Name: imaging replicative domains in ultrastructurally preserved chromatin by electron tomography
Uploaded: 2022-05-20T14:00:00
Description: Watch this Scientific Journal Video about Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography at JoVE.com

This work was supported in part by RSF (grant #17-15-01290) and RFBR (grant #19-015-00273). The authors thank Lomonosov Moscow State University development program (PNR 5.13) and Nikon Center of Excellence in correlative imaging at Belozersky Institute of Physico-Chemical Biology for access to imaging instrumentation.

The protocol is optimized for adherent cells and was tested on HeLa, HT1080, and CHO cell lines.
1. Cell labeling and fixation
<ol>
	<li>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.</li>
	<li>Add EdU (5-ethynyl-2&#39;-deoxyuridine) from 10 mM stock to 10 &#181;M final concentration and place the cells in the incubator for 10 min or longer (depending on the experiment objec...

<ol>
	<li>Rivera-Mulia, J. C., Gilbert, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicating+large+genomes:+divide+and+conquer.">Replicating large genomes: divide and conquer.</a> Molecular Cell. 62 (5), 756-765 (2016).</li><li>M&#233;chali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+DNA+replication+origins:+Many+choices+for+appropriate+answers.">Eukaryotic DNA replication origins: Many choices for appropriate answers.</a> Nature Reviews Molecular and Cell Biology. 11 (10), 728-738 (2010).</li><li>Chagin, V. O., Stear, J. H., Cardoso, M. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superresolution+imaging+reveals+spatiotemporal+propagation+of+human+replication+foci+mediated+by+CTCF-organized+chromatin+structures.">Superresolution imaging reveals spatiotemporal propagation of human replication foci mediated by CTCF-organized chromatin structures.</a> Proceedings of the National Academy of Science. 117 (26), 15036-15046 (2020).</li><li>Leonhardt, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+DNA+replication+factories+in+living+cells.">Dynamics of DNA replication factories in living cells.</a> Journal of Cell Biology. 149 (2), 271-280 (2000).</li><li>Philimonenko, A. A., Hodn&#253;, Z., Jackson, D. 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B., Wu, W., Bian, Q., Kireev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+interphase+large-scale+chromatin+structure+from+analysis+of+engineered+chromosome+regions.">Insights into interphase large-scale chromatin structure from analysis of engineered chromosome regions.</a> Cold Spring Harbor Symposium on Quantitative Biology. 75, 453-460 (2010).</li><li>Lieberman-Aiden, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+mapping+of+long-range+interactions+reveals+folding+principles+of+the+human+genome.">Comprehensive mapping of long-range interactions reveals folding principles of the human genome.</a> Science. 236 (5950), 289-293 (2009).</li><li>Pope, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topologically+associating+domains+are+stable+units+of+replication-timing+regulation.">Topologically associating domains are stable units of replication-timing regulation.</a> Nature. 515 (7527), 402-405 (2014).</li><li>Ou, H. D., Phan, S., Deerinck, T. J., Thor, A., Ellisman, M. H., O'Shea, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChromEMT:+Visualizing+3D+chromatin+structure+and+compaction+in+interphase+and+mitotic+cells.">ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells.</a> Science. 357 (6349), (2017).</li><li>Hoz&#225;k, P., Jackson, D. A., Cook, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+factories+and+nuclear+bodies:+the+ultrastructural+characterization+of+replication+sites+during+the+cell+cycle.">Replication factories and nuclear bodies: the ultrastructural characterization of replication sites during the cell cycle.</a> Journal of Cell Science. 107 (8), 2191-2202 (1994).</li><li>Deng, X., Zhironkina, O. A., Cherepanynets, V. D., Strelkova, O. S., Kireev, I. I., Belmont, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytology+of+DNA+replication+reveals+dynamic+plasticity+of+large-scale+chromatin+fibers.">Cytology of DNA replication reveals dynamic plasticity of large-scale chromatin fibers.</a> Current Biology. 26 (18), 2527-2534 (2016).</li><li>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proceedings of the National Academy of Science. 105 (7), 2415-2420 (2008).</li><li>Gilerovitch, H. G., Bishop, G. A., King, J. S., Burry, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+electron+microscopic+immunocytochemistry+with+silver-enhanced+1.4-nm+gold+particles+to+localize+GAD+in+the+cerebellar+nuclei.">The use of electron microscopic immunocytochemistry with silver-enhanced 1.4-nm gold particles to localize GAD in the cerebellar nuclei.</a> Journal of Histochemistry and Cytochemistry. 43 (3), 337-343 (1995).</li><li>Kireev, I., Lakonishok, M., Liu, W., Joshi, V. 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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...

