Analysis of Histones and Chromatin in Xenopus laevis Egg and Oocyte Extracts

Abstract

Histones are the major protein components of chromatin, the physiological form of the genome in all eukaryotic cells. Chromatin is the substrate of information-directed biological processes, such as gene regulation and transcription, replication, and mitosis. A long-standing experimental model system to study many of these processes is the extract made from the eggs of the anuran Xenopus laevis. Since work in recent years has solidified the importance of post-translational modification of histones in directing biological processes, the study of histones in a biochemically dissectible model such as Xenopus is crucial for the understanding of their biological significance. Here we present a rationale and methods for isolating and studying histones and chromatin in different Xenopus egg and oocyte extracts. In particular, we present protocols for the preparation of: cell-free egg and oocyte extract; nucleoplasmic extract (“NPE”); biochemical purification of maternally-deposited, stored histones in the oocyte and the egg; assembly of pronuclei in egg extract and the isolation of pronuclear chromatin and histones; and an extract chromatin assembly assay. We also demonstrate aspects of the variability of the system to be mindful of when working with extract and the importance of proper laboratory temperature in preparing quality extracts. We expect that these methods will be of use in promoting further understanding of embryonic chromatin in a unique experimental system.

1. Introduction

Chromatin, a macromolecule composed primarily of DNA and protein, is the physiological form of the genome and ultimately the heritable component of eukaryotic cells. Chromatin contains a basic repeating unit called the nucleosome, composed of DNA wrapped around a core octamer of two each of histones H2A, H2B, H3, and H4 [1]. Chromatin is further condensed and utilized by addition of other proteins, including the linker histone H1 [2], trans-acting molecules such as the transcriptional machinery [3], and condensins and cohesins [4]. The histone proteins are highly basic molecules and are frequently modified by a large array of post-translational modifications (PTMs), including: lysine and arginine methylation; lysine acetylation, ubiquitylation, SUMOylation, and ADP-ribosylation; serine, threonine, and tyrosine phosphorylation; and citrullination of methyl-arginine residues [5]. Much evidence now exists to support the hypothesis that the combination of modifications on chromatin-bound histones and the presence and absence of various histone variants contribute to and potentially direct DNA-templated cellular events [6–9]. Therefore the study of chromatin, histones, and their post-translational modifications has become increasingly significant in current research into epigenomics, development, cell-fate, and reprogramming.

While many model systems have been used for the study of chromatin and histones, the vast majority of current work has been conducted in yeast and mammalian cultured cells. Other experimental model systems provide unique opportunities to explore both basic biological phenomena as well as special events at particular life-stages and organism-specific mechanisms. In this piece we focus our attention on a long-standing experimental model whose contributions to our current understanding of fundamental mechanisms underlying the cell cycle are well known, particularly from a biochemical perspective.

The frog Xenopus laevis is a well-established laboratory organism, in part because its macroscopic oocytes and eggs are easily obtainable in large number. These oocytes and eggs can be used for many studies in a variety of approaches, including developmental studies and also biochemical studies in cell-free extracts. Much of the seminal work in understanding biochemical aspects of chromatin and histones was performed in cell-free Xenopus oocyte and egg extracts [10]. However, in recent years fewer research groups have utilized these remarkable extract preparations, in part due to the inability to genetically manipulate the frogs. In our view, this is a minor impediment as the unique capacity to biochemically probe the cell-free extracts and the capacity to utilize defined DNA templates, recombinant proteins, and even the application of heterologous nuclei from cultured cells opens distinct avenues of investigation that are not possible in other models.

Here we present a number of procedures that we utilize to study a variety of aspects of chromatin and histone biology in cell-free extracts of Xenopus oocyte and egg extracts. These procedures include: brief overviews of the preparation of oocyte, egg, and nucleoplasmic extracts (previously published elsewhere) [11–14]; biochemical purification of maternally-deposited, chaperone-bound stored histones in the oocyte and egg extracts; assembly of pronuclei in egg extract and the isolation of chromatin and its constituent histones; and finally a chromatin assembly activity assay. We highlight some previously unreported caveats to the use of Xenopus in the laboratory, including the critical importance of maintaining a low ambient laboratory temperature, and the batch-to-batch variability of extract chromatin assembly activity.

We do note that during the centrifugation of Xenopus eggs and oocytes for preparation of extract, maternal chromatin is pelleted and rendered largely unrecoverable amid debris and insoluble material. Regardless, a single maternal chromatin complement would provide a negligible amount of material to study. Therefore, we do not currently have an approach to study the maternally-deposited epigenome. Despite this limitation, we present procedures for the isolation of pronuclei chromatin and histones from assembled sperm nuclei in egg extract; post-S phase, this mimics an early embryo, pre-mid blastula transition nucleus.

