The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.
During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.
Xenopus laevis egg extract is a powerful and widely applied tool to study complicated cellular events in the simplicity of a cell-free assay. Since their first description by Lohka & Masui 1 they have been extensively used to study mitotic processes such as chromatin condensation 2, spindle assembly 3, nuclear envelope breakdown 4, but also nucleocytoplasmic transport 5 or DNA replication 6. The events taking place at the end of mitosis, necessary for reformation of the interphasic nucleus such as nuclear envelope reformation and nuclear pore complex reassembly are much less understood compared to the early mitotic events but can be similarly studied using Xenopus egg extract 7. We have recently established an assay based on Xenopus egg extract to study chromatin decondensation at the end of mitosis 8, an under-investigated process that awaits its detailed characterization.
In metazoans, chromatin is highly condensed at mitotic entry in order to perform faithfully segregation of the genetic material. To ensure that the chromatin is accessible for gene expression and DNA replication during interphase, it needs to be de-compacted at the end of mitosis. In vertebrates, chromatin is up to fifty-fold more compacted during mitosis compared to interphase 9, in contrast to yeasts where the mitotic compaction is usually much lower, e.g., only two-fold in S. cerevisiae 10. Vertebrate chromatin decondensation has been mostly studied in the context of sperm DNA reorganization after egg fertilization. A molecular mechanism, in which nucleoplasmin, an abundant oocyte protein, exchanges sperm-specific protamines to histones H2A and H2B stored in the egg. This process was also elucidated using Xenopus egg extract 11,12. However, the expression of nucleoplasmin is limited to oocytes 13 and mitotic chromatin does not contain these sperm-specific protamines. Therefore chromatin decondensation at the end of mitosis is nucleoplasmin independent 8.
For the in vitro decondensation reaction we employ extract generated from activated X.laevis eggs and chromatin clusters isolated from synchronized HeLa cells. Treatment of eggs with a calcium ionophore mimics the calcium release into the oocyte generated by sperm entry during fertilization. The calcium wave triggers the cell cycle resumption and the egg, arrested in the second metaphase of meiosis, progresses to the first interphase 14. Therefore, egg extracts prepared form activated eggs represent the mitotic exit/interphase state and are competent to induce events specific for mitotic exit like chromatin decondensation, nuclear envelope and pore complex reformation. For the isolation of mitotic chromatin clusters we used a slightly modified version of the protocol published by Gasser & Laemmli 15, where chromosome clusters are released by lysis from HeLa cells synchronized in mitosis and isolated in polyamine containing buffers by gradient centrifugations.
Mitotic Chromatin Cluster Isolation from HeLa Cells
1. Preparations
2. Synchronization of Cells
3. Mitotic Clusters Isolation
4. Preparations of Buffer for Interphasic Xenopus laevis Egg Extract
NOTE: Xenopus laevis frogs are maintained and treated in accordance with the guidelines and regulations set forth by the Convention of the council of Europe on the protection of vertebrate animals used for experimental and other purposes (EU ratified in 1998) and the German law pertaining to the use of vertebrate animals in research.
5. Protocolfor Interphasic Xenopus laevis Egg Extract
6. Preparation of Buffers for In Vitro Reconstitution of Chromatin Decondensation
7. Protocol for In Vitro Reconstitution of Chromatin Decondensation
8. Preparation of Buffer for Immunofluorescence Staining of In Vitro Reconstituted Chromatin Decondensation Samples
9. Protocol for Immunofluorescence Staining of In Vitro Reconstituted Chromatin Decondensation Samples
NOTE: All following incubations of the coverslips are made in a 24-well plate with at least 250 µl solution per well, if not stated otherwise. In vitro decondensed chromatin samples are more sensitive than fixed cells therefore be careful when adding or removing solutions. It is recommended to use plastic Pasteur pipettes cut angular. For washing steps and secondary antibody incubation place the plate at RT on rocking or rotating platform, moving not faster than 100 rpm.
Time dependence of the decondensation reaction
Figure 1 shows a typical time course of the decondensation assay. The cluster of chromosomes visible at the beginning of the reaction decondenses and merges into a single, round and smooth nucleus. When the egg extract is replaced by sucrose buffer the chromosome cluster remains condensed, which suggest that decondensation activity is present in the egg extract.
Chromatin decondensation is an energy dependent process
The in vitro decondensation reaction can be conveniently manipulated e.g., by addition of inhibitors. In the experiment shown on Figure 2, the non-hydrolyzable ATP or GTP analogs, ATPγS or GTPγS, were added to the reaction. Both inhibit the decondensation showing, that it is an ATP and GTP dependent, active process (Figure 2).
Chromatin decondensation and nuclear envelope reformation can be separated
The decondensation assay was performed in the presence or absence of membranes (Figure 3). Please note that in both conditions chromatin undergoes decondensation, however addition of membranes results in bigger nuclei. Most probably, reformation of the nuclear envelope induces a secondary decondensation step by yet another mechanism dependent on nuclear transport.
