim B.
      • Cai L.
      • Choi H.J.
      • Ohgi K.A.
      • Tran C.
      • Chen C.
      • Chung C.H.
      • Huber O.
      • Rose D.W.
      • et al.
      Transcriptional regulation of a metastasis suppressor gene by Tip60 and beta-catenin complexes.
      ,
      • Rottbauer W.
      • Saurin A.J.
      • Lickert H.
      • Shen X.
      • Burns C.G.
      • Wo Z.G.
      • Kemler R.
      • Kingston R.
      • Wu C.
      • Fishman M.
      Reptin and pontin antagonistically regulate heart growth in zebrafish embryos.
      ). Our readdition experiments suggest that RuvBL1 or RuvBL2 alone can fulfil the RuvB-like dependent functions in chromatin decondensation and thus, in this context, are redundant (Figure 5). Whether this feature is also seen in other RuvB-like-dependent processes remains to be investigated. It is also possible that chromatin decondensation constitutes a unique and probably archetypal process where RuvBL1 and RuvBL2 can substitute for each other.
      Interestingly, RuvB-like ATPases have been implicated in various human cancers and have been speculated to be a promising therapeutic target (for a review, see
      • Huber O.
      • Ménard L.
      • Haurie V.
      • Nicou A.
      • Taras D.
      • Rosenbaum J.
      Pontin and reptin, two related ATPases with multiple roles in cancer.
      ,
      • Nano N.
      • Houry W.A.
      Chaperone-like activity of the AAA+ proteins Rvb1 and Rvb2 in the assembly of various complexes.
      ). Often, the precise function of RuvBL1 and RuvBL2 in pathogenesis is not defined. Whether their role in chromatin decondensation is relevant for this will be an exciting and promising avenue for future research.

      Experimental Procedures

      Cell-free Decondensation of Mitotic Chromatin

      Cytosol was prepared by crushing activated Xenopus laevis eggs by a low-speed centrifugation (20 min at 21,000 × g) to obtain egg extracts, followed by high-speed centrifugations (twice, 12 min at 360,000 × g). Activation of the eggs—which are naturally arrested in the second meiotic metaphase—by treatment with a Ca2+ ionophore induces meiotic exit. Thus, extracts prepared from these eggs represent a postmitotic/interphasic state and are competent to induce late mitotic/interphasic events such as nuclear reformation or DNA replication. The protocol including the preparation of flotation purified membranes is described in detail in
      • Eisenhardt N.
      • Schooley A.
      • Antonin W.
      Xenopus in vitro assays to analyze the function of transmembrane nucleoporins and targeting of inner nuclear membrane proteins.
      . Mitotic chromatin was isolated as in
      • Gasser S.M.
      • Laemmli U.K.
      Improved methods for the isolation of individual and clustered mitotic chromosomes.
      . In vitro chromatin decondensation was induced by incubating approximately 1,000 mitotic chromatin clusters in 18 μl of cytosol from Xenopus egg extracts and 2 μl of flotation purified membranes supplemented with 3 μM 6-dimethylaminopurine, 10 mM ATP, 10 mM creatine phosphate, 0.2 mg/ml creatine kinase, and 0.4 mg/ml glycogen at 20°C. As a negative control, sucrose buffer (250 mM sucrose, 50 mM KCl, 2.5 mM MgCl2, and 10 mM HEPES [pH 7.5]) was used instead of cytosol. At the end of the incubation time, samples were fixed in 0.5 ml 4% paraformaldehyde (PFA) and 0.5% glutaraldehyde in 80 mM PIPES [pH 6.8], 1 mM MgCl2, 150 mM sucrose, and 10 μg/ml DAPI for 30 min on ice. Chromatin was reisolated by centrifugation though a 30% sucrose cushion in PBS (15 min at 2,500 × g) on poly-L-lysine-coated coverslips and mounted in Vectashield (Vector Laboratories). Samples were analyzed using a confocal microscope (FV1000; Olympus; equipped with a photomultiplier [model R7862; Hamamatsu]) with 405, 488, and 559 nm laser lines and a 60× numerical aperture 1.35 oil immersion objective lens using the FluoView software (Olympus) at room temperature. Immunofluorescence and transmission electron microscopy was performed as in
      • Theerthagiri G.
      • Eisenhardt N.
      • Schwarz H.
      • Antonin W.
      The nucleoporin Nup188 controls passage of membrane proteins across the nuclear pore complex.
      .
      For western blot analysis of reisolated chromatin (modified from
      • Hayashihara K.
      • Uchiyama S.
      • Kobayashi S.
      • Yanagisawa M.
      • Matsunaga S.
      • Fukui K.
      Isolation method for human metaphase chromosomes.
      , the decondensation reaction was increased by a factor of ten. At the end of the reaction, samples were immediately layered on top of 1 ml wash buffer—10 mM HEPES [pH 7.5], 50 mM KCl, 14% (v/v) Optiprep (Sigma), 1 mM dithiothreitol, 2.5 mM MgCl2, 0.2 mM spermine, 0.5 mM spermidine, 1 mM ATP, 10 μg/ml 4-(2-aminoethyl)-benzenesulfonylfluoride, 0.2 μg/ml leupeptin, 0.1 μg/ml pepstatin, 0.2 μg/ml aprotinin—and the chromatin was pelleted (30 min at 10,000 × g in a swing-out rotor) and analyzed.
      In depletion experiments, cytosol was incubated twice with antibody-coated beads at a 1.2:1 beads-to-cytosol ratio for 20 min. CSF-arrested extracts were prepared as in
      • Murray A.W.
      Cell cycle extracts.
      and released into interphase by the addition of 1 mM CaCl2.

