Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography

Sophie Sosnovskaya; Amir N. Zakirov; Ekaterina D. Ryumina; Ekaterina Kharybina; Sergei A. Golyshev; Olga S. Strelkova; Oxana A. Zhironkina; Andrei Moiseenko; Anton Orekhov; Igor I. Kireev

doi:10.3791/62803

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Method Article

Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography

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DOI:

10.3791/62803

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May 20th, 2022

Sophie Sosnovskaya^*¹^,² , Amir N. Zakirov^*¹^,² , Ekaterina D. Ryumina^*¹^,³ , Ekaterina Kharybina¹^,² , Sergei A. Golyshev¹ , Olga S. Strelkova¹ , Oxana A. Zhironkina¹ , Andrei Moiseenko² , Anton Orekhov⁴ , Igor I. Kireev¹^,²^,⁵

¹Belozersky Institute of Physico-chemical Biology, Lomonosov Moscow State University, ²Faculty of Biology, Lomonosov Moscow State University, ³Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, ⁴National Research Center, Kurchatov Institute, ⁵V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology, and Perinatology

^* These authors contributed equally

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....