2. Preparation of Extracts

Xenopus laevis extracts are prepared from the oocytes or unfertilized eggs of the female Xenopus frog. Females are primed for egg-laying with injection of pregnant mare serum gonadotropin (PMSG) to induce oocyte maturation, followed by an injection of human chorionic gonadotropin (HCG) to induce the laying of eggs. To date, four egg extracts have been developed and used for the study of a variety of DNA-templated processes, three of which we will discuss here. The first type is a low-speed interphase supernatant (LSS) [15, 16]. This extract has the ability to replicate sperm chromatin through the formation of a transport-competent nucleus. The membrane-free high-speed interphase supernatant (HSS), derived from further clarification of LSS, is replication-incompetent [17], but contains the necessary factors for assembly of the pre-replication complex [18, 19] and has the ability to chromatinize small plasmid DNA. The final egg extract we will discuss is the nucleoplasmic extract (NPE) [20], which is obtained by replicating sperm chromatin in LSS. The resulting nuclei are harvested and crushed by centrifugation to yield a highly concentrated, membrane-free nuclear extract. The addition of this extract to either plasmid or sperm chromatin pre-incubated in HSS results in one semi-conservative round of DNA replication.

In addition to the interphase extracts described above, mitotic extracts (trapped in metaphase II of meiosis) can also be prepared from mature eggs in the presence of EGTA [21, 22]. EGTA serves to chelate the calcium stores released during egg crushing, preventing activation of the extract into S-phase. The addition of calcium leads to degradation of Cyclin B1 and induces this cytostatic-factor (CSF) arrested extract to undergo a complete round of cell cycle, including mitosis. When prepared in the absence of cycloheximide, this extract can reliably execute multiple rounds of cell cycle. While not discussed herein, this extract might allow careful study of changes in chromatin modification state affected during the cell cycle. For protocols for CSF extract preparation, please refer to Murray [23].

Here, we discuss preparation of extracts from both oocytes and laid eggs [24]. While LSS extracts from both oocytes and eggs are able to assemble chromatin, this activity in egg extracts is typically more robust, perhaps due to the maturation of chaperones required for assembly [25]. Unlike egg extracts, extracts prepared from oocytes are incapable of assembling nuclei or initiating replicative synthesis [26], and therefore are unsuitable for the study of chromatin dynamics during DNA replication. However, oocyte extracts display the distinct advantage of being trascriptionally active, evidenced by high levels of transcription from lampbrush chromosomes during oogenesis [27], allowing for the interrogation of chromatin states during gene activation and transcription. In addition, egg extracts have been instrumental in understanding the biochemical nature of the DNA damage signal [28], and can be further used to investigate changes in chromatin structure upon DNA damage and repair [29, 30]. The choice of extract preparation for a particular experiment will depend upon the biological activity that is under investigation. This choice highlights a key feature of the Xenopus cell-free systems in that discrete biological events are dissectible by simply modifying the preparation procedure, allowing for precise biochemical studies in a biological context.

Note that for all extract preparations, beginning with egg laying, we find temperature to be a critical aspect of the procedure. We find that the best quality extracts are prepared from eggs laid at 16 °C. In addition, we find that all egg manipulations are best performed at an ambient laboratory temperature of 22 °C or below, preferably 18 °C, in order to have predictable and reproducible extract activities. This point is especially important for the preparation of nucleoplasmic extract. If this is not possible, we recommend bringing all buffers to final volume with 20% cold dH₂O immediately before use.

All animal use and handling should be done in accordance with an IAUCAC-approved protocol. For protocols regarding frog husbandry [31], handling, and injections, refer to the Xenopus laevis volume of Methods in Cell Biology [32]. For additional protocols regarding the preparation of egg extracts, please refer to Smythe and Newport [11], Tutter and Walter [12], and Lupardus et al [13].

2.1. Oocyte Extract

2.1.1. Materials and Reagents

• Pregnant Mare Serum Gonadotropin (PMSG), Calbiochem #367222

• MS-222, Sigma E10521

• Dissection tools

• 1× Merriam's Buffer: 10 mM Hepes-KOH pH 7.8, 88 mM NaCl, 3.3 mM Ca(NO₃)₂, 1 mM KCl, 0.41 mM CaCl₂, and 0.82 mM MgSO₄

• Dispase II, Roche 04942078001

• Collagenase Type 1A, Sigma C9891

• Sucrose

• Dithiothreitol (DTT, 1 M stock in dH₂O, store at −20 °C)

• 13 × 51 polycarbonate tubes, Beckman #349622

• Ultracentrifuge (Beckman SW-55 rotor)

2.1.2. Procedure for Preparation of Oocyte Extract

Select 1–4 adult female Xenopus frogs that have not laid eggs within the prior 3 months and prime them with 50 units PMSG 3–5 days prior to use in order to maximize the quantity of matured, late stage oocytes. Anesthetize the frogs with MS-222 and sacrifice them according to standard and IAUCAC-approved protocols. Immediately dissect out the entire ovaries, which are found in the abdomen, to a beaker. The ovaries will be easily removed intact, and should contain a large number of obviously mature and large oocytes with clearly pigmented and marked layers; mature stage VI oocytes look almost identical to laid eggs. First, grossly disrupt the ovaries using dissection scissors. Next, disrupt the follicular cell layer with a 3 hr digestion with the mild protease Dispase II (40 mg per 100 mL in 1× Merriam's buffer) at room temperature with very slow stirring with a large stirbar in a beaker (approximately 1 rotation per second). If the stirplate spins too quickly, place a styrofoam tray between the stirplate and the beaker. After this digestion the ovaries should be somewhat dispersed. This step is followed by a 1 hr digestion with collagenase (50 mg per 100 mL in 1× Merriam's buffer). The defolliculated oocytes are then washed extensively with 1× Merriam's containing 200 mM sucrose and 1 mM DTT. The later staged oocytes are denser and will settle to the bottom, while the less dense stage I and II oocytes are mostly lost during the preparation. Finally, transfer the oocytes into 13 × 51-mm Beckman ultracentrifuge tubes with a wide-bore transfer pipet with an equal volume of 1× Merriam's buffer containing 50 mM sucrose. Spin-crush the oocytes at 35,000 rpm (150,000 × g) for 60 min in an SW-55 rotor or equivalent. Remove the middle, clarified layer with a pipette and respin for 30 min at 150,000 × g. Again remove the middle layer, add glycerol to 5% final and flash freeze the extract in appropriately sized aliquots in liquid nitrogen.

