Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography

Summary

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.

Abstract

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.

Introduction

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 replicat....

Protocol

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 cover....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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/)