Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography

Podsumowanie

This protocol presents a technique for high-resolution mapping of replication sites in structurally preserved chromatin in situ that employs a combination of pre-embedding EdU-streptavidin-Nanogold labeling and ChromEMT.

Streszczenie

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, replication, segregation, etc.) remain poorly understood, partially due to the lack of experimental approaches to high-resolution visualization of specific chromatin loci in structurally preserved nuclei. Here we present a protocol for the visualization of replicative domains in monolayer cell culture in situ, by combining EdU labeling of newly synthesized DNA with subsequent label detection with Ag-amplification of Nanogold particles and ChromEM staining of chromatin. This protocol allows for the high-contrast, high-efficiency pre-embedding labeling, compatible with traditional glutaraldehyde fixation that provides the best structural preservation of chromatin for room-temperature sample processing. Another advantage of pre-embedding labeling is the possibility to pre-select cells of interest for sectioning. This is especially important for the analysis of heterogeneous cell populations, as well as compatibility with electron tomography approaches to high-resolution 3D analysis of chromatin organization at sites of replication, and the analysis of post-replicative chromatin rearrangement and sister chromatid segregation in the interphase.

Wprowadzenie

DNA replication is a basic biological process required for faithful copying and transmission of the genetic information during cell division. In higher eukaryotes, DNA replication is subjected to tight spatio-temporal regulation, which is manifested in sequential activation of replication origins¹. Neighboring replication origins firing synchronously form clusters of replicons². At the level of optical microscopy, sites of ongoing DNA replication are detected as replication foci of various number and size. Replication foci display specific patterns of spatial distribution within the cell nucleus depending on the replication timing of the labeled DNA^3,4, which, in turn, is tightly correlated with its gene activity. Thanks to well-defined sequence of DNA replication, strictly ordered in space and time, replicative labeling is a powerful method of precise DNA labeling not only for the study of replication process per se, but also to discriminate a specific DNA sub-fraction with defined transcription activity and compaction level. Visualization of replicating chromatin is usually performed through the detection of major protein components of DNA replication machinery (either by immunostaining or by expression of fluorescent protein tags^5,6) or by the incorporation of modified DNA synthesis precursors^7,8,9,10. Of these, only methods based on the incorporation of modified nucleotides into newly replicated DNA allow for the capture of conformational changes in chromatin during replication, and trace the behavior of replicative domains after their replication is completed.

In higher eukaryotes, DNA packaging into chromatin adds another level of complexity to the regulation of basic genetic functions (transcription, replication, reparation, etc.). Chromatin folding affects accessibility of DNA to regulatory trans-factors and DNA conformational changes (double helix unwinding) required for the template synthesis. Therefore, it is generally accepted that DNA-dependent synthetic processes in the cell nucleus require a structural transition of chromatin from its condensed, repressive state to a more accessible, open conformation. Cytologically, these two chromatin states are defined as heterochromatin and euchromatin. However, there is still no consensus concerning the mode of DNA folding in the nucleus. The hypotheses range from a "polymer melt" model¹¹, where nucleosomal fiber behaves as a random polymer for which the packing density is controlled by phase separation mechanisms, to hierarchical folding models postulating sequential formation of chromatin fiber-like structures of increasing thickness^12,13. Hierarchical folding models recently gained support from molecular approaches based on the analysis of in situ DNA-DNA contacts (chromosome conformation capture, 3C), demonstrating the existence of the hierarchy of chromatin structural domains¹⁴. It is important to note that replication units correlate very well to these chromatin domains¹⁵. The major criticism of these models is based on potential artificial chromatin aggregation caused by sample preparation procedures, such as permeabilization of cell membranes and removal of non-chromatin components, in order to improve chromatin contrast for ultrastructural studies while improving chromatin accessibility for various probes (e.g., antibodies). Recent technical advances in selective DNA staining for electron microscopy by DNA-binding fluorophore-mediated photo-oxidation of diaminobenzidine (ChromEMT⁶) has allowed for the elimination of this obstacle. However, the same considerations hold true for electron microscopy visualization of replicating DNA^17,18. Here we describe a technique that allows for the simultaneous high-resolution ultrastructural mapping of newly synthesized DNA and total chromatin in intact aldehyde-crosslinked cells. The technique combines detection of EdU-labeled DNA by Click-chemistry with biotinylated probes and streptavidin-Nanogold, and ChromEMT.