2.2. Egg Extract

2.2.1. Materials and Reagents

• Pregnant Mare Serum Gonadotropin (PMSG), Calbiochem #367222

• Human Chorionic Gonadotropin (HCG), Sigma CG10

• L-cysteine, Sigma C7352

• 10× MMR (Marc's modified Ringer's): 1 M NaCl, 20 mM KCl, 10 mM MgSO₄, 20 mM CaCl₂, 1 mM EDTA, 50 mM Hepes-KOH pH 7.8

• 10× ELB salt solution: 25 mM MgCl₂, 500 mM KCl, 100 mM Hepes-KOH pH 7.7

• Sucrose, 2.5 M stock in dH₂O, sterile filtered and stored at 4 °C

• Dithiothreitol (DTT, 1 M stock in dH₂O, store at −20 °C)

• Cycloheximide (10 mg/mL in 50% ethanol/dH₂O), Calbiochem #239763; make fresh every time

• ELB-sucrose: 1× ELB salts, 1mM DTT, 50 μg/mL cycloheximide, 250 mM sucrose; make fresh every time

• Aprotinin (10 mg/mL in dH₂O, store at −20 °C), Sigma A1153

• Leupeptin (10 mg/mL in dH₂O, store at −20 °C), Sigma L2884

• Cytochalasin B (5 mg/mL in DMSO, store at −20 °C), Biomol #T-108

• Falcon 2059 15-mL tubes (BD Biosciences)

• Refrigerated centrifuge (Sorvall HB-6 rotor)

• 13 × 51 polycarbonate tubes, Beckman #349622

• Ultracentrifuge (Beckman SW-55 rotor)

2.2.2. Procedure for Preparation of Interphase LSS

Prime 3–4 female Xenopus frogs with 50 units PMSG 3–5 days prior to use. Approximately 18–20 hr before you would like to begin, induce the frogs to lay eggs with an injection of 1000 units HCG. The next day, pool batches containing eggs with good morphology. Discard batches of stringy eggs, and batches exhibiting excessive lysis as indicated by white coat color and cloudy buffer. Transfer the pooled eggs to a 250-mL beaker and dejelly in 4 volumes 2.2% L-cysteine-KOH, pH 7.7, mixing gently with a transfer pipette every 30 sec. Half way though, discard buffer and add fresh cysteine solution. The dejellying should be complete in 5–6 min, observed by the clear jelly coats floating above the eggs before dissolution and the compact packing of dejellied eggs. After dejellying, quickly move through the MMR washes. Wash the eggs 4–5 times each with about 200 mL 0.5× MMR, making sure to add buffer gently, but with sufficient force to fully resuspend the eggs during each wash. Wash the eggs 3–4 times each with about 200 mL ELB-sucrose. In ELB-sucrose, bad eggs are more buoyant than good eggs and can be easily discarded while decanting the buffer. After the final wash has been decanted, pour eggs from the beaker into 2059 Falcon tubes. Allow the eggs to settle and remove excess buffer using a transfer pipette or by aspiration. Spin for 1 min at 1100 rpm (~200 × g) and remove excess buffer. It is important to pack the tubes as full as possible to allow greater recovery of the resulting interphase extract. For each mL of packed eggs, add 0.5 μL each of aprotinin, leupeptin, and cytochalasin B for final concentrations of 5, 5, and 2.5 μg/mL, respectively. Crush the eggs by centrifugation at 10,000 rpm (~16,000 × g) for 20 min in an HB-6 rotor. The centrifuge should be chilled to 4 °C, but the rotor should be kept at room temperature to insure that the eggs are warm when crushed, resulting in complete activation of the extract into S-phase. After this spin, the extract should be kept on ice at all times. Centrifugal fractionation results in a bottom layer containing yolk and pigment, a middle layer of golden cytoplasm, and a top layer of yellow lipid. Remove the cytoplasmic extract from the side of the tube using an 18-gauge needle as described [12], being careful to avoid the dark pigmented layer containing the mitochondria. Combine all LSS in a 15-mL tube. For each mL of recovered LSS, add 1 μL each of aprotinin, leupeptin, and cytochalasin B for final concentrations of 10, 10, and 5 μg/mL, respectively. Also, for each mL of LSS, add 5 μL 10 mg/mL cycloheximide and 1 μL 1M DTT. Invert gently to mix, being careful to avoid the introduction of air bubbles. The expected yield of the LSS interphase extract is 1–3 mL per frog.