Figure 1. Time course of the in vitro decondensation reaction. Mitotic chromatin clusters from HeLa cells were incubated with interphasic Xenopus egg extract. Samples were fixed at indicated time points with 4% PFA and 0.5% glutaraldehyde, stained with DAPI and analyzed by confocal microscopy. Re-printed from Magalska et al. 8. Scale bar is 5 µm. Please click here to view a larger version of this figure.
Figure 2. Chromatin decondensation requires ATP and GTP hydrolysis. Chromatin decondensation was performed in the presence of 10 mM ATPγS, 10 mM GTPγS or control buffer. Samples were fixed with 4% PFA and 0.5% glutaraldehyde at indicated time points and analyzed by confocal microscopy. Re-printed from Magalska et al. 8. Scale bar is 5 µm. Please click here to view a larger version of this figure.
Figure 3. Chromatin decondensation in the presence and absence of membranes. Chromatin decondensation was performed in the absence (A) or presence (B) of floatation purified membranes for 120 min. Samples were fixed with 4% PFA and 0.5 % glutaraldehyde and analyzed by confocal microscopy. Chromatin is stained with DAPI, membranes with DiIC18 (1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate). Scale bar is 5 µm. Please click here to view a larger version of this figure.
Xenopus laevis egg extracts are a very useful tool to faithfully reproduce cellular processes in vitro, and this system was successfully used in the characterization of cell cycle and cell division events 2,3,5,6,17. Due to large stores of nuclear components sequestered in the egg during oogenesis, egg extracts are an excellent source of cellular components. Compared to other approaches like RNAi on mammalian tissue cell lines or genetic manipulation, it offers several advantages: The cell-free system allows studying cellular processes in which cellular viability would be otherwise a limitation. Moreover single steps of complex processes can be analyzed in simple assays. The here presented decondensation assay allows studying molecular mechanisms of postmitotic decondensation with no interference from other mitotic events, respectively. Xenopus egg extracts are easy to manipulate by depletion of specific proteins and addition of inhibitors or mutated proteins 8. For example, Figure 2 shows the result of adding the non-hydrolyzable ATP or GTP analogs, ATPγS and GTPγS to the decondensation assay. By dilution and differential centrifugation of Xenopus eggs components like membranes and cytosol can be separated 16. Figure 3 shows the decondensation assay performed in the presence or absence of membranes. Finally, the cell-free assay can also be used to identify novel factors e.g., by a fractionation approach. Using such a strategy we have identified the AAA+-ATPases RuvBL1/RuvBL2 as crucial decondensation factors 8.
In vitro systems based on X. laevis eggs have been employed with different DNA templates: Forbes et al. showed that injection of phage λ DNA into unfertilized X. laevis eggs induced the assembly of chromatin on naked phage λ DNA. As injection of viral DNA activated the egg, the assembly of chromatin was followed by formation of a nucleus-like structure 18 and similarly λ-phage DNA can be used in combination with egg extracts to generate nucleus like structures in vitro 19. Magnetic beads coated with DNA have been used to study chromatinization of DNA 20 and recruitment of nuclear membranes 21 as well as assembly of a nuclear envelope and pore complexes 22, although it remains open to which extent this resembled a bona fide nuclear re-assembly process. The protocol presented here allows decondensation of isolated mitotic chromatin clusters from HeLa cells using extract generated from activated Xenopus eggs. It thoroughly reconstructs events leading to a reformation of an interphasic nucleus 8. Compared to the widely applied nuclear assembly reaction used to study the formation of the nuclear envelope and the nuclear pore complexes at the end of mitosis, in the decondensation assay HeLa mitotic chromatin clusters instead of sperm DNA are used. Sperm DNA can be assembled into mitotic chromatin or even individual chromosomes upon incubation with extract prepared from unfertilized and non-activated eggs 3. We decided to use mitotic clusters as chromatin source to simplify the procedure and avoid interference from chromatin condensation. In addition, the preparation of the egg extract is slightly modified: For the chromatin decondensation low speed extract cleared by two high speed centrifugation steps in fixed angle rotors are used. Low speed extract can be stored for up to 6 month in liquid nitrogen without losing its activity. In contrast, in the nuclear assembly reactions, cytosol and floated membranes are generated from low speed extracts by dilution and differential high-speed centrifugation before possible freezing (see Eisenhardt et al. 16 for a detailed protocol). In our assay system, addition of membranes allows the formation of a closed nuclear envelope including nuclear pore complexes. The resulting nuclei are competent for nuclear import and export 8. Thus, this system supports both chromatin decondensation and nuclear envelope reformation. Interestingly, chromatin decondensation is also possible in the absence of membranes (Figure 3). However addition of membranes results in slightly bigger nuclei. Most likely, the reformation of the nuclear envelope induces a secondary decondensation step by yet undefined mechanisms, which depends on nuclear import.