Protokół

The protocol is optimized for adherent cells and was tested on HeLa, HT1080, and CHO cell lines.

1. Cell labeling and fixation

1. Plate cells on acid-cleaned coverslips in a 3 cm Petri dish. Grow the cells in the media recommended for the cell line being used to 70% confluency.
2. Add EdU (5-ethynyl-2'-deoxyuridine) from 10 mM stock to 10 µM final concentration and place the cells in the incubator for 10 min or longer (depending on the experiment objective). For shorter pulses (up to 2 min), prepare a Petri dish with pre-warmed fresh culture media supplemented with 10 µM EdU, and transfer coverslips in it, rather than adding EdU to the cells directly.
   NOTE: All subsequent steps are carried out at room temperature if not indicated otherwise.
3. Fix the labeled cells with 2.5% glutaraldehyde, freshly made by diluting 25% stock in 100 mM cacodylate buffer, for 1 h.
   NOTE: The subsequent EdU detection steps are sensitive to fixation timing. Longer fixation times may adversely affect EdU labeling, causing lower signal-to-noise ratio.
4. Remove glutaraldehyde by washing the samples with PBS supplemented with 5 mM MgCl₂ (PBS* thereafter). Wash three times with a 10 min incubation for each wash.
5. Permeabilize plasma membranes with 1% Triton X-100 in PBS* (PBS*T thereafter). Wash twice with a 5 min incubation for each wash.
6. Extensively wash the sample with PBS*. Perform five changes and incubate for 5 min in each change.
7. Quench the residual-free aldehyde groups with 20 mM glycine in PBS*, two times for 10 min each.
8. Block the samples in 1% BSA in PBS* for 30 min.

2. Click-reaction

NOTE: This procedure is modified from a previously published protocol¹⁹.

1. Immediately before use, prepare Click-reaction mix for EdU detection: in a microcentrifuge tube, mix 430 µL of 100 mM Tris-HCl (pH = 8.5), 20 µL of 100 mM CuSO₄, 1.2 µL of biotin-azide (10 mM in DMSO), and 50 µL of 0.5 M ascorbic acid. For quality control of the replicative labeling and Click-procedure at the level of fluorescent microscopy, biotin-azide can be replaced by AlexaFluor 488-azide.
   NOTE: The order of addition of components in the above reaction is important.
2. Perform click-reaction in a moist chamber to minimize the evaporation and concentration shifts in the reaction cocktail. Prepare the moist chamber by placing a wet sheet of filter paper on the bottom of the Petri dish and covering it with a paraffin film. Place the coverslips on the film's surface with the cells facing up and layer 50-100 µL of the reaction cocktail onto the coverslip. The reaction takes 30 min at room temperature.
3. Stop the reaction by washing the sample in 0.1% Triton X-100 in PBS* (PBS*T), five times for 5 min each.
4. Block in 1% BSA in PBS*T for 30 min at room temperature.
5. Prepare streptavidin-Nanogold solution with PBS*T containing 1% BSA. Incubate the sample with streptavidin, conjugated with 1.4 nm Nanogold particles (Nanoprobes) in 1% BSA + PBS*T overnight at +4 °C in the newly prepared moist chamber.
6. Stop the reaction and wash the sample in PBS*T, five times for 10 min each.
7. Stabilize biotin streptavidin complex by postfixing in 1% glutaraldehyde in PBS* for 30 min.
8. Remove glutaraldehyde by intense washing with deionized water and wash the sample in PBS*, five times for 20 min each.
9. Quench free aldehyde groups with freshly prepared 1 mg/mL NaBH₄ in water, two times for 10 min each. During incubation, bubbles of H₂ are formed which can lift the coverslips. Carefully push them down with the tweezers.
10. Wash with deionized water five times for 5 min each.
    NOTE: For quality control at the labeling step, it is advisable to perform control labeling with fluorescently labeled streptavidin (Figure 2). This would provide an estimate of labeling intensity and background level before switching to tedious and artifact-prone subsequent steps.

3. Ag-amplification

NOTE: This procedure is modified from Gilerovitch et al., 1995 (see ^20,21).