2.2.3. Procedure for Preparation of HSS

The LSS interphase extract can be clarified by further centrifugation to yield high speed supernatant, or HSS. Transfer fresh LSS prepared as described in section 2.2.2 to 13 × 51 mm polycarbonate centrifuge tubes (Beckman #349622), or an appropriately sized tube. Centrifuge at 52,000 rpm (~260,000 × g) in an SW-55 rotor for 90 min at 4 °C (alternatively, centrifuge at 55,000 rpm in a TLS-55 rotor for 60 min at 4 °C). Following the centrifugation, remove the top layer of lipids by gently touching the surface with the bulb of a plastic transfer pipette and slowly lifting. Aspirate residual lipids with either a thin gel-loading tip or a pulled glass capillary and low vacuum. Use a wide-bore P200 tip to transfer the clear HSS to a new ultracentrifuge tube. Avoid the cloudy lower layer containing membranes, as well as the further sedimented mitochondrial and glycogen-containing layers. Centrifuge as before, adjusting the spin time to 30 min. After the final spin, aspirate any remaining lipids with a thin gel-loading tip. The remaining clear HSS is then recovered and mixed thoroughly by pipetting. Freeze the extract as one-use 50 μL aliquots in liquid nitrogen and store at −80 °C, where the extract will be stable for years.

2.3. Nucleoplasmic Extract

The LSS interphase extract can alternatively be used as a precursor to the nucleoplasmic extract, NPE. NPE is obtained by replicating sperm chromatin in LSS and harvesting the highly concentrated extract from the resulting pronuclei. Expected yields for NPE are 10–20 μL per mL of LSS, thus, for a typical preparation of NPE, we prepare LSS from a minimum of 15–20 frogs, scaling the dejellying and wash steps accordingly. Here, we find maintaining an ambient temperature below 22 °C, preferably 18 °C, critical during egg manipulation. Additionally, we find that maintaining a controlled temperature of 21–22 °C during nuclear swelling results in reduced instances of nuclear apoptosis (evidenced by the appearance of dispersed DAPI-stained chromatin fragments while monitoring nuclei swelling), and enhanced activity of the recovered extracts.

2.3.1. Materials and Reagents

• Nocodazole (5 mg/mL in DMSO, store at −20 °C)

• Adenosine triphosphate (ATP, 0.2 M in dH₂O, pH to 7.0, store at −20 °C), Sigma A7699

• Phosphocreatine (1 M in dH₂O, store at −20 °C), Sigma P7936

• Creatine Kinase (5 mg/mL in 10 mM Hepes-KOH pH 7.5, 50% glycerol, 50 mM NaCl, store at −20 °C), Sigma C3755

• Falcon 2063 tubes (BD Biosciences)

• DAPI stain: 7.5% formaldehyde, 0.2 M sucrose, 10 mM Hepes-KOH pH 7.8, 10 μg/mL DAPI (store at −20 °C in amber tube)

• 5 × 20 mm polyallomer tubes, Beckman #342630

• 5 × 20 mm tube adapter for TLS-55 rotor, Beckman #358614

• Ultracentrifuge (Beckman TLS-55 rotor)

2.3.2. Procedure for Isolation of NPE

Prepare LSS as described in section 2.2.2, scaling accordingly. To the LSS, add nocodazole to 3.3 μg/mL and 10% volume ELB-sucrose. The addition of nocodazole inhibits microtubule polymerization and subsequent binding to nuclei, allowing the nuclei to float to the top of the tube in a later centrifugation step. Mix 10x by gentle inversion. Avoid introducing air bubbles, as they lead to oxidation of the extract. Spin LSS in Falcon 2059 tubes for an additional 15 min at 10,000 rpm (~16,000 × g) in an HB-6 rotor at 4 °C. Following the centrifugation, remove residual lipids by first gently touching the surface with the bulb of a plastic transfer pipette and slowly lifting, then by aspiration with a thin gel-loading tip. Next, separate LSS from the residual pigment layer by gently decanting LSS into a new tube, combining all fractions. It is crucial to avoid the mitochondria-containing pigment layer; to do so, approximately 1 mL of LSS must not be decanted. For each mL of extract, add 10 μL 0.2 M ATP, 20 μL 1 M phosphocreatine, and 1 μL 5 mg/mL creatine kinase. Mix 10× by gentle inversion. Add demembranated Xenopus sperm chromatin [11, 23]. First, distribute 4.5 mL LSS to 5 mL Falcon 2063 tubes. Allow LSS to warm to room temperature (3–5 min). While the LSS is warming, thaw the sperm chromatin. Thoroughly mix a 1 mL aliquot of LSS with 90 μL sperm chromatin at 220,000/μL by pipetting 15 times with a P1000 pipette. Transfer the sperm chromatin/LSS mix back to the Falcon 2063 tube and mix 10 times by gentle inversion. The final sperm concentration is 4,400/μL. Note, the optimal final concentration resulting in the greatest volume of nuclei formation must be determined empirically for each sperm chromatin preparation as described [13].