For the isolation of mitotic chromatin clusters from HeLa cells, a modified version of the protocol established by Gasser and Laemmli 15 was used. Synchronized mitotic cells are lysed in a buffer containing the non-ionic detergent digitonin and by mechanic forces. The chromatin is isolated as clusters that contain all chromosomes from one nucleus. The crucial difference compared to single chromosome isolation protocols is the fact that the cells are not hypotonically swollen but cooled down to 4 °C before lysis. This prevents the disconnection of the individual chromosomes 15,23. Compared to the protocol published by J.R. Paulson 23 who recognized the advantage of the isolation of whole chromatin clusters, Gasser & Laemmli used EDTA-containing polyamine buffers instead of Mg2+ based buffers to reduce the activity of kinases, nucleases, proteases and phosphatases and by this decrease the amount of protein and DNA modifications occurring during the isolation process 15. Additionally, using a colloidal silica particles gradient during differential centrifugation highly reduces cytoplasmic contamination. The protocol can also be used to isolate mitotic chromatin clusters from Chinese hamster ovary and mouse cells 15.
Altogether, our protocol faithfully reconstitutes chromatin decondensation as it happens at the end of mitosis. The ATP dependence of the in vitro chromatin decondensation can be at least in part explained by the involvement of RuvBL1/2 but also another AAA+-ATPase, p97, which removes the mitotic kinase Aurora B from the chromatin during mitotic exit 24. Why the process requires GTP hydrolysis is one of the open questions that we intend to answer using this setup.
The authors have nothing to disclose.
This work was supported by the German Research Foundation and the ERC (AN377/3-2 and 309528 CHROMDECON to W.A.) and a PhD Fellowship of the Boehringer Ingelheim Fonds to A.K.S. Figure 1 & 2 are reprinted from Developmental Cell 31(3), Magalska et al., RuvB-like ATPases function in chromatin decondensation at the end of mitosis, 305-318, 2014, with kind permission from Elsevier.
spermine tetrahydrochloride | Fluka analytical | 85610-25G | |
spermidine trihydrochloride | Sigma | S2501-5G | |
high-purity digitonin | Millipore | 300410-1GM | toxic |
PMSF | Applichem | A0999,0100 | toxic |
thymidine | Calbiochem | 6060 | |
nocodazole | Calbiochem | 487928 | toxic |
37 % formaldehyde solution | Roth | 7398-1 | toxic |
trypan blue solution (0.4%) | Sigma | T8154 | toxic |
1,4-dithiothreitol (DTT) | Roth | 6908.2 | |
AEBSF hydrochloride | Applichem | A1421,0001 | |
pepstatin | Roth | 2936.1/2/3 | |
leupeptin | Roth | CN334 | |
aprotinin | Roth | A162.3 | |
Percoll (colloidal silica particles solution) | GE Healthcare | 17-0891-01 | |
glutamine | Gibco | 25030-024 | |
Penicillin-Streptomycin | Gibco | 15140-122 | |
75 cm² tissue culture flasks | Greiner Bio-one | 658175 | |
heat-inactivated fetal bovine serum (FBS) | Gibco | 10500-064 | |
Homogenizer (40 mL tissue grinder) | Wheaton | 357546 | |
Neubauer chamber | Assistent | 441/1 | |
Oak Ridge Centrifuge Tubes, polycarbonate (50 ml) | Nalgene | 3118-0050 | |
100 µm cell strainer, nylon | BD Falcon | 352360 | |
cytochalasin B | Applichem | A7657,0010 | toxic |
cycloheximide | Roth | 8682.3 | toxic |
L-cystein | Merck | 1,028,381,000 | |
hCG available as Ovogest | MSD | 1431593 | |
PMSG available as Intergonan | MSD | 1431015 | |
A23187 (mixed calcium-magnesium-salt) | Enzo | ALX-450-002-M010 | toxic |
syringe needles (1.20 x 40 mm, 18 G x 1 1/2") | Braun | 4665120 | |
ATP | Serva | 10920.03 | |
GTP, 2 Na x 3 H20 | Roth | K056.1/2/3/4 | |
creatine phosphat disodium salt | Calbiochem | 2380 | |
creatine phosphokinase | Sigma | C3755-35KU | |
DMAP | Sigma | D2629-1G | |
DAPI | Roche | 10236276001 | |
PFA | Sigma | P-6148 | toxic |
centrifugation tubes for TLA 100 (7 x 10 mm, 5/16 x 13/16 in.) | Beckman Coulter | 343775 | |
"Cell-Saver" (tips with wide opening, 1000 µL) | Biozym | 729065 | |
50 % glutaraldehyde solution, grade I | Sigma alderich | G7651-10 mL | toxic |
0.1 % (w/v) poly-L-lysine solution | Sigma | P8920-100 mL | |
flat-bottom tubes (6 mL, 16.0/55 mm) | Greiner Bio-one | 145211 | |
Vectashield mounting medium | Vector laboratories | H1000 | |
tubes for TLA120 (11 x 34 mm, 7/16 x 1 3/8 in.) | Beckman Coulter | 343778 | |
"Cell-Saver" (tips with wide opening, 200 µL) | Biozym | 729055 | |
12 mm coverslips | Thermo Scientific | 0784 #1 |