1. Resuspend 50 g of acacia powder in 100 mL of deionized water. Dissolve for 3 days, degas, and filter through four layers of cheesecloth. Store frozen in 5 mL aliquots in 50 mL tubes. Thaw immediately before use.
2. Add 2 mL of 1 M MES (pH = 6.1) to 5 mL of thawed aliquot of acacia powder solution to make 7 mL of 0.28 M MES (pH = 6.1) in acacia powder solution. Mix by slowly rocking the tube for 30 min. Wrap the tube with foil to protect from light.
3. Simultaneously with step 3.2., wash coverslips with washing buffer (50 mM MES, pH = 5.8, 200 mM sucrose), three times for 10 min each.
4. Prepare the fresh solution of N-propyl gallate (NPG). In a 15 mL tube, dissolve 10 mg of NPG in 250 µL of 96% ethanol, and adjust to 5 mL with deionized water.
5. Put 36 mg of silver lactate in a foil-wrapped 15 mL tube.
6. After step 3.2. is completed, add 1.5 mL of NPG solution to acacia powder mix and rock for another 3 min.
   NOTE: All the subsequent steps are carried out in a darkroom. Use non-actinic safelight (yellow or red).
7. Equilibrate the sample with washing buffer and proceed to the dark room. Simultaneously, add 5 mL of deionized water to silver lactate and dissolve by vigorous shaking for 1-2 min.
8. Add 1.5 mL of silver lactate solution to acacia powder mix, rock slowly for 1 min. Avoid bubble formation, as oxygen in the mix will slow down the reaction.
9. Drain the solution from the dish with coverslips and pour 3 mL of silver lactate-acacia powder mix on the cells. Rock the dish several times and incubate for 2-5 min. Incubation time depends on acacia powder batch and the temperature. This should be defined experimentally.
   NOTE: A longer incubation time may result in unspecific silver binding.
10. To stop the reaction, drain the reaction mix and wash the dish extensively with three to five changes of deionized water, followed by three more changes for 5 min each.
11. Check the staining under the brightfield microscope. Good staining should be light to dark brown with a very faint yellowish background.
    ​NOTE: If the staining is not developing, it is possible to repeat immediately with the already made mix (the mix is active for about 15 min if kept in the dark). However, the staining in this case might develop too fast.

4. Gold toning

NOTE: In order to protect silver nanoparticles from dissolution by OsO₄ oxidation in the subsequent procedures, an impregnation with gold is used at this step (Sawada, Esaki, 1994)²².

1. Rinse coverslips with deionized water.
2. In order to protect silver nanoparticles from dissolution by OsO₄ oxidation, incubate the samples in 0.05% tetrachloroauric acid for 2 min in the dark.
3. Wash thoroughly with deionized water for 10 min.
   ​NOTE: At this stage additional contrast is added to the DNA containing material. The color of the sample should change from brown to black.

5. ChromEM

NOTE: This protocol was modified from Ou et al., 2017¹⁶.

1. Incubate the coverslips and saturate the samples in 10 µM DRAQ5 in PBS for 10 min in the dark.
2. Prepare 0.2% diaminobenzidine tetrahydrochloride (DAB) solution in 50 mM Tris or PBS:
   dissolve DAB in 90% of final volume by stirring vigorously in the dark, then adjust the pH to 7.0-7.4 with NaOH. Adjust the volume to 100% and filter through a 0.22 µM filter, store wrapped in foil.
3. Add DAB solution to cells (2 mL per 3 cm Petri dish) and incubate for 1 min.
4. Move the coverslip into the glass bottom Petri-dish and place it to the stage of an inverted microscope. Irradiate the sample (several fields of view) on an inverted microscope with metal-halide light source and Cy5 filter set (640 nm, 1 W/cm2 at focal plane) through Plan Apo λ 40х (NA = 0.95) lens for 10 min.
   ​NOTE: An indication of successful DAB photoconversion is complete DRAQ5 photobleaching and dark DAB precipitate formation in irradiated nuclei. However, the latter is not always apparent over intense EdU-silver-gold signal (especially when extended EdU pulses are utilized).
5. Wash with deionized water three times for 5 min each.