Incubate the nuclear assembly reaction at 21–22 °C in a controlled temperature incubator or water bath, inverting the tubes gently every 10 min. After 30 min of incubation, remove 1 μL assembly reaction into 1 μL DAPI stain and observe the swelling pronuclei using a fluorescence microscope on the 40× objective. Using phase-contrast brightfield illumination, a nuclear envelope should be apparent. The DAPI staining should reveal a diffuse and somewhat rounded appearance of the swelling pronucleus. Continue observing the nuclear swelling at 60 min, and then every 10 min thereafter. When the swollen pronuclei have a punctuated DAPI staining, indicating recondensation of chromatin and the end of S-phase, the nuclei are ready for isolation (typically between 60 and 90 min). To harvest nuclei, place the Falcon 2063 tubes containing the assembly reactions in Falcon 2059 tubes containing a 3 mL dH2O cushion. Centrifuge the assembly for 2 min at 10,000 rpm (~16,000 × g) in an HB-6 rotor at 4 °C. Following this spin, the tubes should be kept on ice at all times. Carefully harvest the nuclei layer, which should be approximately 3–4 mm thick and quite viscous. The nuclei layer will appear as a translucent white layer, while the underlying cytoplasm is golden in color. Use a wide-bore or cut-off pipette tip to slowly withdraw nuclei, 20 μL at a time, and transfer nuclei to a polyallomer 5 × 20 mm Beckman ultracentrifuge tube. Begin withdrawing nuclei from the outside of the tube, moving towards the center while rotating. Allow at least 30 min for this step. Once all apparent nuclei have been recovered in this manner, gently harvest the brown viscous layer underlying the nuclei (~0.5 mL) and transfer to an 0.6 mL microfuge tube. Check the Falcon 2063 tube for additional nuclei and harvest. In the meantime, centrifuge the 0.6 mL microfuge tube for 2–3 min at 16,000 × g. Recover the nuclei layer from this sample, if present.

To collect the nucleoplasmic extract, centrifuge nuclei for 30 min at 55,000 rpm (260,000 × g) in a TLS-55 swing-bucket rotor with tube adapters at 2 °C. The resulting extract should appear clear, topped with a thin lipid layer. Carefully aspirate any lipids present using a thin gel-loading tip. Harvest the clear NPE, taking care to avoid the chromatin-containing pellet. Gently mix the NPE to ensure a uniform extract. Freeze 10 μL NPE aliquots in liquid nitrogen and store at −80 °C. The extract can tolerate at least one additional round of freezing and thawing without significant loss of activity. Finally, if the chromatin-containing pellet is to be saved for analysis of histone content, it should be frozen in liquid nitrogen and stored at −80 °C.

After preparation, each batch of NPE must be tested for activity. We typically test the ability of NPE to replicate a small plasmid or sperm chromatin that has been pre-incubated in HSS (see section 5.2.2). Extracts are tested at ratios relative to HSS of 2:1, 1.5:1, and 1:1, and the ratio used for future experiments is the smallest ratio resulting in complete replication.

3. Isolation of Maternally-deposited, Stored Histones

Xenopus eggs contain a store of nonchromatinized predeposition histones that are complexed with a number of chaperones. These histones are incorporated into chromatin during the 12 cell cycles prior to the mid-blastula transition, the point at which active transcription begins [33–37]. Our recent studies suggest that these histones carry distinct patterns of histone modification that can be correlated with the embryonic pluripotent state [38, 39]. The protocol below relies on the polyanionic structure of heparin, mimicking the negatively charged DNA polymer, to isolate the basic chaperone-complexed histone proteins from a highly concentrated egg extract. These histones can then be further purified by HPLC [40], and analyzed for post-translational modification state using mass spectrometry or immunoblotting with modification-specific antibodies [38, 39].

3.1. Materials and Reagents

• 10× ELB salt solution: 25 mM MgCl₂, 500 mM KCl, 100 mM Hepes-KOH pH 7.7

• Dithiothreitol (DTT, 1 M stock in dH₂O, store at −20 °C)

• Heparin Sepharose CL-6B, GE Healthcare 17-0467-01

• Heparin binding buffer: 1× ELB salts, 10% glycerol, 1 mM EDTA, 1 mM DTT

• Heparin wash buffer: 1× ELB salts, 0.5 M NaCl 10% glycerol, 1 mM EDTA, 1 mM DTT

• Heparin elution buffer: 1× ELB salts, 2 M NaCl 10% glycerol, 1 mM EDTA, 1 mM DTT

• Complete protease inhibitors, EDTA-free, Roche 11 873 850 001

• PhosSTOP phostphatase inhibitors, Roche 04 906 837 001

• Butyric acid, Aldrich B103500

• β-glycerophosphate, Calbiochem 35675

• Sodium Fluoride, Fisher S299

• Phenylmethanesulphonyl fluoride (PMSF, 100 mM stock in isopropanol), Sigma P7626

• Econo-Pac chromatography columns, Bio-Rad #732-1010

• β-mercaptoethanol (βME), Fisher O34461

• 100% trichloroacetic acid (TCA), Fisher A322

• Oakridge centrifuge tubes, Nalgene #3119-0050

• Refrigerated centrifuge (Sorvall SS-34 rotor)

• Coomassie Brilliant Blue stain: 40% methanol, 10% acetic acid, 0.025% Coomassie R-250 in dH₂O

3.2. Procedure for Isolation of Stored Histones

Prepare 3–4 mL LSS egg extract as described in section 2.2.2. During the crushing step, prepare Heparin Sepharose resin. Add 0.5 g Heparin Sepharose CL-6B to 10 mL dH2O in a 50 mL Falcon tube for a final bed volume of ~2 mL. Mix gently at room temperature until resin is hydrated, and then spin for 3 min at low speed (250 × g), discarding the supernatant. Next, gently wash the resin twice for 15 min each with 50 mL Heparin binding buffer. This step removes any additives that are necessary for freeze-drying the Heparin Sepharose. Also, equilibration in the low ionic strength of the binding buffer will allow heparin-histone binding to occur while reducing non-specific interactions.