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 DNA3,4, 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 tags5,6) or by the incorporation of modified DNA synthesis precursors7,8,9,10. 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 &#34;polymer melt&#34; 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 thickness12,13. 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 DNA17,18. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5-ethynyl-2`-deoxyuridine (EdU)</td>
			<td>Thermo Fisher</td>
			<td>A10044</td>
			<td></td>
		</tr>
		<tr>
			<td>2-(4-Morpholino)ethane Sulfonic Acid (MES)</td>
			<td>Fisher Scientific</td>
			<td>BP300-100</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 555-azide</td>
			<td>Termo Fisher</td>
			<td>A20012</td>
			<td></td>
		</tr>
		<tr>
			<td>biotin-azide</td>
			<td>Lumiprobe</td>
			<td>C3730</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumine</td>
			<td>Boval</td>
			<td>LY-0080</td>
			<td></td>
		</tr>
		<tr>
			<td>DDSA</td>
			<td>SPI-CHEM</td>
			<td>26544-38-7</td>
			<td></td>
		</tr>
		<tr>
			<td>DMP-30</td>
			<td>SPI-CHEM</td>
			<td>90-72-2</td>
			<td></td>
		</tr>
		<tr>
			<td>DRAQ5</td>
			<td>Thermo Scientific</td>
			<td>62251</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy resin monomer</td>
			<td>SPI-CHEM</td>
			<td>90529-77-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde (25%, EM Grade)</td>
			<td>TED PELLA, INC</td>
			<td>18426</td>
			<td></td>
		</tr>
		<tr>
			<td>Gum arabic</td>
			<td>ACROS Organics</td>
			<td>258850010</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Panreac</td>
			<td>141396.1209</td>
			<td></td>
		</tr>
		<tr>
			<td>NaBH4</td>
			<td>SIGMA-ALDRICH</td>
			<td>213462</td>
			<td></td>
		</tr>
		<tr>
			<td>NMA</td>
			<td>SPI-CHEM</td>
			<td>25134-21-8</td>
			<td></td>
		</tr>
		<tr>
			<td>N-propyl gallate</td>
			<td>SIGMA-ALDRICH</td>
			<td>P3130</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>MP Biomedicals</td>
			<td>2810305</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver lactate</td>
			<td>ALDRICH</td>
			<td>359750-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-AlexaFluor 488 conjugate</td>
			<td>Termo Fisher</td>
			<td>S11223</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-Nanogold conjugate</td>
			<td>Nanoprobes</td>
			<td>2016</td>
			<td></td>
		</tr>
		<tr>
			<td>tetrachloroauric acid</td>
			<td>SIGMA-ALDRICH</td>
			<td>HT1004</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane (Tris)</td>
			<td>CHEM-IMPEX INT&#39;L</td>
			<td>298</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fluka Chemica</td>
			<td>93420</td>
			<td></td>
		</tr>
		<tr>
			<td>Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Coater</td>
			<td>Hitachi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Copper single slot grids</td>
			<td>Ted Pella</td>
			<td>1GC10H</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy5 fluorescence filter set (Ex620/60 DM660 Em700/75)</td>
			<td>Nikon</td>
			<td>Cy5 HQ</td>
			<td>Alternatives: Zeiss, Leica, Olympus</td>
		</tr>
		<tr>
			<td>Diamond knife Ultra Wet 45o</td>
			<td>Diatome</td>
			<td>DU</td>
			<td>Alternatives: Ted Pella</td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Nikon</td>
			<td>Ti-E</td>
			<td>Alternatives: Zeiss, Leica, Olympus</td>
		</tr>
		<tr>
			<td>High-tilt sample holder</td>
			<td>Jeol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotator</td>
			<td>Biosan</td>
			<td>Multi Bio RS-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission electron microscope operating at 200 kV in EFTEM mode, with high-tilt goniometer</td>
			<td>Jeol</td>
			<td>JEM-2100</td>
			<td>Alternatives: FEI, Hitachi</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Ted Pella</td>
			<td>523</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica</td>
			<td>UltraCut-E</td>
			<td>Alternatives: RMC</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image acquisition</td>
			<td>Open Source</td>
			<td>SerialEM (https://bio3d.colorado.edu/SerialEM/)</td>
			<td></td>
		</tr>
		<tr>
			<td>Image processing</td>
			<td>Open Source</td>
			<td>IMOD (https://bio3d.colorado.edu/imod/)</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging replicative domains in ultrastructurally preserved chromatin by electron tomography

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,&#160;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.

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...

Watch this Scientific Journal Video about Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography at JoVE.com

Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography

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.

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, ...

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This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...