6. Dehydration and epoxy resin embedding

1. In a microcentrifuge tube, prepare partially reduced osmium tetraoxide solution by mixing 2% aqueous solution of K4Fe(CN)6 with an equal amount of 2% aqueous solution of OsO₄, to get 1% final concentration of both components. Incubate coverslips in reduced OsO₄ for 1 h and wash in deionized water three times for 5 min each.
   NOTE: At this stage the reduced osmium compound reacts with DAB depositions and increases the contrast of DNA.
   CAUTION: Osmium is toxic, work under a fume hood and handle waste according to local safety regulations.
2. Dehydrate the samples in a series of graded ethanol solutions in increasing percentages: 50% EtOH - 3 x 10 min; 70% EtOH - 3 x 10 min, 80% EtOH - 3 x 10 min, 96% EtOH - 3 x 10 min, 100% EtOH - 3 x 10 min.
3. For infiltration and embedding use the resin formula with the component volumetric ratio as follows: epoxy resin monomer:DDSA:MNA:DMP-30 = 9:6:4:0.23 (w/w). This formula is miscible with ethanol.
   1. Incubate the coverslip in the ethanol-resin mix (3 parts 100% EtOH:1 part epoxy resin) for 30 min.
   2. Incubate the coverslip in the ethanol-resin mix (1 part 100% EtOH:1 part epoxy resin) for 2 h.
   3. Incubate the coverslip in the ethanol-resin mix (1 part 100% EtOH:3 parts epoxy resin) overnight.
   4. Replace ethanol-resin mix with freshly prepared pure resin. Incubate in pure resin mix for 8 h. Open the dish to let remnants of ethanol to evaporate.
   5. Transfer coverslips into a new dish with pure resin and incubate for 2 h at 37-42 °C.
   6. Fill a silicon mold with freshly prepared resin. Place the coverslips with the cells facing down on appropriate silicon mold filled with epoxy resin. Cure the resin for 24 h at 37 °C, then at 60 °C for at least 2 days.
      CAUTION: Components of epoxy resin mix are potential carcinogens. Wear gloves and work under the fume hood.
4. Remove the resin slab from the mold, clean the surface of the coverslip with the scalpel or razor blade. To remove the coverslip, drop the coverslip into liquid nitrogen and then transfer the slab into the boiling water. Repeat if necessary.
5. Locate the irradiated area under a stereomicroscope and cut it out from the slab with a hacksaw or any other suitable instrument. Pre-warm the slab at 70 °C on a hot plate, if needed.
6. Fasten the cut-out into an ultramicrotome sample holder for final block trimming. Using the razor, prepare the pyramid shaped sample. Mount the holder with pyramid onto the ultramicrotome and install the knife.
   1. Trim the block so that the irradiated cells bearing EdU label are included. Prepare a semi-thin section (250 nm thickness) suitable for electron tomography.
      NOTE: Section thickness can be adjusted to suit the experimental requirements.
7. Detach section from the knife's edge and place them on the single-slot grids. For electron tomography, 250 nm sections are picked onto 1 mm single-slot grids and after drying these sections can be carbon-coated from both sides. No additional contrasting is necessary.
   NOTE: Since the sections already contain high contrast Au-Ag particles, there is no need to add fiducial gold particles to the surface of the section.
8. Examine the grids in a transmission electron microscope at low magnification (about 600x) to locate the cells with appropriate replication patterns.
   ​NOTE: This protocol generates labeling density high enough for easy detection of replication foci even at low magnification. It is advisable first to create a map of the section for subsequent navigation and angular projections collection for tomography. Switch to higher magnification and take high-resolution images.