Prepare the extract for binding to Heparin Sepharose by diluting to 50 mL with Heparin binding buffer containing freshly added protease, phosphatase, and histone deacetylase (HDAC) inhibitors. At this step, when all extract proteins are present, we use Roche complete protease and phosphatase inhibitor tablets. Throughout the protocol, we use 10 mM butryic acid as general inihibitor of most HDACs. Apply the diluted extract to the equilibrated Heparin Sepharose and incubate in batch at 4 °C with gentle rotation for 2–4 hr. Centrifuge the resin at 4 °C for 3 min at 250 × g and reserve the supernatant as flow through for further analysis. If the histone-depleted extract will be used for additional protein purifications, it should be frozen in liquid nitrogen and stored at −80 °C. The Heparin Sepharose is next washed to remove unbound and non-specifically bound material. Wash the resin twice with 50 mL Heparin binding buffer, and then twice with 50 mL Heparin wash buffer, containing freshly added protease, phosphatase, and HDAC inhibitors. For the washes, we use 1 mM PMSF, 25 mM β-glycerophosphate, and 20 mM NaF as protease and phosphatase inhibitors. The resin is washed for 15 min at 4 °C with gentle rotation and then centrifuged at 250 × g for 3 min at 4 °C. Reserve each wash for later analysis.

After the final wash, pour the resin into a Bio-Rad Econo-Pac chromatography column at 4 °C. Elute histones with 25 mL Heparin elution buffer. Dialyze the eluent against 2 L dH₂O with 5 mM βME at 4 °C for 2 hr. Replace the dialysis buffer and dialyze overnight at 4 °C. Due to the reduction of salt concentration, some swelling which increases the sample volume is expected. The soluble histones are then precipitated with trichloroacetic acid (TCA) as described [40]. Briefly, histones are diluted with 100% TCA to 25% final concentration and incubated on ice in an Oakridge centrifuge tube for 30 min. The duration of this incubation may be extended to overnight. Centrifuge at 20,000 × g for 20 min at 4 °C in a pre-chilled SS-34 or equivalent rotor. Use a pipette to carefully remove the supernatant. The histone pellet at this stage may not be visible and may be smeared on the tube wall, therefore note the outside wall of the tube during centrifugation. Remove salts by carefully washing the histone pellet with ice-cold acetone without disturbing it. This acetone wash will solubilize excess acid without dissolving the protein pellet. Centrifuge 20,000 × g for 10 min at 4 °C and then repeat the acetone wash. At this point, the precipitated histones may be visible as a white pellet. Air dry the histone pellet for 20 min at room temperature. Resuspend the pellet in 1 mL dH₂O with 3 mM βME and transfer to a 1.5 mL microfuge tube. Reserve 10 μL for analysis. Freeze the sample in liquid nitrogen and lyophilize. Store the dried histones at −80 °C.

To assess histone purity, separate the reserved samples using 15% SDS-PAGE electrophoresis. Stain the gel with Coomassie Brilliant Blue (Figure 1A). As histones isolated from extracts are difficult to fully resolve by SDS-PAGE, we also recommend immunoblotting with general, modification-independent, antibodies against the four core histones (Figure 1B).

Figure 1.

Analysis of histones isolated from egg extracts and pronuclei. (A) Samples from histone purification from LSS were resolved on 15% SDS-PAGE and stained with Coomassie Brilliant Blue dye. FT - flow through (1 μL), W - wash (5 μL each), E - eluate after TCA precipitation (2 μL). (B) Two microliters of histones after TCA precipitation were immunoblotted with antibodies against H3 (Abcam Ab1791, 1:10,000), H2B (Abcam Ab64164, 1:2000), H2A (α-H2A.X–F [30], 1:2000), and H4 (polyclonal antibody directed against full-length H4 protein, 1:5000). (C) Histones purified from pronuclei were resolved on 15% SDS-PAGE and stained with Coomassie Brilliant Blue dye.

4. Assembly of Pronuclei and Isolation of Histones

Pronuclei, or haploid sperm nuclei (or in the case of Xenopus laevis a diploid, as these frogs have a tetraploid genome), are readily prepared by incubating demembranated sperm in interphase egg extract. The extract decondenses the sperm chromatin, assembles a nuclear envelope and imports nuclear factors, and promotes a single, complete round of DNA replication. The protocol below facilitates isolation of not only histones deposited during replication, but also other chromatin-associated proteins, allowing further understanding of binding requirements for various DNA-templated events. Isolated histones can be further purified by HPLC [40], and analyzed for post-translational modification state using mass spectrometry or immunoblotting with modification-specific antibodies [38, 39].