      Quantification of In Vitro Chromatin Decondensation

      Chromatin boundaries were defined by an intensity threshold, and the total chromatin area was calculated. For the smoothness analysis, the perimeter of the boundary was used to estimate the surface roughness as a ratio of the perimeter squared over area. To analyze chromatin homogeneity, chromosomes were defined using an edge-finding algorithm (the largest eigenvalue of the structure tensor; ImageJ plugin FeatureJ, http://www.imagescience.org/meijering/), and the sum of the chromosomes’ areas was computed and normalized to the total area within the boundary. To minimize the statistical effects of very irregularly shaped (highly condensed) chromatin, a maximum of 20% (in roughness/relative area) above the fully decondensed state was adopted for both analyses. Surface smoothness and internal homogeneity were defined as the differences from the maximal roughness and maximal relative area, respectively. The fully condensed state was set to zero, and the maximal decondensed state to one and all other values were normalized accordingly.

      Fractionation of Xenopus Egg Extracts

      Xenopus egg cytosol was subjected to sequential fractionation to allow the identification of factors involved in chromatin decondensation. The fractions obtained were then tested in the in vitro assay described earlier for decondensation activity. First, cytosolic egg extract was fractionated by ammonium sulfate precipitation. Proteins that precipitated in 20% ammonium sulfate (fraction A) and those that did not precipitate (fraction B) were separated. Fraction B was then precipitated by increasing the ammonium sulfate concentration to 50%. Both fractions were resuspended in sucrose buffer. Fraction A was then applied to a Hi-Trap-Q-HP-Sepharose column (GE Healthcare) and eluted using a step gradient of 500 mM KCl. The decondensation-active fraction (P1) was further separated on a Superose 6 PC3.2/30 column (GE Healthcare) in sucrose buffer. Fractions were eluted at a 1.5–2.0 ml retention volume. For the decondensation assay, fractions A and B obtained from ammonium sulfate precipitation—as well as the flowthrough, P1, and P2 from the ion exchange—were dialyzed against sucrose buffer. The decondensation assay was always performed in the presence of fraction B in a 1:4 volume ratio. Active fractions eluted from the size exclusion column (G13–G15) were analyzed by mass spectrometry (described in the Supplemental Information).

      Pronuclear Assembly Assay

      For pronuclear assembly, cytosol from Xenopus egg was incubated with 1,000 sperm heads prepared from Xenopus testis (
      • Gurdon J.B.
      Injected nuclei in frog oocytes: fate, enlargement, and chromatin dispersal.
      ) for 10 min at 20°C to allow for sperm chromatin decondensation. To start the reaction, floated DiIC18 (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)-labeled membranes (
      • Antonin W.
      • Franz C.
      • Haselmann U.
      • Antony C.
      • Mattaj I.W.
      The integral membrane nucleoporin pom121 functionally links nuclear pore complex assembly and nuclear envelope formation.
      ), 10 mM ATP, 10 mM creatine phosphate, 0.2 mg/ml creatine kinase, and 0.4 mg/ml glycogen were added. For depletions, cytosol was incubated twice with antibody-coated beads at a 1.2:1 bead-to-cytosol ratio for 20 min.

      Miscellaneous

      Statistical analysis was performed with the IBM-SPSS Statistics 21 software. Live cell imaging, nuclear import and ATPase assays, production of recombinant proteins, and a description of the antibodies used can be found in the Supplemental Experimental Procedures.

      Author Contributions

      A.M. and W.A. designed the experiments. A.M., A.K.S., A.S., and W.A. performed decondensation experiments; A.M. and D.M.-A. performed live-cell imaging; F.Z. designed and wrote the image analysis software for the decondensation measurements; R.S. purified recombinant RuvBL1/2 complexes; H.S. performed electron microscopy; J.M. performed mass spectrometry; and W.A. wrote the manuscript.

      Acknowledgments

      This work was supported by the German Research Foundation and the European Research Council (AN377/3-1 and 309528 CHROMDECON to W.A.) and by a PhD Fellowship of the Boehringer Ingelheim Fonds to A.K.S. We are grateful to A. Konopka (Nencki Institute of Experimental Biology) for help with the statistical analysis; K. Feldmeier for advice on the ATPase activity measurements (Max Planck Institute [MPI] for Developmental Biology); C. Liebig (Light Microscopy Facility of the MPI for Developmental Biology) for suggestions on image acquisition and analysis; and I. Poser in the lab of A. Hyman (MPI of Molecular Cell Biology and Genetics) for providing stable HeLa BAC cell lines (funded by the MitoSys project, European Community’s Seventh Framework Programme [FP7/2007-2013], grant agreement 241548). A.M. is especially thankful for continuous support by G. Wilczyński (Nencki Institute of Experimental Biology).

      Supplemental Information

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      Linked Article

      • RUVs Drive Chromosome Decondensation after Mitosis
          Strzelecka et al.
        Developmental CellNovember 10, 2014
        • In Brief
          Condensation of chromosomes during mitosis is required for their segregation into daughter cells but must be reversed to allow for postmitotic functions. In this issue of Developmental Cell, Magalska et al. (2014) show that the ATPases RuvBL1/2 drive postmitotic chromatin decondensation, demonstrating that this is an active process.
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