7. Electron tomography

1. Insert the grid into the transmission electron microscope with a high-tilt holder. Load the sample into the electron microscope and locate the region of interest.
2. Align the microscope in EFTEM mode in near parallel illumination mode. Tune the energy filter with 20 eV energy-selecting slit placed at zero loss peak.
3. Pre-irradiate the tomography acquisition area at a dose rate of 40 e/Ų/s for at least 1.5-2 min, which corresponds to the total dose at least of 3000 e/Ų.
4. Adjust eucentric height with automatic task in SerialEM. Check autofocus task to ensure that it is working correctly at zero tilt angle and -0.8 mkm target defocus value.
5. Tilt the sample holder to -60° with the Walk-Up task.
6. Set up the tomography acquisition from -60 to +60° with step 2.0°. Exposure should be set dependent on the tilt angle to keep the average intensity on the camera. Limit the image shift in case it causes an energy shift and thus slit misalignment. The tomography data should be saved as .mrc file.
7. Import the .mrc file into IMOD software for tomography reconstruction. Remove the outlier pixel values due to X-rays.
8. Align the tilt series. Perform initial cross-correlation, then manually mark 10-12 gold particles as fiducials. Track the fiducial model and inspect that every fiducial is tracked correctly through the tilt series. Create sample tomograms (slices) and manually mark the thin section borders to prevent the possible tilt of the reconstructed density inside the volume. The mean residual error for fine alignment was less than 1.2 pix.
9. Reconstruct the tomogram with a filtered back projection algorithm and then trim the output to fit the region of interest into the final volume.

Wyniki

Replication foci in mammalian cell nuclei display distinct patterns of distribution within the nucleus depending on S-phase progression. These patterns correlate with transcriptional activity of the loci being replicated. Since the method presented here utilizes a rather strongfixation procedure, it is fairly straightforward to use replicative pulse labeling for specific detection of chromatin loci in various transcriptional states, even under conditions offering best structural preservation of chromatin obtained by room...

Dyskusje

The method described here has several advantages over previously published protocols. First, the use of Click-chemistry for labeling replicated DNA eliminates the necessity of DNA denaturation prerequisite for BrdU detection with antibodies, thus better preserving chromatin ultrastructure.

Second, utilization of biotin as a secondary ligand that is generated after glutaraldehyde fixation and proper quenching of unbound aldehyde groups minimizes chemical modification of the target, thus improv...

Materiały

Name	Company	Catalog Number	Comments
Reagent
5-ethynyl-2`-deoxyuridine (EdU)	Thermo Fisher	A10044
2-(4-Morpholino)ethane Sulfonic Acid (MES)	Fisher Scientific	BP300-100
AlexaFluor 555-azide	Termo Fisher	A20012
biotin-azide	Lumiprobe	C3730
Bovine Serum Albumine	Boval	LY-0080
DDSA	SPI-CHEM	26544-38-7
DMP-30	SPI-CHEM	90-72-2
DRAQ5	Thermo Scientific	62251
Epoxy resin monomer	SPI-CHEM	90529-77-4
Glutaraldehyde (25%, EM Grade)	TED PELLA, INC	18426
Gum arabic	ACROS Organics	258850010
Magnesium chloride	Panreac	141396.1209
NaBH4	SIGMA-ALDRICH	213462
NMA	SPI-CHEM	25134-21-8
N-propyl gallate	SIGMA-ALDRICH	P3130
PBS	MP Biomedicals	2810305
Silver lactate	ALDRICH	359750-5G
Streptavidin-AlexaFluor 488 conjugate	Termo Fisher	S11223
Streptavidin-Nanogold conjugate	Nanoprobes	2016
tetrachloroauric acid	SIGMA-ALDRICH	HT1004
Tris(hydroxymethyl)aminomethane (Tris)	CHEM-IMPEX INT'L	298
Triton X-100	Fluka Chemica	93420
Instruments
Carbon Coater	Hitachi
Copper single slot grids	Ted Pella	1GC10H
Cy5 fluorescence filter set (Ex620/60 DM660 Em700/75)	Nikon	Cy5 HQ	Alternatives: Zeiss, Leica, Olympus
Diamond knife Ultra Wet 45o	Diatome	DU	Alternatives: Ted Pella
Fluorescent microscope	Nikon	Ti-E	Alternatives: Zeiss, Leica, Olympus
High-tilt sample holder	Jeol
Rotator	Biosan	Multi Bio RS-24
Transmission electron microscope operating at 200 kV in EFTEM mode, with high-tilt goniometer	Jeol	JEM-2100	Alternatives: FEI, Hitachi
Tweezers	Ted Pella	523
Ultramicrotome	Leica	UltraCut-E	Alternatives: RMC
Software
Image acquisition	Open Source	SerialEM (https://bio3d.colorado.edu/SerialEM/)
Image processing	Open Source	IMOD (https://bio3d.colorado.edu/imod/)