4.1. Materials and Reagents

• Falcon 2059 15-mL tubes (BD Biosciences)

• 50× Energy mix: 0.5 M phosphocreatine, 0.5 mg/mL creatine kinase, 100 mM ATP, 100 mM MgCl₂, 250 mM Hepes-KOH pH 7.8, 50 mM DTT (store at −20 °C in 10 μL aliquots)

• Torrey Pines ECHOtherm peltier incubator, IC20

• DAPI stain: 7.5% formaldehyde, 0.2 M sucrose, 10 mM Hepes-KOH pH 7.8, 10 μg/mL DAPI (store at −20 °C in amber tube)

• 10× ELB salt solution: 25 mM MgCl₂, 500 mM KCl, 100 mM Hepes-KOH pH7.7

• ELB-CIB buffer: 1× ELB salts, 250 mM sucrose, 1 mM DTT, 1 mM EDTA, 1 mM spermidine, 1 mM spermine, 0.1% Triton X-100

• Sodium butyrate

• PhosSTOP phosphatase inhibitors, Roche 04 906 837 001

• Complete protease inhibitors, EDTA-free, Roche 11 873 850 001

4.2. Isolation of Pronuclear Chomatin and Histones

First, prepare at least 2–3 mL of LSS egg extract, as described in section 2.2.2. Add demembranated sperm to a final concentration of 12,000/mL in a Falcon 2059 tube, with addition of 50× energy mix, thoroughly mix and incubate at 22°C. The Torrey Pines Echotherm peltier incubator is useful for maintaining tubes at this temperature. After 30 min of incubation, remove 1 μL into 1 μL DAPI stain and observe the swelling pronuclei in a fluorescence direct-view microscope on the 40× objective. Using phase-contrast brightfield illumination, a nuclear envelope should be apparent. The DAPI staining should reveal a diffuse and somewhat rounded appearance of the swelling pronucleus. Continue observing the nuclear swelling at 60 minutes, 75 and 90 minutes. When the swollen pronuclei have a punctuated DAPI staining, indicating recondensation of chromatin and the end of S-phase, the nuclei are ready for isolation (typically between 60 and 90 minutes).

The pronuclear chromatin is isolated by disrupting the nuclear envelope and sedimenting the chromatin through a sucrose cushion. First, dilute each 2 mL of nuclei suspension to 10 mL total with ELB-CIB buffer containing freshly added 10 mM sodium butyrate, 1× phosphatase inhibitors and 1× protease inhibitors. Mix thoroughly with a pipet and incubate on ice for 10 min to effect nuclear lysis. Underlay the suspension with 1 mL of ELB-CIB containing 0.5 M sucrose, and isolate the chromatin via centrifugation at 4000 rpm for 5 min. Discard the entire supernatant by carefully decanting, being careful not to dislodge the whitish slightly loose pellet of chromatin at the bottom of the tube. Wash the resulting pellet once with 1 mL ELB-CIB + 250 mM KCl by pipetting up and down. Respin the pellet in a microcentrifuge at 13,000 rpm for 2 min. Discard the supernatant entirely and acid-extract the histones in 0.4N H₂SO₄, as described [40]. The purity of the resulting histones is then analyzed by SDS-PAGE (Figure 1C). Alternatively, prior to acid extraction, the recovered chromatin sample can be analyzed for chromatin-bound proteins directly by SDS-PAGE.

5. Analysis of Extract Activity

The ability of the HSS/NPE extract system to replicate sperm chromatin or small plasmid DNA makes this an advantageous system for the study of many replication related processes. Plasmid DNA added to HSS is first chromatinized with stored histones and then loaded with the pre-replication complex [17–19]. This structure then undergoes one round of complete DNA replication upon addition of NPE [20]. While a functional HSS/NPE system displays robust replication activity, batch-to-batch variability requires that each new preparation of extract be independently assayed. We routinely test new batches of HSS for the ability to chromatinize small plasmid DNA, and for the ability of NPE to replicate small plasmids pre-incubated in HSS. Each new batch is tested directly against a previously validated extract.

5.1. Materials and Reagents

• Phosphocreatine (1 M in dH₂O, store at −20 °C), Sigma P7936

• Creatine Kinase (5 mg/mL in 10 mM Hepes-KOH pH 7.5, 50% glycerol, 50 mM NaCl, store at −20 °C), Sigma C3755

• Adenosine triphosphate (ATP, 0.2 M in dH₂O, pH to 7.0, store at −20 °C), Sigma A7699

• 50× Energy Mix: 0.5 M phosphocreatine, 0.5 mg/mL creatine kinase, 100 mM ATP, 100 mM MgCl₂, 250 mM Hepes-KOH pH 7.8, 50 mM DTT (store at −20 °C in 10 μL aliquots)

• Nocodazole (5 mg/mL in DMSO, store at −20 °C)

• pG5ML or other small plasmid DNA, relaxed with Topoisomerase I

• α³²P-dATP (10 μCi/μL, 3000 Ci/mmol)

• Stop buffer: 10 mM Tris-Cl pH 8.0, 0.5% SDS, 20 mM EDTA

• Proteinase K, Roche 03 115 828 001

• Phenol/chloroform/isoamylalcohol

• Ethanol, 200 proof

• GlycoBlue, Ambion AM9515

• DNA loading buffer

• RNase A

• 1× TAE

• 0.5× TBE

• agarose

• 100% trichloroacetic acid (TCA), Fisher A322

• Gel dryer

• Phoshorimager and screen

5.2.1. Analysis of Plasmid Chromatinization in HSS

When assaying the ability of an HSS extract to chromatinize a small plasmid, there are several factors to consider. First, we and others find that histone stores in the extract are sufficient for the chromatinization of 20–25 ng plasmid DNA per μL of extract [41]. We typically use the maximum concentration in a chromatinization assay to facilitate analysis. Next, we use plasmid DNA that has previously been relaxed with Topoisomerase I. This allows facile discrimination between the free DNA and the faster-migrating chromatinized states (Figure 2A); this migration difference is due to the negative supercoiling induced upon plasmid chromatinization. In these assays, we typically use pG5ML, a 5.5-kb plasmid containing the 5S rRNA positioning sequence, but any other small plasmid, such as pBluescript, will suffice.

Figure 2.

Egg extracts exhibit batch-to-batch variability in chromatinization and replication assays. (A) Relaxed pG5ML was incubated in two independent preparations of HSS, HSS-1 and HSS-2, and analyzed at the indicated time points. The mobility of supercoiled (i.e., chromatinized) (I), relaxed (II), and linear (III) forms of pG5ML are indicated. (B) Supercoiled pG5ML was incubated in either HSS-1 or HSS-2 for 30 or 60 min. Following the addition of 1 vol NPE, replication was measured at the indicated time points.

Quickly thaw 25 μL HSS and place on ice. Add 0.5 μL 50× Energy Mix, 0.35 μL nocodazole at 5 mg/mL, and 625 ng relaxed plasmid DNA. Incubate the extract at 22 °C, taking 5 μL time points into 200 μL stop buffer at 0, 10, 30, 60, and 90 min. Add proteinase K to a final concentration of 500 ng/μL and digest protein at 55 °C for 30 min. At this point, the DNA is phenol/chloroform extracted, followed by ethanol precipitation with GlycoBlue. Resuspend the dried samples in 1× DNA loading dye containing bromophenol blue and RNase A at a conentration of 25 μg/mL. After 10 min at room temperature, electrophorese the samples on an 0.8% agarose gel in 0.5× TBE at 4 °C at a rate of ~3 V/cm until the bromophenol blue dye front has migrated 6–8 cm. Stain the gel in 2 μg/mL ethidium bromide in 0.5× TBE for 30 min, rinse in H₂O, and image (Figure 2A). Note that batch-to-batch variability in HSS extracts is sometimes observed, with the two batches shown exhibiting different kinetics of plasmid chromatinization.

5.2.2. Replication of Plasmid DNA in the HSS/NPE System

Quickly thaw HSS and place on ice. For every 10 μL HSS, add 0.2 μL 50× Energy Mix and 0.14 μL nocodazole at 5 mg/mL. Add plasmid DNA to a final concentration anywhere from 1–20 ng/μL. Aliquot 3 μL for each replication reaction, add 0.1 μL α³²P-dATP to each, and incubate at 22 °C for 30 min. During this time, quickly thaw NPE and place on ice. For every 10 μL NPE, add 0.2 μL 50× Energy Mix and 0.2 μL 1 M DTT, and warm to 22 °C. Add 3 μL NPE to each HSS aliquot and incubate at 22 °C. Take 1 μL time points into 20 μL stop buffer at 10, 20, 30, 45, and 60 min. Add proteinase K to a final concentration of 500 ng/μL and digest protein at 55 °C for 30 min. Add DNA loading buffer and electrophorese the samples on an 0.8% agarose gel in 1× TAE at 10 V/cm until the bromophenol blue has migrated 5–6 cm. Remove the portion of the gel below the dye front to reduce background signal from unincorporated nucleotides. Fix the gel in 10% TCA for 30 min, and then extract liquid from the gel by placing it between several layers of Whatman paper and paper towel topped with a 750 g weight. Complete gel dehydration onto Whatman paper using a gel dryer, and visualize the gel by autoradiography (Figure 2B). A robust HSS/NPE system will completely replicate a plasmid population in 20 min.

The protocol above describes a 1:1 ratio of NPE to HSS. When testing the activity of a new batch of NPE, we recommend assaying NPE to HSS ratios of 2:1, 1.5:1, and 1:1, performing future assays with the lowest ratio resulting in complete replication. Also, while we typically observe more variation in NPE preparation, here we show that the quality of the HSS preparation also contributes to a successful replication assay (Figure 2B). While the first batch tested is able to chromatinize a small plasmid, albeit at a lower rate (Figure 2A), this batch of HSS in unable to support DNA replication, most likely due to a defect in pre-replication complex assembly on the chromatinized plasmid.

6. Concluding Remarks

Xenopus laevis oocyte and egg extracts provide a cell-free system that can faithfully recapitulate many nuclear events. The ability to biochemically manipulate these extracts with defined DNA templates and recombinant proteins allows a distinct advantage in studying the role of histone modifications and variants in a number of chromatin-templated processes. Here, we present a number of protocols for preparation of extracts, as well as biochemical purification of both pre-deposition maternal histones and assembled pronuclear histones. We also stress the importance of temperature while manipulating oocyte and egg extracts.

We report batch-to-batch variability in extract activities, first demonstrated in the kinetics of HSS-mediated chromatin assembly on small plasmid DNA (Figure 2A). This variability is also demonstrated by the inability of certain batches of HSS to support DNA replication (Figure 2B), most likely due to a defect in pre-replication complex assembly on the chromatinized plasmid. While the mechanisms resulting in these deficiencies are unknown, the striking differences observed demonstrate the necessity of testing each preparation of extract for desired activity.